Embed Size (px)
Citation preview
University of Groningen
Shiga toxin-producing Escherichia coli (STEC) from Humans in the NetherlandsFerdous, Mithila
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.
Document VersionPublisher's PDF, also known as Version of record
Publication date:2017
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):Ferdous, M. (2017). Shiga toxin-producing Escherichia coli (STEC) from Humans in the Netherlands: Noveldiagnostic approach, molecular characterization and phylogenetic background. [Groningen]: University ofGroningen.
CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.
Download date: 26-06-2020
1
Mithila Ferdous
Shiga Toxin-Producing Escherichia coli (STEC) from Humans in the Netherlands
Novel Diagnostic Approach, Molecular Characterization and
Phylogenetic Background
2
The work described in this thesis was performed in the Department of Medical Microbiology
of the University Medical Center Groningen, the Netherlands, in collaboration with regional
partners specifically CERTE laboratory for infectious diseases. The printing of the thesis was
financially supported by Groningen University Institute for Drug Explorations (GUIDE) and
Royal Netherlands Society for Microbiology (KNVM) - division microbial typing.
ISBN : 978-90-367-9480-0 (printed version)
ISBN : 978-90-367-9479-4 (electronic version)
Cover : Growth of Shiga Toxin-Producing Escherichia coli (STEC) in CHROMagar STEC medium
under UV light; in the magnifying glass comparison of different STEC genomes using blast ring
image generator, and a picture of E. coli plush toy (GIANTmicrobes®).
Printing : CPI Koninklijke Wöhrmann B.V., Zutphen, The Netherlands
Copyright : Mithila Ferdous, 2017. No part of this publication may be reproduced or transmitted in
any form or by any means without the permission of the author.
3
Shiga Toxin-Producing Escherichia coli (STEC) from Humans in the Netherlands
Novel Diagnostic Approach, Molecular Characterization and
Phylogenetic Background
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 13 February 2017 at 14.30 hours
by
Mithila Ferdous born on 18 November 1984
in Kushtia, Bangladesh
4
Supervisor
Prof. dr. A.W. Friedrich
Co-supervisor
Dr. J.W.A. Rossen
Assessment Committee
Prof. dr. J.M. van Dijl
Prof. dr. H.J. Verkade
Prof. dr. E.J. Kuijper
5
To my parents
6
Paranimfen
Sigrid Rosema
Monika Chlebowicz
7
Contents
Chapter 1. General Introduction and Scope of the Thesis 9
Chapter 2. Assessing the Public Health Risk of Shiga Toxin-Producing Escherichia
coli by Use of a Rapid Diagnostic Screening Algorithm
31
Chapter 3. Molecular Characterization and Phylogeny of Shiga Toxin-Producing
Escherichia coli Isolates Obtained from Two Dutch Regions Using Whole
Genome Sequencing
55
Chapter 4. Comprehensive Characterization of Escherichia coli O104:H4 Isolated
from patients in the Netherlands
81
Chapter 5. The Mosaic Genome Structure and Phylogeny of Shiga Toxin-Producing
Escherichia coli O104:H4 is Driven by Short-term Adaptation
99
Chapter 6. Is shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or
Enterohemorrhagic Escherichia coli? Comprehensive Molecular
Analysis Using Whole Genome Sequencing
121
Chapter 7. Virulence, Antimicrobial Resistance Properties and Phylogenetic Background
of Non-H7 Enteropathogenic Escherichia coli O157
141
Chapter 8. General Discussion, Summary and Future Perspectives 161
Appendices
Nederlandse Samenvatting
Acknowledgements
Biography
List of Publication
173
177
181
183
8
9
CHAPTER 1
General Introduction and Scope of the Thesis
Chapter 1
10
GENERAL INTRODUCTION
Escherichia coli (E. coli)
Escherichia coli is a facultative anaerobic, gram-negative rod that lives in the intestinal tract of
human and animals. E. coli was first described by the German-Austrian pediatrician Theodor
Escherich in 1885, as Bacterium coli commune, which he isolated from the feces of new-born (1). E.
coli typically colonizes the gastrointestinal tract of human infants within a few hours after birth. The
bacterium is ingested with foods or water or obtained directly from other individuals handling the
infant and can adhere to the mucus overlying the large intestine (2, 3).
The relationship between E. coli and the host can be defined as commensalism, as the
normal E. coli microbiota provides some benefits to its host by preventing colonization of other
pathogens through the production of bacteriocins and several other mechanisms (4). However, in
specific situations, for example, when E. coli colonizes other organs and tissues, or when the host
immune system has been suppressed by drug treatment or by other illnesses, E. coli can become an
opportunistic pathogen and cause infection. Highly-adapted E. coli strains are capable of causing a
variety of different intestinal and extraintestinal, sometimes life-threatening diseases with diarrhea
and urinary tract infection being the most common ones (3). Recent genome analyses indicated that
acquisition and loss of genes has contributed to the emergence of pathogroups in E. coli (5, 6).
Diarrheagenic E. coli
The most common group of pathogenic E. coli is the group of Diarrheagenic E. coli (DEC) referring to
the strains that can cause diarrhea or other gastrointestinal diseases. Based on their specific
virulence factors and phenotypic traits they can be divided into six main categories (3) (Table1).
Shiga toxin-producing E. coli (STEC)
Overview
STEC refers to those strains of E. coli that produce at least one member of a potent cytotoxin family
called Shiga toxin (Stx). STEC is one of the major food-borne pathogens associated with outbreaks
and sporadic cases of diarrhea and severe diseases, including hemorrhagic colitis (HC) and hemolytic
uremic syndrome (HUS) in human (7, 16). Enterohemorrhagic E. coli (EHEC), a subpopulation of STEC,
can cause bloody diarrhea and some even hemolytic uremic syndrome (HUS) and these are known as
HUS-associated E. coli or HUSEC (17). Karmali and colleagues first identified STEC as the infectious
agent responsible for HUS after correlating E. coli infection in patients with diarrhea and HUS with
the presence of the toxin (16, 18). Treatment of STEC infection is mainly restricted to supportive care
Introduction and Scope of the Thesis
11
including balancing fluid level and electrolytes, and monitoring the possible development of
microangiopathic complications, such as HUS (19). Antibiotic therapy is considered to be not
beneficial as several antibiotics have been observed to induce the expression and release of Stx (20,
21).
Epidemiology of STEC infection
STEC emerged as human pathogens for the first time in the USA in the early 1980s during large-scale
outbreaks of HC and HUS caused by shigatoxigenic, non-sorbitol fermenting (NSF) strains of E. coli
O157:H7 (22-24). Ever since, strains of NSF E. coli O157:H7 have been epidemiologically,
microbiologically and clinically important worldwide. In addition, a new lineage, a sorbitol fermenting
(SF), non-motile O157:NM, was identified as the cause of outbreaks of HUS in Germany in 1988 (25,
26). Currently, over 450 O:H serotypes of human and non-human origin have been detected among
STEC (27). Besides O157, serotypes O26, O45, O103, O111, O121, and O145 have been involved in
outbreaks and severe illness, and are considered as the “top (or big) six STEC” (28).
Ruminants and primarily cattle are the most important natural reservoir of STEC. STEC is usually not
pathogenic for cattle, although some serogroups can cause diarrhea in calves (29). STEC can be found
in many other reservoirs, e.g. water, soil, meat, fruit and vegetable products that are contaminated
with ruminants’ fecal material. Furthermore, person to person transmission is a well-documented
phenomenon during outbreaks and is probably also a reason for a significant portion of sporadic
cases (30).
Chapter 1
12
Table 1. Charateristics of six Diarrheagenic E. coli groups
Diarrheagenic E. coli Groups
Type of diarrhea Major virulence factors Other specifications Reference
Enterotoxigenic E. coli (ETEC)
Acute, self-limited, secretory diarrhea in children and in travelers from industrialized to developing countries
Toxin: Heat-labile enterotoxin (LT), Heat-stable enterotoxin (ST), Adhesin : Fimbrae, Autotransporter EtpA
First recognized in the 1960s (7-10)
Enteroaggregative E. coli (EAEC)
Persistent diarrhea in children
Adhesin : Aggregative adherence fimbriae (AAF), Dispersin Toxin : Autotransporter protease Pic, Shigella enterotoxin 1 (ShET1), EAEC heat-stable enterotoxin 1, Plasmid-encoded enterotoxin (Pet)
First described in 1987. Adhere to HEp-2 cells in an aggregative (AA) pattern
(11, 12)
Enteropathogenic E. coli (EPEC)
Infant diarrhea in developing countries
Described below in detail First strain of E. coli causing outbreaks of infantile diarrhea described in the 1940s
(3)
Enteroinvasive E. coli (EIEC)
Dysentery, Watery diarrhea, Invasive inflammatory colitis
Plasmid-borne type III secretion system, encoded by mxi and spa genes, enables the insertion of a pore containing IpaB and IpaC proteins into host cell membranes
First described in 1944 (3, 13)
Shiga Toxin-producing E. coli (STEC)
Diarrhea, Bloody diarrhea, Hemorrhagic colitis, Hemolytic uremic syndrome
Described below in detail
Diffusely adherent E. coli (DAEC)
Diarrhea in immunologically naive or malnourished children
Fimbrial and afimbrial (Afa) adhesins, collectively designated Afa–Dr adhesins
Defined by a pattern of diffuse adherence (DA) on HeLa and HEp-2 cells in which the bacteria uniformly cover the entire cell surface
(14, 15)
Introduction and Scope of the Thesis
13
Major Virulence Factors of STEC
Toxins
(a) Shiga toxins (Stx)
The most important virulence factor of STEC is the Shiga toxin (Stx). It is named after Kiyoshi Shiga,
who first described the bacterial origin of dysentery caused by Shigella dysenteriae (34). In E. coli this
toxin was first described in 1977 as a vero cytotoxin and named as VT because of its toxic effect to
vero cells (kidney epithelial cells extracted from an African green monkey) in culture (18). Afterwards
the name shiga-like toxin and eventually shiga toxin was used by several investigators around the
world (35).
Structure and Function of Stx
The toxin is a multisubunit protein composed of one molecule of an A subunit StxA, (molecular
weight 32 kDa) responsible for the toxic activity of the protein, and five molecules (each has a
molecular weight of 7.7 kDa) of the B subunit StxB (Figure 1a) (36). The B subunit binds to
globotriaosylceramide-3 (Gb3) (Figure 1b) (37) present on specific cells and thereby determining the
site pathophysiology in the host. The A subunit exhibits an RNA N-glycosidase activity against the 28S
rRNA that inhibits host protein synthesis and induces apoptosis (38, 39). In humans, Stx released by
the bacteria binds to endothelial cells expressing Gb3, allowing its absorption into the bloodstream
and dissemination to other organs (38). Renal glomerular endothelium expresses high levels of Gb3
in humans, and Stx production results in acute renal failure, thrombocytopenia, and
microangiopathic hemolytic anemia, all typical characteristics of HUS (23).
Figure 1. Shiga toxin structures. a) Shiga toxin with one A subunit (StxA), cleaved into fragments A1 and A2, and five B fragments that constitute the homopentameric B subunit (StxB). b) A ribbon diagram of Shiga toxin, highlighting globotriaosylceramide (Gb3- shown in a ball and a stick representation) binding sites on StxB. c) An enlargement of StxA at the site of the furin cleavage site (Arg25-Met252), and showing the disulphide bond between Cys242 and Cys261, linking the A1 and A2 fragments. The figure is reproduced with permission from the Nature publishing group (39).
Chapter 1
14
Stx subtyping
Stxs from E. coli are classified into two major types, Stx1 and Stx2 which share approximately 60%
amino acid identity (40). Stx1 appears to be more homogeneous and is almost identical to Stx from S.
dysenteriae type 1 (41). Stx2 comes in different variants that may display a few amino acid changes
influencing the disease outcome (40, 42) (Table 2). Recently, a new nomenclature for Stx variants or
subtypes was proposed by Scheutz et al (35) which is used in this thesis.
Table 2. Features of different Stx subtypes with their classical (old) and latest (new) nomenclature
Latest nomenclature according to Scheutz et al., 2012 (35)
Classical typing as described in references (42-45)
Disease association
Stx1 Stx1a Stx1 Presence of Stx1 alone (without Stx2) is mostly associates with mild diarrhea or asymptomatic carrier Stx1c Stx1c
Stx1d Stx1d
Stx2 Stx2a Stx2 More often associated with HUS
Stx2b Stx2d Associated with diarrhea or asymptomatic carrier
Stx2c Stx2c More often associated with HUS
Stx2d Stx2dact*
Stx2e Stx2e Associated with pig edema disease, not common in human
Stx2f Stx2f Initially feral pigeons were the natural reservoir, but nowadays emerging in human
Stx2g Stx2g Mostly isolated from cattle and water, not common in human
*Stx2dact refers to the toxin type which has the capacity to be activated by elastase present in intestinal mucus. According
to the nomenclature by Sheutz et al., Stx2d includes the activatable Stx2 toxins and the non activatable Stx2d are
designated as Stx2b.
Stx lost E. coli
Sometimes E. coli of O157:H7/NM and other serotypes without an stx gene are excreted from the
feces of patients with HUS or diarrhea and the strains share other virulence genes common in STEC
and belong to the same MLST (Multilocus sequence typing) clonal complex as corresponding STEC
serotypes. These isolates are considered as Stx lost variants which might have lost the Stx encoding
bacteriophage during the course of infection or during culturing procedures in the laboratory (46-48).
(b) CDT (cytolethal distending toxin)
Although CDT has been associated mainly with EPEC and necrotoxigenic E. coli, it was later detected
in the majority of STEC O157:NM strains (49). CDT is encoded by three genes, cdtA, cdtB, and cdtC,
Introduction and Scope of the Thesis
15
which are required for its cytotoxicity and is involved in chromatin disruption, which leads to G2/M-
phase growth arrest of the target cell and ultimately cell death (50).
(c) Enteroaggregative heat-stable toxin
Enteroaggregative heat-stable toxin (EASTI) is one of the toxins produced by the EAEC but it is also
found in STEC strains (51), where it may contribute to the initial phase of watery diarrhea (52).
Pathogenicity islands (PAIs)
(a) Locus of Enterocyte Effacement (LEE)
One of the most important characteristics of STEC is the ability to produce attaching and effacing
(A/E) lesions as does the EPEC on a variety of cell types (7, 53). These lesions are characterized by the
intimate adherence of bacteria to the enterocyte, dissolution of the brush border at the site of
bacterial attachment, and disruption of the cellular cytoskeleton (54). This adherence is mediated by
the outer membrane protein intimin encoded by the eae gene, which is part of a PAI named the locus
of enterocyte effacement (LEE) (55). The LEE of STEC comprises 43,359 bp and contains 41 genes. The
open reading frames (ORFs) that are not present in the EPEC LEE fall within a putative prophage,
designated 933L, that is located next to the selC locus. Besides eae, the tir gene encoding the
translocated intimin receptor (Tir), and the cesT gene encoding the Tir chaperone are required for
adhesion to the host cell (54, 56). LEE also contains a type three-secretion system (TTSS), necessary
to translocate bacterial proteins towards the enterocyte (55, 57), espADB genes (EPEC secreted
proteins) encoding translocator proteins that form a conduit through which the TTSS delivers
effector proteins to the host cell (58). There are also non-LEE encoded effector (nle) proteins
translocated by the TTSS, including Cif (carried on a lambdoid phage), EspI/NleA (carried by a
prophage CP- 933P) and TccP/EspFu (carried on prophage CP-933U) (59). The proteins located on the
LEE and the prophages are shown in Figure 2.
(b) The High pathogenicity island (HPI)
The HPI was first described in pathogenic Yersinia species and later found in non O157 STEC isolates.
HPI encodes the pesticin receptor FyuA and the siderophore yersiniabactin. There is a hypothesis
that this island can contribute to the fitness of the strains in certain ecological niches (46, 60).
(c) Tellurite Resistance and Adherence conferring Island (TAI)
A 35-kb PAI has been detected in STEC O157:H7 and other STEC strains including serotypes that do
not contain the eae gene (61). This island contains a gene that encodes a novel bacterial adherence-
conferring protein similar to the iron-regulated gene A (IrgA) of Vibrio cholerae and was therefore
Chapter 1
16
termed Iha (IrgA homologous adhesin). The tellurite resistance locus was found adjacent to the iha
gene (46).
Pathogenecity island O#122
Most STEC strains contain a 23 Kb PAI termed O#122. PAI O#122 carries efa1/lifA, a 10 kb virulence
gene that is involved with in adhesion to cultured cells and the repression of the host lymphocyte
activation response (62).
EHEC Large Plasmid
Most of the STEC strains possess a large plasmid, 75 to 100 kb in size, encoding additional virulence
factors. This has best been described for STEC O157:H7, carryinga 90 kb plasmid pO157 (46)
containing several putative virulence determinants described below.
Figure 2. Genetic organization of the STEC/EPEC LEE and STEC prophages CP-933U, CP-933K, and CP-933P. Different colors of genes encode proteins with different functions as mentioned in the legend. In addition, the effector proteins e.g., espI, espJ, nleD carried by the phages are shown. The figure is reproduced with permission from the American Society of Microbiology (63).
(a) Enterohemolysin
One of the well-known virulence genes encoded on pO157 is hly, encoding hemolysin (Ehly). Ehly is a
member of the repeats in toxin (RTX) family of toxins and could contribute to disease through lysis of
erythrocytes and release of hemoglobin as a potential source of iron. E. coli of serogroups O157, O26
and O111 commonly produce Ehly and it is therefore a useful epidemiological marker for potential
Stx-producing strains (52).
Introduction and Scope of the Thesis
17
(b) Proteases
EspP is a 104-kDa protein secreted by E. coli O157 and has proteolytic activity against human
coagulation factor V which could result in a decreased coagulation reaction leading to prolonged
bleeding (52). StcE, a zinc metalloprotease is secreted by the closely linked etp type II secretion
system on pO157. Expression of the stcE gene is up-regulated by the regulator Ler (64).
(c) Catalase-peroxidase
Another pO157-encoded determinant is the KatP (catalase-peroxidase), which is produced in
addition to the two chromosomally encoded catalases or hydroperoxidases of E. coli (65). Catalases
are part of the bacterial defense mechanisms against oxidative stress, while the peroxidases are
haem-binding acceptors (30).
(d) toxB
A homologue of the efa-1/lifA gene designated toxB is present on pO157 (66). It is important for full
expression of adherence by affecting the production and secretion of EspA, EspB, and Tir (67).
Adhesion
The fimbrial adhesins of STEC include long polar fimbriae (Lpf) (68), SfaA (69), SfpA (70), and StcA
(69). Recently, a type IV pilus, called the hemorrhagic coli pilus, has been identified that is involved in
adherence and biofilm formation (5). Among other adhesins, Saa (encoded by the saa gene) (71) is
produced by strains of certain serotypes of LEE-negative STEC (e.g., O113:H21 and O91:H21),
including some strains that have been isolated from patients with HUS. Saa is significantly more
associated with STEC from cattle than with those from humans and could be involved in the initial
bacterial adherence (72). E. coli common pilus (ECP), composed of a 21-kDa pilin subunit EspA, is a
pilus-adherence factor that is crucial for the virulence of E. coli O157 in humans.
Differences in virulence factors between STEC and EPEC
Most of the virulence factors of EPEC may also be present in STEC (Table 3) (59, 69, 73). Only
virulence factors unique to EPEC are described below.
LEE PAI
A hallmark phenotype of EPEC is the ability to produce A/E lesions like many STEC strains (7, 53)
caused by genes on LEE. The LEE of EPEC comprises 35,624bp and contains the same 41 genes as
STEC. However, EPEC LEE lack the prophage 933L and sequences of genes, as e.g., espA, espB, espD,
Chapter 1
18
eae and tir in STEC and shows a high variability of 15%, 26%, 19%, 13% and 33%, respectively
compared to these genes in STEC (6, 55, 57).
Localized adherence of EPEC and Bundle-Forming Pili
EPEC adheres to epithelial cells in vitro in a so-called localized-adherence (LA) pattern. The principal
factor responsible for the LA phenotype is a surface appendage known as the bundle-forming pilus
(BFP) (encoded by the bfp gene cluster), a member of the type IV fimbria family (encoded on a ∼92-
kb IncFII plasmid named EPEC adherence factor (EAF) (74-76). EPEC isolates containing this plasmid
are named typical EPEC (tEPEC), whereas EAF negative EPEC are referred to as atypical EPEC (aEPEC)
(77).
The bfp gene cluster contains 14 genes, the most important one being bfpA, which encodes the
major structural subunit of Bfp, “bundlin” (78, 79). The transcriptional regulators Per (plasmid-
encoded regulator) up-regulates the expression of BFP.
EspC PAI
EPEC secretes a 110-kDa protein EspC, located on a region termed as espC PAI (80). EspC possesses
enterotoxic activity and most likely plays an accessory role in EPEC pathogenesis, presumably as an
enterotoxin (81). The espC island also contains a putative virulent locus ORF3 similar to VirA
of Shigella flexneri (80).
Table 3. Major virulence factors of EPEC
Virulence factor category Virulence factor Present in EPEC Present in STEC
Adherence BFP Yes No Intimin Yes Yes Lymphostatin/LifA Yes Yes Paa Yes Yes
Type 1 fimbrae Yes Yes
E. coli factor for adherence (Efa)
Yes Yes
Type III trnslocated proteins Tir,EspA,EspB,EspD,EspF-I, Map, NleA-C, Cif
Yes Yes
Toxin CDT Yes Yes
EAST Yes Yes
Regulation Ler Yes Yes
Per Yes No
Protease EspC Yes No
Introduction and Scope of the Thesis
19
Diagnosis and Typing of STEC
Most clinical laboratories use conventional culture methods to screen for STEC in human feces. STEC
O157:H7 can be easily distinguished from most E. coli by their inability to ferment sorbitol. To isolate
O157 STEC, different selective and differential medium such as sorbitol-MacConkey agar (SMAC),
cefixime tellurite-sorbitol MacConkey agar (CT-SMAC), or CHROMagar O157 are used where the
O157 colonies are colorless on SMAC or CT-SMAC and are mauve or pink on CHROMagar O157 (82-
84). Most other STEC serotypes ferment sorbitol and are therefore difficult to differentiate from
other E. coli. For this, non-culture methods as Stx detection and Stx activity assays may be used (85,
86). Recently, PCR-based methods targeting the stx1 and stx2 genes are used for diagnosis and
confirmation of STEC infection, resulting in rapid and improved detection rates (87). Other assays
including determination of the O group, virulence factors such as intimin and enterohemolysin, and
differentiating among the subtypes of Stx have been developed (35, 88). For typing of STEC different
DNA fingerprinting methods e.g., Pulsed-Field Gel Electrophoresis (PFGE), Amplified-Fragment Length
Polymorphism (AFLP), Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) are used
(89, 90). Among the sequence based typing methods, MLST is the most reliable method to determine
genetic relatedness of epidemiologically-unrelated isolates, but has limited discriminatory power
(91).
The primary advantage of nonculture assays is that they can be used to detect all serotypes of STEC
and provide results more quickly than culture. However, with nonculture based assays the organism
is not isolated for subsequent serotyping and detailed characterization limiting the ability of
physicians to predict the potential severity of the infection in the patient (e.g., risk for HUS) (92, 93).
A reliable verification of STEC infections is only possible using a combination of culture, molecular,
and serological or toxicological detection methods (94).
Whole genome sequencing technology
Now-a-days using massively parallel (or “next-generation”) DNA sequencing technologies, it is
possible to examine the complete or nearly complete genomes of bacterial isolates within days (95).
Indeed, the rapid advances in sequencing technology and bioinformatics tools during the last decade
have initiated a transition from classical conservation genetics to conservation genomics (96, 97). A
number of (online) tools are available for the successful application of WGS data (Table 4). WGS
provides the opportunity for a step-change in diagnostic microbiological practice with (in the long
term) little or no increase in overall costs (98). The high resolution discriminatory power of WGS
makes it suitable for molecular typing, genetic profiling, outbreak investigation and population
Chapter 1
20
structure analysis (99, 100). A schematic representation of the use and applications of WGS is
presented in Figure 3.
Sequencing the STEC Genome
The complete genome sequence of an STEC O157 strain, isolated from a large outbreak that occurred
in 1996 in Sakai City, Japan (referred to as O157 Sakai), and of another O157 strain (EDL933) provided
the first opportunity to perform a direct comparison of the genomes of E. coli strains at the DNA
sequence level (101, 102). Approximately 4.1 million base pairs of “backbone” sequences are
conserved between the genomes, but these stretches are punctuated by hundreds of sequences
present in one strain but not in the other (73). During the E. coli O104:H4 outbreak in Germany
several studies indicated that bench top sequencing platforms could generate data with sufficient
speed to have an important effect on clinical and epidemiologic problems (103).
STEC-ID-net study
A multicenter prospective study, STEC-ID-net, was performed during a 12-month period (April 2013-
March 2014) in two regions of the Netherlands; Groningen, located in the north, and Rotterdam,
located in the south-west part of the country. Two regional MMLs (Certe, Groningen and Star-MDC,
Rotterdam), the University Medical Center, Groningen (UMCG), the National Institute for Health and
Environment (RIVM), and the public health services (PHSs) of Groningen, Drenthe and Rotterdam-
Rijnmond participated in the study. The study was performed to formulate evidence-based
recommendations for optimizing diagnostics, notification and surveillance of STEC in the
Netherlands. A total of 23,153 stool samples from patients with presumed infectious gastroenteritis
from different parts of the Netherlands were screened using qPCR targeting the stx1/stx2 (marker for
STEC) and escV genes (marker for EPEC) by the regional MMLs. In total 425 (1.9%) feces samples
were positive for stx1 and/or stx2 gene using direct PCR on feces. Patients that tested positive for stx
and/or escV, were interviewed by phone or e- mail by the PHSs (104). Eventually, 132 STEC and 976
EPEC isolates were obtained from the study. The research described in this thesis consists of spin-off
studies performed on the STEC and part of the EPEC isolates obtained within the STEC-ID-net study.
Introduction and Scope of the Thesis
21
Figure 3. Overview of the approaches and applications of whole genome sequencing used in this thesis. The workflow described here is starting from a bacterial isolate and the duration of sequencing may vary from 1-2 days depending on the sequenced fragment lengths.
Chapter 1
22
Table 4. Different WGS data analysis tools used in this study
Name of the WGS analysis tool*
Application Link / Accession
CLC genomics
workbench
Sequence Assembly CLC bio A/S, Aarhus, Denmark
ABACAS Orientation of assembled sequence http://abacas.sourceforge.net
Mauve Multiple Genome Alignment http://gel.ahabs.wisc.edu/mauve
Rast Genome annotation http://rast.nmpdr.org/rast.cgi?page=Jobs&logout=
1
Virulence Finder Determination of virulence gene https://cge.cbs.dtu.dk/services/VirulenceFinder/
VFDB Determination of virulence gene http://www.mgc.ac.cn/VFs/main.htm
SerotypeFinder Determination of O and H type
encoding gene
https://cge.cbs.dtu.dk/services/SerotypeFinder/
MLST finder Determination of sequence type https://cge.cbs.dtu.dk/services/MLST/
ResFinder Determination of antibiotic resistance
gene
https://cge.cbs.dtu.dk/services/ResFinder/
SpeciesFinder Prediction of bacterial species https://cge.cbs.dtu.dk/services/SpeciesFinder/
Phagege Search Tool
(PHAST)
Prediction of bacteriophages http://phast.wishartlab.com/
Blast Ring Image
Generator (BLAST)
Genome comparison https://sourceforge.net/projects/brig/
Easyfig Genome comparison http://mjsull.github.io/Easyfig/
Artemis comparison
Tool (ACT)
Genome comparison http://www.sanger.ac.uk/science/tools/artemis-
comparison-tool-act
Seqsphere MLST+ Phylogenetic relationship Ridom GmbH, Münster, Germany
RAxML Phylogenetic relationship https://github.com/stamatak/standard-RAxML
Other tools Several http://seqanswers.com/wiki/Softwarehttp://molbi
ol-tools.ca/Genomics.htm
*Mainly the tools used in this thesis are mentioned here, but many more tools are available and can be found following the
links mentioned in the last row.
SCOPE OF THE THESIS
STEC are considered as emerging food-borne pathogen worldwide. Since the large outbreak in
Germany with a rare STEC O104:H4, the necessity for continuous epidemiological and microbiological
surveillance and a rapid and reliable diagnosis of STEC in the Netherlands seems obvious. We
hypothesized that a better insight into the (molecular) characteristics of STEC improves their
diagnosis and predict their pathogenic potential. Therefore, in this thesis we established and
implemented modern molecular assays, i.e. whole genome sequencing (WGS) for diagnosis and
detailed characterization of STEC isolates obtained from patients in the Netherlands. We also
hypothesized that STEC with the potential to cause severe disease outcome belong to particular
phylogenetic groups. Furthermore, we wanted to see the evolutionary relationship of
Introduction and Scope of the Thesis
23
Enteropathogenic E. coli (EPEC) serotype O157 with the clinically well-known STEC O157 strains
known to cause severe illness to human.
At present it is still not possible to fully define human pathogenic STEC that may cause HUS or bloody
diarrhea. The concept of seropathotyping proposed by Karmali et al. was not applicable for isolates
not been fully serotyped. Therefore, a molecular approach combining serotypes as well as genes
encoding virulence factors additional to stx genes, was proposed by European Food Safety Authority
(EFSA) to perform a quantitative risk assessment relating the presence of virulence genes and/or
serogroup to a particular disease outcome. This molecular approach, with a slight modification was
used in our study described in chapter 2. In this chapter, a rapid screening algorithm including both
molecular and conventional methods of STEC detection was applied directly to stool samples of
patients with gastrointestinal complaints. Moreover, we tried to discriminate infections with less-
virulent STEC from those with clinical relevance and risk for public health based on the proposed
molecular approach.
Chapter 3 describes the overall molecular characterization and diversity of 132 STEC isolates
obtained from two different regions of the Netherlands using high throughput (WGS). In this chapter,
we tried to find out a potential association of certain virulence factors of STEC strains with a clinical
picture of the patient and to observe if the isolates responsible for relatively severe disease outcome
belong to a particular phylogenetic background.
E. coli O104:H4 isolates very similar to the outbreak strains were isolated in the Netherlands from an
sporadic case of HUS which is described in chapter 4. Several phenotypic and genotypic laboratory
methods were used to distinguish virulence and antibiotic resistance properties of these isolates
from the German 2011 O104:H4 outbreak strains. Eventually WGS was used to reveal their
phylogenetic relationship. In Chapter 5, a comprehensive investigation was performed on the
genomes of 23 STEC O104:H4 isolates, including previously reported outbreak- and non-outbreak-
related isolates, to get insight into their evolutionary history. It also describes the role of mobile
genetic elements, e.g., plasmids and bacteriophages in the rapid evolution of the outbreak clone.
STEC O157:H7 has been most often associated with more severe forms of the disease and is
considered to be one of the major food-borne pathogens across the world. Several studies have
shown that E. coli O157:H7 often exist in nature in a toxin negative form and therefore, if only the stx
gene is considered for screening in the laboratory, the stx negative but eae positive isolates would be
considered as EPEC. In chapter 6, we performed a detailed molecular and phylogenetic comparison
of stx positive and negative E. coli O157:H7 by using WGS and we were able to prove that these stx
negative isolates of E. coli are in fact members of the STEC O157:H7 group that either have lost the
Chapter 1
24
stx gene or are ready to acquire the Stx converting bacteriophage. In chapter 7, virulence, antibiotic
resistance properties and phylogenetic analysis of E. coli O157 isolates other than the H7 flagellar
type are described. The aim of this chapter was to show that E. coli O157 groups are of diverse
phylogenetic backgrounds that have acquired the O157 antigen biosynthesis gene cluster. We also
showed that the presence of different mobile genetic elements in different H types have contributed
to their virulence and antibiotic resistance properties.
REFERENCES
1. Escherich, T. 1885. Die Darmbakterien des Neugeborenen und Säuglings. Fortschr. Med. 3:515.
2. Hooper, LV, Gordon, JI. 2001. Commensal host-bacterial relationships in the gut. Science. 292:1115-1118.
3. Kaper, JB, Nataro, JP, Mobley, HL. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2:123-140.
doi: 10.1038/nrmicro818 [doi].
4. Tenaillon, O, Skurnik, D, Picard, B, Denamur, E. 2010. The population genetics of commensal Escherichia
coli. Nat. Rev. Microbiol. 8:207-217. doi: 10.1038/nrmicro2298 [doi].
5. Croxen, MA, Finlay, BB. 2010. Molecular mechanisms of Escherichia coli pathogenicity. Nat. Rev.
Microbiol. 8:26-38. doi: 10.1038/nrmicro2265 [doi].
6. Schmidt, MA. 2010. LEEways: tales of EPEC, ATEC and EHEC. Cell. Microbiol. 12:1544-1552. doi:
10.1111/j.1462-5822.2010.01518.x [doi].
7. Nataro, JP, Kaper, JB. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.
8. Qadri, F, Svennerholm, AM, Faruque, AS, Sack, RB. 2005. Enterotoxigenic Escherichia coli in developing
countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin. Microbiol. Rev.
18:465-483. doi: 18/3/465 [pii].
9. Sack, RB, Gorbach, SL, Banwell, JG, Jacobs, B, Chatterjee, BD, Mitra, RC. 1971. Enterotoxigenic
Escherichia coli isolated from patients with severe cholera-like disease. J. Infect. Dis. 123:378-385.
10. Gaastra, W, Svennerholm, AM. 1996. Colonization factors of human enterotoxigenic Escherichia coli
(ETEC). Trends Microbiol. 4:444-452. doi: 0966842X96100688 [pii].
11. Okhuysen, PC, Dupont, HL. 2010. Enteroaggregative Escherichia coli (EAEC): a cause of acute and
persistent diarrhea of worldwide importance. J. Infect. Dis. 202:503-505. doi: 10.1086/654895 [doi].
12. Kaur, P, Chakraborti, A, Asea, A. 2010. Enteroaggregative Escherichia coli: An Emerging Enteric Food
Borne Pathogen. Interdiscip. Perspect. Infect. Dis. 2010:254159. doi: 10.1155/2010/254159 [doi].
13. Sansonetti, P. 2002. Host-pathogen interactions: the seduction of molecular cross talk. Gut. 50 Suppl 3:III2-
8.
14. Mansan-Almeida, R, Pereira, AL, Giugliano, LG. 2013. Diffusely adherent Escherichia coli strains
isolated from children and adults constitute two different populations. BMC Microbiol. 13:22-2180-13-22. doi:
10.1186/1471-2180-13-22 [doi].
Introduction and Scope of the Thesis
25
15. Scaletsky, IC, Fabbricotti, SH, Carvalho, RL, Nunes, CR, Maranhao, HS, Morais, MB, Fagundes-
Neto, U. 2002. Diffusely adherent Escherichia coli as a cause of acute diarrhea in young children in Northeast
Brazil: a case-control study. J. Clin. Microbiol. 40:645-648.
16. Karmali, MA, Petric, M, Lim, C, Fleming, PC, Arbus, GS, Lior, H. 1985. The association between
idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis.
151:775-782.
17. Mellmann, A, Bielaszewska, M, Kock, R, Friedrich, AW, Fruth, A, Middendorf, B, Harmsen, D,
Schmidt, MA, Karch, H. 2008. Analysis of collection of hemolytic uremic syndrome-associated
enterohemorrhagic Escherichia coli. Emerg. Infect. Dis. 14:1287-1290. doi: 10.3201/eid1408.071082 [doi].
18. Konowalchuk, J, Speirs, JI, Stavric, S. 1977. Vero response to a cytotoxin of Escherichia coli. Infect.
Immun. 18:775-779.
19. Hickey, CA, Beattie, TJ, Cowieson, J, Miyashita, Y, Strife, CF, Frem, JC, Peterson, JM, Butani, L,
Jones, DP, Havens, PL, Patel, HP, Wong, CS, Andreoli, SP, Rothbaum, RJ, Beck, AM, Tarr, PI. 2011.
Early volume expansion during diarrhea and relative nephroprotection during subsequent hemolytic uremic
syndrome. Arch. Pediatr. Adolesc. Med. 165:884-889. doi: 10.1001/archpediatrics.2011.152 [doi].
20. Bell, BP, Griffin, PM, Lozano, P, Christie, DL, Kobayashi, JM, Tarr, PI. 1997. Predictors of hemolytic
uremic syndrome in children during a large outbreak of Escherichia coli O157:H7 infections. Pediatrics.
100:E12.
21. Zhang, X, McDaniel, AD, Wolf, LE, Keusch, GT, Waldor, MK, Acheson, DW. 2000. Quinolone
antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J. Infect. Dis.
181:664-670. doi: JID990644 [pii].
22. Karmali, MA, Steele, BT, Petric, M, Lim, C. 1983. Sporadic cases of haemolytic-uraemic syndrome
associated with faecal cytotoxin and cytotoxin-producing Escherichia coli in stools. Lancet. 1:619-620.
23. Karmali, MA, Petric, M, Lim, C, Fleming, PC, Steele, BT. 1983. Escherichia coli cytotoxin, haemolytic-
uraemic syndrome, and haemorrhagic colitis. Lancet. 2:1299-1300. doi: S0140-6736(83)91167-4 [pii].
24. Riley, LW, Remis, RS, Helgerson, SD, McGee, HB, Wells, JG, Davis, BR, Hebert, RJ, Olcott, ES,
Johnson, LM, Hargrett, NT, Blake, PA, Cohen, ML. 1983. Hemorrhagic colitis associated with a rare
Escherichia coli serotype. N. Engl. J. Med. 308:681-685. doi: 10.1056/NEJM198303243081203 [doi].
25. Karch, H, Wiss, R, Gloning, H, Emmrich, P, Aleksic, S, Bockemuhl, J. 1990. Hemolytic-uremic
syndrome in infants due to verotoxin-producing Escherichia coli. Dtsch. Med. Wochenschr. 115:489-495. doi:
10.1055/s-2008-1065036 [doi].
26. Ammon, A, Petersen, LR, Karch, H. 1999. A large outbreak of hemolytic uremic syndrome caused by an
unusual sorbitol-fermenting strain of Escherichia coli O157:H-. J. Infect. Dis. 179:1274-1277. doi: JID980967
[pii].
27. Blanco, JE, Blanco, M, Alonso, MP, Mora, A, Dahbi, G, Coira, MA, Blanco, J. 2004. Serotypes,
virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human
patients: prevalence in Lugo, Spain, from 1992 through 1999. J. Clin. Microbiol. 42:311-319.
28. Bosilevac, JM, Koohmaraie, M. 2012. Predicting the presence of non-O157 Shiga toxin-producing
Escherichia coli in ground beef by using molecular tests for Shiga toxins, intimin, and O serogroups. Appl.
Environ. Microbiol. 78:7152-7155. doi: 10.1128/AEM.01508-12 [doi].
29. Dorn, CR, Francis, DH, Angrick, EJ, Willgohs, JA, Wilson, RA, Collins, JE, Jenke, BH, Shawd, SJ.
1993. Characteristics of Vero cytotoxin producing Escherichia coli associated with intestinal colonization and
diarrhea in calves. Vet. Microbiol. 36:149-159.
Chapter 1
26
30. Welinder-Olsson, C, Kaijser, B. 2005. Enterohemorrhagic Escherichia coli (EHEC). Scand. J. Infect. Dis.
37:405-416. doi: G10T320874113T16 [pii].
31. Ogura, Y, Ooka, T, Iguchi, A, Toh, H, Asadulghani, M, Oshima, K, Kodama, T, Abe, H, Nakayama,
K, Kurokawa, K, Tobe, T, Hattori, M, Hayashi, T. 2009. Comparative genomics reveal the mechanism of the
parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli. Proc. Natl. Acad. Sci. U. S. A.
106:17939-17944. doi: 10.1073/pnas.0903585106 [doi].
32. Cooper, KK, Mandrell, RE, Louie, JW, Korlach, J, Clark, TA, Parker, CT, Huynh, S, Chain, PS,
Ahmed, S, Carter, MQ. 2014. Comparative genomics of enterohemorrhagic Escherichia coli O145:H28
demonstrates a common evolutionary lineage with Escherichia coli O157:H7. BMC Genomics. 15:17-2164-15-
17. doi: 10.1186/1471-2164-15-17 [doi].
33. Caprioli, A, Morabito, S, Brugere, H, Oswald, E. 2005. Enterohaemorrhagic Escherichia coli: emerging
issues on virulence and modes of transmission. Vet. Res. 36:289-311. doi: 10.1051/vetres:2005002 [doi].
34. Yabuuchi, E. 2002. Bacillus dysentericus (sic) 1897 was the first taxonomic rather than Bacillus dysenteriae
1898. Int. J. Syst. Evol. Microbiol. 52:1041. doi: 10.1099/00207713-52-3-1041 [doi].
35. Scheutz, F, Teel, LD, Beutin, L, Pierard, D, Buvens, G, Karch, H, Mellmann, A, Caprioli, A, Tozzoli,
R, Morabito, S, Strockbine, NA, Melton-Celsa, AR, Sanchez, M, Persson, S, O'Brien, AD. 2012.
Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx
nomenclature. J. Clin. Microbiol. 50:2951-2963. doi: 10.1128/JCM.00860-12 [doi].
36. Paton, JC, Paton, AW. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli
infections. Clin. Microbiol. Rev. 11:450-479.
37. Lingwood, CA, Law, H, Richardson, S, Petric, M, Brunton, JL, De Grandis, S, Karmali, M. 1987.
Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J. Biol. Chem.
262:8834-8839.
38. Sandvig, K. 2001. Shiga toxins. Toxicon. 39:1629-1635. doi: S0041010101001507 [pii].
39. Johannes, L, Romer, W. 2010. Shiga toxins--from cell biology to biomedical applications. Nat. Rev.
Microbiol. 8:105-116. doi: 10.1038/nrmicro2279 [doi].
40. Fuller, CA, Pellino, CA, Flagler, MJ, Strasser, JE, Weiss, AA. 2011. Shiga toxin subtypes display
dramatic differences in potency. Infect. Immun. 79:1329-1337. doi: 10.1128/IAI.01182-10 [doi].
41. Zhang, W, Bielaszewska, M, Kuczius, T, Karch, H. 2002. Identification, characterization, and distribution
of a Shiga toxin 1 gene variant (stx(1c)) in Escherichia coli strains isolated from humans. J. Clin. Microbiol.
40:1441-1446.
42. Friedrich, AW, Bielaszewska, M, Zhang, WL, Pulz, M, Kuczius, T, Ammon, A, Karch, H. 2002.
Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J.
Infect. Dis. 185:74-84. doi: JID010725 [pii].
43. Friedrich, AW, Borell, J, Bielaszewska, M, Fruth, A, Tschape, H, Karch, H. 2003. Shiga toxin 1c-
producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease.
J. Clin. Microbiol. 41:2448-2453.
44. Beutin, L, Miko, A, Krause, G, Pries, K, Haby, S, Steege, K, Albrecht, N. 2007. Identification of human-
pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and
molecular typing of Shiga toxin genes. Appl. Environ. Microbiol. 73:4769-4775. doi: AEM.00873-07 [pii].
45. Zheng, J, Cui, S, Teel, LD, Zhao, S, Singh, R, O'Brien, AD, Meng, J. 2008. Identification and
characterization of Shiga toxin type 2 variants in Escherichia coli isolates from animals, food, and humans. Appl.
Environ. Microbiol. 74:5645-5652. doi: 10.1128/AEM.00503-08 [doi].
Introduction and Scope of the Thesis
27
46. Karch, H. 2001. The role of virulence factors in enterohemorrhagic Escherichia coli (EHEC)--associated
hemolytic-uremic syndrome. Semin. Thromb. Hemost. 27:207-213. doi: 10.1055/s-2001-15250 [doi].
47. Feng, P, Dey, M, Abe, A, Takeda, T. 2001. Isogenic strain of Escherichia coli O157:H7 that has lost both
Shiga toxin 1 and 2 genes. Clin. Diagn. Lab. Immunol. 8:711-717. doi: 10.1128/CDLI.8.4.711-717.2001 [doi].
48. Bielaszewska, M, Kock, R, Friedrich, AW, von Eiff, C, Zimmerhackl, LB, Karch, H, Mellmann, A.
2007. Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm? PLoS One.
2:e1024. doi: 10.1371/journal.pone.0001024 [doi].
49. Friedrich, AW, Lu, S, Bielaszewska, M, Prager, R, Bruns, P, Xu, JG, Tschape, H, Karch, H. 2006.
Cytolethal distending toxin in Escherichia coli O157:H7: spectrum of conservation, structure, and endothelial
toxicity. J. Clin. Microbiol. 44:1844-1846. doi: 44/5/1844 [pii].
50. Lara-Tejero, M, Galan, JE. 2001. CdtA, CdtB, and CdtC form a tripartite complex that is required for
cytolethal distending toxin activity. Infect. Immun. 69:4358-4365. doi: 10.1128/IAI.69.7.4358-4365.2001 [doi].
51. Savarino, SJ, Fasano, A, Watson, J, Martin, BM, Levine, MM, Guandalini, S, Guerry, P. 1993.
Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable
toxin. Proc. Natl. Acad. Sci. U. S. A. 90:3093-3097.
52. Law, D. 2000. Virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli. J. Appl.
Microbiol. 88:729-745. doi: jam1031 [pii].
53. Moon, HW, Whipp, SC, Argenzio, RA, Levine, MM, Giannella, RA. 1983. Attaching and effacing
activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun.
41:1340-1351.
54. Jerse, AE, Yu, J, Tall, BD, Kaper, JB. 1990. A genetic locus of enteropathogenic Escherichia coli
necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. U. S.
A. 87:7839-7843.
55. Elliott, SJ, Sperandio, V, Giron, JA, Shin, S, Mellies, JL, Wainwright, L, Hutcheson, SW, McDaniel,
TK, Kaper, JB. 2000. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both
LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli.
Infect. Immun. 68:6115-6126.
56. Kenny, B, DeVinney, R, Stein, M, Reinscheid, DJ, Frey, EA, Finlay, BB. 1997. Enteropathogenic E. coli
(EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell. 91:511-520. doi: S0092-
8674(00)80437-7 [pii].
57. Elliott, SJ, Wainwright, LA, McDaniel, TK, Jarvis, KG, Deng, YK, Lai, LC, McNamara, BP,
Donnenberg, MS, Kaper, JB. 1998. The complete sequence of the locus of enterocyte effacement (LEE) from
enteropathogenic Escherichia coli E2348/69. Mol. Microbiol. 28:1-4.
58. Kenny, B, Ellis, S, Leard, AD, Warawa, J, Mellor, H, Jepson, MA. 2002. Co-ordinate regulation of
distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules.
Mol. Microbiol. 44:1095-1107.
59. Garmendia, J, Frankel, G, Crepin, VF. 2005. Enteropathogenic and enterohemorrhagic Escherichia coli
infections: translocation, translocation, translocation. Infect. Immun. 73:2573-2585. doi: 73/5/2573 [pii].
60. Karch, H, Schubert, S, Zhang, D, Zhang, W, Schmidt, H, Olschlager, T, Hacker, J. 1999. A genomic
island, termed high-pathogenicity island, is present in certain non-O157 Shiga toxin-producing Escherichia coli
clonal lineages. Infect. Immun. 67:5994-6001.
Chapter 1
28
61. Tarr, PI, Bilge, SS, Vary, JC,Jr, Jelacic, S, Habeeb, RL, Ward, TR, Baylor, MR, Besser, TE. 2000. Iha:
a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal
island of conserved structure. Infect. Immun. 68:1400-1407.
62. Klapproth, JM, Scaletsky, IC, McNamara, BP, Lai, LC, Malstrom, C, James, SP, Donnenberg, MS.
2000. A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation. Infect. Immun.
68:2148-2155.
63. Garmendia, J, Frankel, G, Crepin, VF. 2005. Enteropathogenic and enterohemorrhagic Escherichia coli
infections: translocation, translocation, translocation. Infect. Immun. 73:2573-2585. doi: 73/5/2573 [pii].
64. Grys, TE, Siegel, MB, Lathem, WW, Welch, RA. 2005. The StcE protease contributes to intimate
adherence of enterohemorrhagic Escherichia coli O157:H7 to host cells. Infect. Immun. 73:1295-1303. doi:
73/3/1295 [pii].
65. Brunder, W, Schmidt, H, Karch, H. 1996. KatP, a novel catalase-peroxidase encoded by the large plasmid
of enterohaemorrhagic Escherichia coli O157:H7. Microbiology. 142 ( Pt 11):3305-3315. doi:
10.1099/13500872-142-11-3305 [doi].
66. Farfan, MJ, Torres, AG. 2012. Molecular mechanisms that mediate colonization of Shiga toxin-producing
Escherichia coli strains. Infect. Immun. 80:903-913. doi: 10.1128/IAI.05907-11 [doi].
67. Tatsuno, I, Horie, M, Abe, H, Miki, T, Makino, K, Shinagawa, H, Taguchi, H, Kamiya, S, Hayashi, T,
Sasakawa, C. 2001. toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full
epithelial cell adherence phenotype. Infect. Immun. 69:6660-6669. doi: 10.1128/IAI.69.11.6660-6669.2001
[doi].
68. Doughty, S, Sloan, J, Bennett-Wood, V, Robertson, M, Robins-Browne, RM, Hartland, EL. 2002.
Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-
negative strains of enterohemorrhagic Escherichia coli. Infect. Immun. 70:6761-6769.
69. Spears, KJ, Roe, AJ, Gally, DL. 2006. A comparison of enteropathogenic and enterohaemorrhagic
Escherichia coli pathogenesis. FEMS Microbiol. Lett. 255:187-202. doi: FML119 [pii].
70. Friedrich, AW, Nierhoff, KV, Bielaszewska, M, Mellmann, A, Karch, H. 2004. Phylogeny, clinical
associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic
Escherichia coli O157:H-. J. Clin. Microbiol. 42:4697-4701. doi: 42/10/4697 [pii].
71. Paton, AW, Srimanote, P, Woodrow, MC, Paton, JC. 2001. Characterization of Saa, a novel
autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli
strains that are virulent for humans. Infect. Immun. 69:6999-7009. doi: 10.1128/IAI.69.11.6999-7009.2001 [doi].
72. Batisson, I, Guimond, MP, Girard, F, An, H, Zhu, C, Oswald, E, Fairbrother, JM, Jacques, M, Harel,
J. 2003. Characterization of the novel factor paa involved in the early steps of the adhesion mechanism of
attaching and effacing Escherichia coli. Infect. Immun. 71:4516-4525.
73. Donnenberg, MS, Whittam, TS. 2001. Pathogenesis and evolution of virulence in enteropathogenic and
enterohemorrhagic Escherichia coli. J. Clin. Invest. 107:539-548. doi: 10.1172/JCI12404 [doi].
74. Baldini, MM, Kaper, JB, Levine, MM, Candy, DC, Moon, HW. 1983. Plasmid-mediated adhesion in
enteropathogenic Escherichia coli. J. Pediatr. Gastroenterol. Nutr. 2:534-538.
75. Giron, JA, Ho, AS, Schoolnik, GK. 1991. An inducible bundle-forming pilus of enteropathogenic
Escherichia coli. Science. 254:710-713.
76. Donnenberg, MS. 1999. Interactions between enteropathogenic Escherichia coli and epithelial cells. Clin.
Infect. Dis. 28:451-455. doi: 10.1086/515159 [doi].
Introduction and Scope of the Thesis
29
77. Trabulsi, LR, Keller, R, Tardelli Gomes, TA. 2002. Typical and atypical enteropathogenic Escherichia
coli. Emerg. Infect. Dis. 8:508-513. doi: 10.3201/eid0805.010385 [doi].
78. Donnenberg, MS, Giron, JA, Nataro, JP, Kaper, JB. 1992. A plasmid-encoded type IV fimbrial gene of
enteropathogenic Escherichia coli associated with localized adherence. Mol. Microbiol. 6:3427-3437.
79. Stone, KD, Zhang, HZ, Carlson, LK, Donnenberg, MS. 1996. A cluster of fourteen genes from
enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Mol. Microbiol. 20:325-337.
80. Mellies, JL, Navarro-Garcia, F, Okeke, I, Frederickson, J, Nataro, JP, Kaper, JB. 2001. espC
pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin. Infect. Immun. 69:315-324.
doi: 10.1128/IAI.69.1.315-324.2001 [doi].
81. Blum-Oehler, G, Dobrindt, U, Janke, B, Nagy, G, Piechaczek, K, Hacker, J. 2000. Pathogenicity islands
of uropathogenic E. coli and evolution of virulence. Adv. Exp. Med. Biol. 485:25-32.
82. March, SB, Ratnam, S. 1986. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7
associated with hemorrhagic colitis. J. Clin. Microbiol. 23:869-872.
83. Church, DL, Emshey, D, Semeniuk, H, Lloyd, T, Pitout, JD. 2007. Evaluation of BBL CHROMagar
O157 versus sorbitol-MacConkey medium for routine detection of Escherichia coli O157 in a centralized
regional clinical microbiology laboratory. J. Clin. Microbiol. 45:3098-3100. doi: JCM.00426-07 [pii].
84. Zadik, PM, Chapman, PA, Siddons, CA. 1993. Use of tellurite for the selection of verocytotoxigenic
Escherichia coli O157. J. Med. Microbiol. 39:155-158. doi: 10.1099/00222615-39-2-155 [doi].
85. Gilmour, MW, Chui, L, Chiu, T, Tracz, DM, Hagedorn, K, Tschetter, L, Tabor, H, Ng, LK, Louie, M.
2009. Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using
conventional and molecular methods. J. Med. Microbiol. 58:905-911. doi: 10.1099/jmm.0.007732-0 [doi].
86. Qin, X, Klein, EJ, Galanakis, E, Thomas, AA, Stapp, JR, Rich, S, Buccat, AM, Tarr, PI. 2015. Real-
Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical
Samples. J. Clin. Microbiol. 53:2148-2153. doi: 10.1128/JCM.00115-15 [doi].
87. de Boer, RF, Ott, A, Kesztyus, B, Kooistra-Smid, AM. 2010. Improved detection of five major
gastrointestinal pathogens by use of a molecular screening approach. J. Clin. Microbiol. 48:4140-4146. doi:
10.1128/JCM.01124-10 [doi].
88. Perelle, S, Dilasser, F, Grout, J, Fach, P. 2004. Detection by 5'-nuclease PCR of Shiga-toxin producing
Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's most
frequent clinical cases. Mol. Cell. Probes. 18:185-192. doi: 10.1016/j.mcp.2003.12.004 [doi].
89. Heir, E, Lindstedt, BA, Vardund, T, Wasteson, Y, Kapperud, G. 2000. Genomic fingerprinting of
shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE)
and fluorescent amplified-fragment-length polymorphism (FAFLP). Epidemiol. Infect. 125:537-548.
90. Lindstedt, BA, Heir, E, Vardund, T, Kapperud, G. 2000. A variation of the amplified-fragment length
polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme
combination BglII-MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates. FEMS Microbiol.
Lett. 189:19-24. doi: S0378-1097(00)00245-7 [pii].
91. European Food Safety Authority. 2011. Urgent advice on the public health risk of Shiga-toxin producing
Escherichia coli in fresh vegetables. 9(6):2274. doi: doi:10.2903/j.efsa.2011.2274.
92. Cornick, NA, Jelacic, S, Ciol, MA, Tarr, PI. 2002. Escherichia coli O157:H7 infections: discordance
between filterable fecal shiga toxin and disease outcome. J. Infect. Dis. 186:57-63. doi: JID011279 [pii].
Chapter 1
30
93. Mancini, N, Carletti, S, Ghidoli, N, Cichero, P, Burioni, R, Clementi, M. 2010. The era of molecular and
other non-culture-based methods in diagnosis of sepsis. Clin. Microbiol. Rev. 23:235-251. doi:
10.1128/CMR.00043-09 [doi].
94. Baljer, G, Wieler, LH. 1999. Animals as a source of infections for humans--diseases caused by EHEC.
Dtsch. Tierarztl. Wochenschr. 106:339-343.
95. Salipante, SJ, SenGupta, DJ, Cummings, LA, Land, TA, Hoogestraat, DR, Cookson, BT. 2015.
Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology. J. Clin.
Microbiol. 53:1072-1079. doi: 10.1128/JCM.03385-14 [doi].
96. Allendorf, FW, Hohenlohe, PA, Luikart, G. 2010. Genomics and the future of conservation genetics. Nat.
Rev. Genet. 11:697-709. doi: 10.1038/nrg2844 [doi].
97. Ouborg, NJ, Pertoldi, C, Loeschcke, V, Bijlsma, RK, Hedrick, PW. 2010. Conservation genetics in
transition to conservation genomics. Trends Genet. 26:177-187. doi: 10.1016/j.tig.2010.01.001 [doi].
98. Koser, CU, Ellington, MJ, Cartwright, EJ, Gillespie, SH, Brown, NM, Farrington, M, Holden, MT,
Dougan, G, Bentley, SD, Parkhill, J, Peacock, SJ. 2012. Routine use of microbial whole genome sequencing
in diagnostic and public health microbiology. PLoS Pathog. 8:e1002824. doi: 10.1371/journal.ppat.1002824
[doi].
99. Sabat, AJ, Budimir, A, Nashev, D, Sa-Leao, R, van Dijl, J, Laurent, F, Grundmann, H, Friedrich, AW,
ESCMID Study Group of Epidemiological Markers (ESGEM). 2013. Overview of molecular typing methods
for outbreak detection and epidemiological surveillance. Euro Surveill. 18:20380. doi: 20380 [pii].
100. Carrico, JA, Sabat, AJ, Friedrich, AW, Ramirez, M, ESCMID Study Group for Epidemiological
Markers (ESGEM). 2013. Bioinformatics in bacterial molecular epidemiology and public health: databases,
tools and the next-generation sequencing revolution. Euro Surveill. 18:20382. doi: 20382 [pii].
101. Hayashi, T, Makino, K, Ohnishi, M, Kurokawa, K, Ishii, K, Yokoyama, K, Han, CG, Ohtsubo, E,
Nakayama, K, Murata, T, Tanaka, M, Tobe, T, Iida, T, Takami, H, Honda, T, Sasakawa, C, Ogasawara,
N, Yasunaga, T, Kuhara, S, Shiba, T, Hattori, M, Shinagawa, H. 2001. Complete genome sequence of
enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res.
8:11-22.
102. Perna, NT, Plunkett, G,3rd, Burland, V, Mau, B, Glasner, JD, Rose, DJ, Mayhew, GF, Evans, PS,
Gregor, J, Kirkpatrick, HA, Posfai, G, Hackett, J, Klink, S, Boutin, A, Shao, Y, Miller, L, Grotbeck, EJ,
Davis, NW, Lim, A, Dimalanta, ET, Potamousis, KD, Apodaca, J, Anantharaman, TS, Lin, J, Yen, G,
Schwartz, DC, Welch, RA, Blattner, FR. 2001. Genome sequence of enterohaemorrhagic Escherichia coli
O157:H7. Nature. 409:529-533. doi: 10.1038/35054089 [doi].
103. Rohde, H, Qin, J, Cui, Y, Li, D, Loman, NJ, Hentschke, M, Chen, W, Pu, F, Peng, Y, Li, J, Xi, F, Li,
S, Li, Y, Zhang, Z, Yang, X, Zhao, M, Wang, P, Guan, Y, Cen, Z, Zhao, X, Christner, M, Kobbe, R, Loos,
S, Oh, J, Yang, L, Danchin, A, Gao, GF, Song, Y, Li, Y, Yang, H, Wang, J, Xu, J, Pallen, MJ, Wang, J,
Aepfelbacher, M, Yang, R, E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium. 2011. Open-
source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N. Engl. J. Med. 365:718-724. doi:
10.1056/NEJMoa1107643 [doi].
104. Kooistra-Smid AM , de Boer RF, Friesema IHM, Petrignani M, Waegemaeker CHFM, Niessen W,
Croughs PD, Ott A, Rossen JWA, Friedrich AW. 2016. Association between virulence factors of Shiga toxin-
producing Escherichia coli with severity of illness in humans. Poster at the 26th European Congress of Clinical
Microbiology and Infectious Diseases (ECCMID), Amsterdam, the Netherlands.
31
CHAPTER 2
Assessing the Public Health Risk of Shiga Toxin-Producing
Escherichia coli by Use of a Rapid Diagnostic Screening Algorithm
Richard F. de Boera,b, Mithila Ferdousc, Alewijn Ottb,c, Henk R. Scheperb, Guido J. Wisselinka,
Max E. Heckd, John W. Rossenc, Anna M.D. Kooistra-Smida, b, c
aDepartment of Research & Development, Certe Laboratory for Infectious Diseases, Groningen, the
Netherlands bDepartment of Medical Microbiology, Certe Laboratory for Infectious Diseases, Groningen, the
Netherlands cDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen,
Groningen, the Netherlands dCenter of Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
Keywords
culture, diagnostic algorithm, gastroenteritis, real-time multiplex PCR, STEC, virulence factors
J Clin Microbiol (2015) 53:1588 –1598.
Chapter 2
32
ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) is an enteropathogen of public health concern because
of its ability to cause serious illness and outbreaks. In this prospective study, a diagnostic screening
algorithm to categorize STEC infections into risk groups was evaluated. The algorithm consists of
prescreening stool specimens with real-time PCR (qPCR) for the presence of stx genes. The qPCR-
positive stool samples were cultured in enrichment broth and again screened for stx genes and
additional virulence factors (escV, aggR, aat, bfpA) and O serogroups (O26, O103, O104, O111, O121,
O145, O157). Also, PCR-guided culture was performed with sorbitol MacConkey agar (SMAC) and
CHROMagar STEC medium. The presence of virulence factors and O serogroups was used for
presumptive pathotype (PT) categorization in four PT groups. The potential risk for severe disease
was categorized from high risk for PT group I to low risk for PT group III, whereas PT group IV consists
of unconfirmed stx qPCR positive samples. In total, 5,022 stool samples of patients with
gastrointestinal symptoms were included. The qPCR detected stx genes in 1.8% of samples. Extensive
screening for virulence factors and O serogroups was performed on 73 samples. After enrichment,
the presence of stx genes was confirmed in 65 samples (89%). By culture on selective media, STEC
was isolated in 36% (26/73 samples). Threshold cycle (CT) values for stx genes were significantly
lower after enrichment compared to direct qPCR (P<0.001). In total, 11 (15%), 19 (26%), 35 (48%),
and 8 (11%) samples were categorized into PT groups I, II, III, and IV, respectively. Several virulence
factors (stx2, stx2a, stx2f, toxB, eae, efa1, cif, espA, tccP, espP, nleA and/or nleB, tir cluster) were
associated with PT groups I and II, while others (stx1, eaaA, mch cluster, ireA) were associated with
PT group III. Furthermore, the number of virulence factors differed between PT groups (analysis of
variance, P<0.0001). In conclusion, a diagnostic algorithm enables fast discrimination of STEC
infections associated with a high to moderate risk for severe disease (PT groups I and II) from less-
virulent STEC (PT group III).
Diagnostic Algorithm for STEC Risk Assessment
33
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen frequently identified as causative
agent of acute diarrheal disease in humans. The outcomes of STEC infections may range from
asymptomatic carriage and mild diarrhea to severe disease, such as hemorrhagic colitis (HC) and
hemolytic-uremic syndrome (HUS) (1–3). Based on pathogenic properties, a subgroup of STEC is also
designated enterohemorrhagic E. coli (EHEC); this subgroup of stx-positive strains also contains the
locus of enterocyte effacement (LEE) pathogenicity island (4). EHEC belongs to certain serotypes that
are frequently associated with outbreaks and life-threatening illnesses (5). Worldwide, the most
common EHEC serotype both in outbreaks and in sporadic cases of severe disease is E. coli O157:H7
(4, 6, 7). Consequently, public health and regulatory responses have been focused mainly on this
serotype. However, due to increased surveillance with tests able to target all serotypes of STEC,
evidence is accumulating that 30% to 60% of EHEC infections are caused by non-O157 strains (8, 9).
To aid in assessing the public health risks associated with STEC, an empirical seropathotype (SPT)
classification of strains was proposed by Karmali et al. (5), based upon the reported frequency of
STEC serotypes in human illness, their known association with outbreaks, and the severity of the
outcome. Serotypes classified as SPT A (O157:H7 and O157:nonmotile [NM]) or SPT B (O26:H11/NM,
O103:H2, O111:NM, O121:H19, and O145:NM) have been associated with outbreaks and severe
disease; however, SPT A is more frequently reported. SPT C comprises serotypes (e.g., O91:H21,
O113:H21, O5:NM, O104:H21, O121:NM, and O165:H25) that have been associated with sporadic
cases of severe disease but not with outbreaks. SPT D includes STEC serotypes reported to cause
sporadic disease that are associated with diarrhea but not severe disease. Serotypes included in SPT
E have not been associated with human illness.
The identification of non-O157 EHEC serotypes remains challenging because of a lack of phenotypical
characteristics that can distinguish these strains from less-virulent STEC serotypes and other E.
coli that share the same environment. Furthermore, of all confirmed STEC infections in the European
Union during 2007 to 2010, more than 85% of the isolates were not fully serotyped (9). As SPT
classification requires fully serotyped isolates, the identification of non-O157 EHEC serotypes proves
to be a major obstacle. Also, the 2011 O104:H4 EHEC outbreak has demonstrated that the
emergence of new virulent strains is another limitation of the SPT classification proposed by Karmali
et al., as these strains cannot be assigned to a specific SPT group (10, 11).
While it remains unclear which virulence factors (VF) precisely define STEC pathogenicity, the STEC
serotypes that carry VF genes in addition to stx genes are more likely to be associated with HC and
HUS (9, 12). These strains usually carry the LEE, a pathogenicity island (PAI) containing genes
responsible for the characteristic attaching and effacing (A/E) lesions (4, 13). In addition, they can be
Chapter 2
34
characterized by non-LEE-encoded effector (nle) genes, which are harbored on other PAIs in the
bacterial chromosome (5, 14, 15), and virulence plasmids encoding EHEC-hemolysin (EHEC-hlyA) that
are widely distributed among EHEC of different serotypes (16–18). Enteroaggregative E. coli (EAEC)-
STEC hybrid strains of serotypes other than O104:H4, such as O111:H2, O86:NM, O59:NM, and
Orough:NM, have also been associated with sporadic cases and outbreaks of HUS and (bloody)
diarrhea, advocating the incorporation of EAEC virulence markers for the categorization of STEC (19–
21). However, no single VF or combination of virulence factors precisely defines the potential of a
STEC strain to cause more severe disease. While the stx subtypes stx2a and stx2c and the LEE-
positive strains are associated with a high risk of more serious illness (9, 22–24), other virulence gene
combinations (even in E. coli strains that lack the stx genes) may also be associated with severe
disease, including HC and HUS (12, 25–27). Furthermore, patient characteristics and infectious doses
also determine the outcome of disease (28).
Although the current approaches for detecting STEC in clinical microbiology laboratories still mainly
rely on a conventional culture (e.g., sorbitol MacConkey agar [SMAC] or cefixime tellurite [CT-SMAC])
and to a lesser extent on Stx toxin-based assays, a trend toward PCR-based methods for the rapid
detection of STEC (stx1 and stx2 genes) has been observed in recent years, resulting in improved
detection rates (29, 30). Enhanced detection and reporting of STEC infections have as a drawback an
increased workload for community health services, and the clinical and public health relevance of
PCR findings solely based on the detection of the stx genes is unclear (31). Therefore, diagnostic
approaches that can categorize STEC while avoiding the limitations of the SPT classification of Karmali
et al. are needed.
In this study, we describe a rapid screening algorithm, including both molecular and conventional
methods, to determine the pathogenic potential of STEC. The aim is to discriminate infections with
less-virulent STEC from those with clinical relevance and risk for public health.
MATERIALS AND METHODS
Patient specimens.
Our laboratory serves a population of about 1 million inhabitants, including both community and
hospitalized patients. From September 2012 through December 2012 a total of 5022 stool samples
were prospectively screened for presence of enteric bacterial, protozoan and viral pathogens. The
samples originated from patients (n=4714) with infectious gastroenteritis (IG) included in their
differential diagnosis. Their mean age was 39 years (range, 0 to 101 years) and 1985 (42.1%) patients
were males. Clinical information addressing symptoms, use of antibiotics, and travelling history were
obtained from the request form filled out by physicians. On receipt, all stool specimens were
Diagnostic Algorithm for STEC Risk Assessment
35
routinely examined by molecular methods (real-time PCR [qPCR]) for the presence of Campylobacter
jejuni, Salmonella enterica, Shiga toxin-producing Escherichia coli (STEC), Shigella spp., enteroinvasive
E. coli (EIEC), Cryptosporidium parvum, C. hominis, Dientamoeba fragilis, Giardia lamblia, and
Entamoeba histolytica. Upon specific request of physicians, examination for the presence of
adenovirus (EIA), rotavirus (EIA), norovirus (qPCR), and Clostridium difficile toxins A and B (EIA) was
also performed.
Design of the diagnostic algorithm and STEC risk assessment.
The algorithm consists of qPCR for detection of the stx genes (stx1 and stx2) on stool samples, as
described previously (30). qPCR stx-negative stool samples were regarded as STEC negative. In case
of a stx-qPCR positive result, the stool sample was enriched in brilliant green bile (BGB) broth
followed by DNA extraction, and multiplex qPCR for the detection of VF (stx1, stx2, stx2f, escV, aggR
and aat genes), and O-serogroup determination (wzxO26, wzxO103, wzxO104, wbdO111, wzxO121, ihpO145
and rfbO157). In order to obtain an isolate, qPCR positive samples were cultured directly and after
enrichment on STEC selective media. Virulence determinants and O-serogroups were confirmed by
qPCR on suspicious colonies (or streaks) grown on STEC selective media, and by seroagglutination.
Attempts to obtain an isolate were made up to a maximum of 5 colonies per agar plate. A schematic
overview of the diagnostic algorithm is presented in Figure 1.
The risk assessment of STEC infections was performed using a molecular approach, as described
previously (9). It delivers a scheme that describes the presumptive categorization of STEC according
to their potential risk, using the presence of genes encoding VF additional to the presence of the stx
genes. Categorization of stx PCR positive samples is based upon the presence of the VF escV (LEE-
positive), and/or aggR/aat (pAA-positive) and on detection of O-serogroups that are most frequently
associated with severe human disease and outbreaks, e.g. O26, O103, O104, O111, O121, O145, and
O157. The potential risk for diarrhea and severe disease has been categorized as pathotype (PT)
group I (high risk for diarrhea and severe disease) to PT group III (moderate risk for diarrhea/low risk
for severe disease), while PT group IV consists of stx PCR positive samples that are not confirmed
after enrichment (Table 1). In case an isolate was obtained and fully serotyped, a classification of the
STEC isolate into a seropathotype (SPT) was made as described previously (5). Stx subtyping and
genetic characterization of cultured isolates was performed in order to confirm the validity of the
proposed molecular-based PT approach for risk assessment of STEC infections.
Chapter 2
36
PCR guided culture.
For culture of STEC selective media SMAC and CHROMagar STEC (CHROMagar Microbiology, Paris,
France) were used (24h at 35°C), directly and after enrichment in BGB broth for approximately 16h at
35°C. Identification of STEC and/or EHEC O157- suspicious colonies (non-sorbitol fermenting colonies
on SMAC and mauve non-fluorescent colonies on CHROMagar STEC) and STEC/EHEC non-O157
(mauve fluorescent colonies on CHROMagar STEC) was carried out by detection of virulence genes
and serogenotyping with qPCR, performing an indole reaction and serological typing (serogroup
O157 only). All genotypically/biochemically identified E. coli were confirmed using the VITEK 2
system (bioMérieux, Boxtel, The Netherlands). All culture and identification media were produced by
Mediaproducts BV, Groningen, The Netherlands, whereas the E. coli O157 agglutination serum was
from Oxoid, Basingstoke, Hampshire, England. Resistance profiling was performed with the VITEK 2
system. Furthermore, of qPCR positive samples with Ct<35, five E. coli colonies cultured on SMAC
agar were sub-cultured and sent to the National Institute for Public Health and the Environment
(RIVM, Bilthoven, The Netherlands) for genotying (stx1, stx2, stx2f, eae, EHEC-hlyA, and O157) and
O:H-serotyping of isolates, as part of STEC national surveillance.
Figure 1 The STEC diagnostic algorithm consists of both molecular and conventional methods; when stx genes
are detected with direct qPCR, the stool sample is enriched. DNA is isolated from the enriched broth and
screened for the presence of stx genes and additional VF and O serogroups. VF and O serogroups are confirmed
by qPCR on suspicious colonies grown on selective media and by seroagglutination. STEC isolates were fully
serotyped (O:H typing) at the RIVM.
Diagnostic Algorithm for STEC Risk Assessment
37
Table 1. Proposed molecular approach for the presumptive categorization of STEC based on
enriched BGB PCR results
NA; not applicable a escV gene, marker for presence of the LEE PAI; aggR/aat genes, markers for the presence of the pAA plasmid
carried by EAEC; NA, not applicable.
Molecular assays
(i) Specimen preparation and DNA extraction
Specimen preparation followed by DNA extraction using the automated NucliSens easyMAG
(bioMérieux, Boxtel, The Netherlands) according to the manufacturer’s instructions was performed
as previously described (30). Briefly, for DNA extraction from stool, 100 µl fecal suspension and 50 µl
of enriched selenite broth was used as input. For DNA extraction from enriched BGB broth, 100 µl
was used as input. In addition, approximately 6000 copies of the Phocine herpes virus 1 (PhHV),
which served as an internal control (IC), were co-purified. DNA was eluted in 110 μl of elution buffer.
For confirmation of suspicious colonies by qPCR, DNA from isolates was extracted by heat lysis for 10
min at 95°C in NucliSens easyMAG elution buffer. For genetic characterization by microarray, the
DNA extraction was performed using a DNeasy blood and tissue kit (QIAGEN, GmbH Germany) from
the overnight culture of the pure isolates according to the manufacturer’s instructions.
(ii) Real-time PCR
Real-time amplification was carried out on an AB 7500 sequence detection system (Applied
Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) as described previously (30). Each 25 µl
reaction consisted of 5 µl template DNA, 1x TaqMan Universal PCR Master Mix, and 2.5 µg bovine
serum albumin (Roche Diagnostics Netherlands B.V., Almere, The Netherlands). The primers and
probes used for detection of virulence determinants and O-serogroup specific gene targets are listed
PT group
Direct PCR
stx genes present
Enriched BGB PCR stx genes present
Additional genesa Serogroups
Potential risk
Diarrhea HUS/HC
I Yes Yes escV-positive or aggR and/or aat
positive
O26, O103, O104, O111, O121, O145,
O157 High High
II Yes Yes escV-positive or aggR and/or aat
positive Any other serogroup High Moderate
III Yes Yes escV negative
and aggR and/or aat negative
Any serogroup Moderate Low
IV Yes No NA NA NA NA
Chapter 2
38
in the supplementary table S1. Reactions were run under the following conditions: 50°C for 2 min,
95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min. In every PCR run a
negative extraction control (NEC) and positive extraction control (PEC) or PCR mix control (PMC) was
included. A real-time PCR was considered inhibited when the Ct value for the PhHV exceeded the
mean Ct value for uninhibited specimens + 2 standard deviations.
(iii) Stx subtyping
DNA isolates of the BGB broth and/or confirmed STEC isolates were sent to the University Medical
Center Groningen (UMCG, Groningen, The Netherlands) for stx subtyping. Subtyping of the stx1 and
stx2 gene was performed as described previously (32). Briefly, for stx1 subtyping, a triplex PCR was
performed, each 25 μl reaction consisted of 2.5 μl of PCR buffer 10x (Qiagen), 1 μl of MgCl2 25 mM
(Qiagen), 0.5 μl of 10 mM dNTP mix (Applied Biosystems), 0.25 μl Hotstar Polymerase 5U/µl
(Qiagen), 2 μl of each of two primers for stx1a, 1 μl of each of the four primers for stx1c and stx1d
(stock solution of all primers was 5 μM), and 5 μl of template DNA.
For stx2 subtyping PCR, each 20 μl reaction consisted of 2.5 μl of PCR buffer 10x (Qiagen), 0.8 μl of
MgCl2 25 mM (Qiagen), 0.4 μl of 10 mM dNTP mix (Applied Biosystem), 0.2 μl Hotstar Polymerase
5U/µl (Qiagen), 1.25 µl of each of the primers and 5 μl of template DNA. Stx2c and stx2e subtyping PCR
was performed as a duplex PCR as well as stx2f and stx2g. Reactions were run under the following
conditions: 95°C for 15 min followed by 35 cycles of 94°C for 50 s, 64°C (hybridization was at 66°C for
stx2d) for 40 s and 72°C for 60 s, with a final extension at 72°C for 3 min.
(iv) Genetic characterization by DNA microarray
Confirmed STEC isolates were sent to the University Medical Centre Groningen for genetic
characterization using an E. coli genotyping combined assay kit according to the manufacturer’s
protocol (Clondiag, Alere Technologies, GmbH, Jena, Germany). The E. coli oligonucleotide arraystrips
contain gene targets for the identification of virulence genes, antimicrobial resistance genes and
DNA-based serotyping genes. Briefly, multiplex linear DNA amplification and labeling was performed
in a total volume of 10 µl containing 3.9 µl of 2x labeling Buffer, 1 µl of E. coli labeling primer mix, 0.1
µl of DNA Polymerase and 5 µl of genomic DNA (100-200 ng/µl). Reactions were run under the
following conditions: 96°C for 5 min followed by 45 cycles of 50°C for 20 s, 72°C for 30 s and 96°C for
20 s.
The hybridization and washing steps were performed using the Hybridization plus kit according to the
manufacturer’s protocol. Visualization of hybridization was achieved using the ArrayMate instrument
(CLONDIAG GmbH, Jena, Germany) and signals of the array spots were analyzed automatically.
Ambiguous called signals were re-checked visually in order to obtain a definite interpretation when
possible. In case any signal remained inconclusive, they were regarded as negative.
Diagnostic Algorithm for STEC Risk Assessment
39
Statistical analysis.
We used the Fisher exact method to test if the presence or absence of VF was associated to certain
PT groups and whether growth of suspicious colonies on CHROMagar STEC was associated with
presence of the escV gene (LEE-positive) (JavaStat). Median Ct values of subgroups were compared
using the Wilcoxon rank sum test with NCSS version 2007 (NCSS statistical software, Kaysville, UT,
USA). One-way analysis of variance (ANOVA) was used to compare the total number of VF present in
isolated strains that were assigned to PT groups. For all tests statistical significance was indicated by
a two-tailed p<0.05.
Furthermore, cluster analysis of VF with construction of dendrograms was performed with
Bionumerics version 4.6 (Applied Maths NV, Sint-Martens-Latem, Belgium) using the Dice correlation
and the unweighted-pair group method using average linkages (UPGMA).
RESULTS
Detection frequency of stx genes in patient specimens.
A total of 5,022 stool specimens from 4,714 patients were examined, using direct qPCR for detection
of the stx genes. In total, 90 samples (84 patients) were positive for the stx genes (1.8%). The
diagnostic algorithm was applied on all samples, but for only 73 samples (70 patients) all screening
data were available; therefore the remaining 17 samples were excluded for analysis. Direct qPCR for
the stx genes was confirmed by qPCR on “enriched BGB” in 65 samples (89%). In the remaining 8
samples (11%) no stx genes could be detected after broth enrichment, although in one sample the
virulence factors aggR/aat/escV and O104 serogroup were detected. These 8 samples initially had a
relatively high Ct-value (Ct 34) in the direct qPCR.
The stx ΔCt values for “enriched BGB” PCR and direct qPCR (ΔCt = CtBGB – Ctdirect) ranged from Ct +9 to
–21. In 55/65 (85%) of the “enriched BGB” samples the ΔCt 0, indicative for the presence of viable
STEC; Ct values of “enriched BGB” PCR (mean Ct value = 23.1) were significantly lower compared to
Ct values of direct qPCR (mean Ct value = 29.6) (Wilcoxon rank sum, p<0.001) (Figure 2A).
The additional virulence genes escV, aggR/aat and bfpA were detected in 49% (n=36), 6% (n=4), and
6% (n=4) of qPCR positive samples, respectively. The O145, O26, O157, O104, O121 and O111
serogroups were detected in 11.0% (n=8), 8% (n=6), 4% (n=3), 3% (n=2), 3% (n=2), and 1% (n=1) of
qPCR positive samples, respectively (Table 2).
PCR guided culture.
The PCR guided culture yielded a positive result in 42.5% (31/73) of direct qPCR positive samples. A
STEC isolate was obtained in 35.6% (26/73) of the samples; from one sample two STEC isolates were
Chapter 2
40
obtained. Two additional samples (3%) were streak PCR positive for stx genes. The serotypes that
were identified are listed in Table 2. Using the Karmali seropathotype concept, one STEC isolate
(O157:H7) could be assigned to SPT group A, six STEC isolates (4x O26:H11; 2x O145:NM) to SPT
group B, and two STEC isolates (O117:H7 and O146:H21) to SPT group D. The other 17 STEC isolates
(65.4%) could not be assigned to a SPT group. One isolate and one streak PCR positive sample could
not be serotyped.
From the remaining three culture positive samples an enteropathogenic E. coli (EPEC) (n=2; O88:H25
and ONT:H31) or EAEC (n=1; O104:H4) was isolated. The isolation yield of the SMAC medium was
higher (21/73) compared to the CHROMagar STEC medium (15/73), although 5 isolates and one
streak PCR positive sample were only identified with CHROMagar STEC. Furthermore, the growth of
suspicious colonies on CHROMagar STEC was highly associated with presence of the escV gene (LEE-
positive) detected by the “enriched BGB” PCR (19/23 vs 17/50) (Fisher exact; p<0.0001).
Ct values (stx1/stx2) of samples in which the PCR guided culture remained negative were significantly
higher than in samples with positive guided culture (Wilcoxon rank sum, p<0.0003). This difference in
Ct value between the PCR guided culture negative and positive group remained significant, when
comparing Ct values of enriched BGB PCR (Wilcoxon rank sum, p<0.001). The distribution of Ct values
of enriched BGB PCR on which guided culture was performed are shown in Figure 2B.
Figure 2. Direct comparison of stx CT values for direct qPCR versus stx CT values for enriched BGB PCR (A). The
solid line represents the hypothetical identical performance between both methods. The stx CT values for
enriched BGB PCR were significantly lower (Wilcoxon rank sum, P0.001). Distribution of CT values for STEC
isolates that were positive according to enriched BGB qPCR (B). The black bars represent the number of stool
specimens positive in the PCR-guided culture. The dashed bars represent the additional qPCR-positive stool
specimens.
Diagnostic Algorithm for STEC Risk Assessment
41
Table 2. Overall results of risk categorization of STEC-positive stool samples
No. of samples by PT group
qPCR Stx subtyping Serotyping of cultured isolates
a
Total no. of virulence factors by DNA array
Serogenotype Additional virulence
factors
I (n=11)
1 O157 escV stx1a + stx2c O157:H7 32
1 O157 escV, aggR/aat stx2c Not cultured
1 O26 escV stx1a + stx2b + stx2c O26:H11 35
1 O26 escV stx2a O26:H11 31
2 O26 escV (n=2) stx1a O26:H11 29/29
1 O26 / O121 / O145 escV stx2b + stx2c O26 (streak)b -
1 O145 escV stx1a + stx2a O145:NM 28
1 O145 escV stx2 not typable O145:NMc 34
1 O157 / O26 / O145 escV stx2a + stx2d + stx2e Not cultured -
1 O121 / O111 / O145 escV stx1a Not cultured -
II (n=19)
2 escV (n=2) stx1a + stx2a O165:NM 33 O182:H25 25
4 escV (n=4), aggR/aat (n=1)
stx1a Not cultured -
1 escV stx2a O182:H25 27
1 escV stx2a + stx2c Not typedd 33
1 escV stx2c Not cultured -
1 escV stx2f O63:H6 (n=1) 18
1 escV stx2f O125:H6 (n=1) 16
1 escV, bfpA stx2f O88:H25 (EPEC)e 15
6 escV (n=6) stx2f Not cultured -
1 escV stx1c Not cultured
III (n=35)
3 escV (n=1), bfpA (n=1) stx1a O91:NM (n=3)f 9/9/10
2 stx1a O91:H14 (n=2) 9/11
1 stx1a ONT:NM 8
1 escV stx1a O117:H7 f 4
1 stx1a Culture positive (streak)
-
11 O145 (n=1) stx1a Not cultured -
1 escV, bfpA stx1c + stx2b O128:H2f 17
1 stx1c + stx2b O76:H19 20 1 aggR/aat stx1c + stx2b O146:H21
f 19
1 stx1c + stx2b Not cultured -
1 stx2b O7:H6 2 1 stx2b ONT:H31 (aEPEC)
e 12
1 stx2b Not cultured -
2 stx2c ONT:H28 (n=1) 11
1 stx2d Not cultured
1 stx2e Not cultured 1 stx1c Not cultured
1 stx1 not typable O16:H5 13
3 O145 (n=1) escV, bfpA (n=1) Not typable Not cultured
IV (n=8)
1 O104 escV, aggR and/or aat Not typed O104:H4 (EAEC)e 10
7 O145/O104 (n=1) Not typed Not cultured - a NM, non-motile; ONT, O-serogroup O1 to O187 negative. b PCR-positive culture by screening DNA isolated from a loopful of bacterial growth of the first streaking area of culture plates. c Coinfection with an ONT:H45 (stx2f positive) isolate. This isolate was not genetically characterized. d The isolate could not be recultured after transportation to the RIVM for genotyping/serotyping. e Isolates did not contain stx genes and were designated EPEC (O88:H25; escV, bfpA), atypical EPEC (aEPEC [ONT:H31; escV]), and EAEC (O104:H4; aggR and/or aat). f The isolates did not contain the additional virulence factors escV or aggR and/or aat.
Chapter 2
42
Stx subtyping of clinical samples.
Subtyping of stx genes was performed on DNA isolates of enriched BGB broths that were PCR
positive for stx genes. Of these 65 positive samples, 30 (46%) were stx1 positive, 26 (40%) were stx2
positive, and 9 (14%) were stx1 and stx2 positive. Two stx1 subtypes (stx1a and stx1c) and six stx2
subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, and stx2f) were detected with a total of 15 different stx1 and
stx2 subtype combinations. The most frequently detected subtype variants were stx1a (40%), stx2f
(14%), stx1c + stx2b (6%), stx2c (6%), stx2b (5%), and stx1a + stx2a (5%), accounting for 49 samples (75%).
For three samples subtyping results remained negative, although the DNA load seemed to be
sufficient. For two of these samples subtyping remained negative after DNA isolation from the
obtained STEC isolate. For an additional three samples no stx subtype could be obtained due to low
DNA load (all Ct ≥32 in enriched BGB PCR).
Risk categorization of STEC and distribution of virulence factors between PT groups.
Samples were presumptively categorized in four pathotype (PT) groups based on the “enriched BGB”
PCR results. A total of 11 samples (15%), 24 samples (33%), 30 samples (41%), and 8 samples (11%)
were categorized in PT group I, group II, group III, and group IV respectively. However, based on the
presence of the additional virulence factor bfpA and screening of VF in the cultured isolates, a total
of 5 samples (7%) were re-categorized from PT group II to PT group III; 4 STEC isolates did not contain
the escV gene (O91:NM, O117:H7, O128:H2 and ONT:H31) and in one sample there was no
correlation in Ct value for stx (Ct=39) and the other VF escV (Ct=19) and bfpA (Ct=18). The final risk
categorization was 11 samples (15%), 19 samples (26%), 35 samples (48%), and 8 samples (11%) for
PT group I, group II, group III, and group IV, respectively (Table 2). The presence of stx genes for
samples categorized in PT group IV could not be confirmed after enrichment, thereby excluding them
from further analysis.
The studied virulence factors (VF) differed with respect to their distribution among the different
pathotype groups (Table 3). Compared to the PT group that is associated with a moderate risk for
diarrhea and low risk for severe disease (III), PT groups that are associated with a high risk for
diarrhea and higher risk for severe disease (I + II, combined) exhibited a significant higher prevalence
of various VF analyzed (specifically, stx2, stx2a, stx2f, toxB, eae, efa1, cif, espA, tccP, espP, nleA/B, and
the tir cluster). Although not significant, stx2c was more prevalent in PT group I + II (OR 4.1 [95%CI =
0.7 – 32.7]). Inversely, stx1, the mch cluster, ireA and eaaA were significantly more prevalent in PT
group III (Table 3). Interestingly, the adhesion-encoding gene iha was present in all PT group I isolates
and almost all isolates in PT group III (92%), however there was no significant association between
Diagnostic Algorithm for STEC Risk Assessment
43
presence of iha and PT groups. Furthermore, all LEE-positive STEC isolates contained the EHEC-hlyA
gene, with exception of the two stx2f STEC isolates. Noteworthy, certain VF were also highly
associated with PT group I (specifically, stx2c, toxB, eae, efa1, cif, tccP, nleA and/or nleB, katP and the
tir cluster).
The total number of VF present in STEC isolates also showed a significant non-random distribution
between PT groups (Table 2); the number of VF differed significantly between PT group I (VFmean 31
[95%CI = 27 – 35], PT group II (VFmean 25 [95%CI = 21 – 30]), and PT group III (VFmean 11 [95%CI = 8 –
13]) (ANOVA, p<0.0001, F = 38.5). Interestingly, the total number of VF present in the two cultured
stx2f STEC isolates (O63:H6, and O125:H6) that were categorized in PT group II, were considerably
lower compared to other 4 STEC isolates categorized in this PT group (Table 2). By cluster analysis of
potential VF, escV-positive and escV-negative isolates were separated into two main clusters (Figure
3). The escV-negative cluster included all PT group III isolates and a stx-negative isolate EAEC
O104:H4 (PT group IV). The escV-positive cluster included all PT group I + II isolates and two stx-
negative EPEC isolates (O88:H25 and ONT:H31) that clustered in a distinct branch with the two stx2f
positive STEC isolates.
Clinical symptoms of patients.
Diarrhea was reported by 80%, 44%, 57%, and 75% of patients in PT groups I, II, III, and IV,
respectively. Bloody diarrhea was reported by 20%, 6%, 3%, and 0% of patients in PT groups I, II, III,
and IV, respectively. Patients in PT group I presented significantly more often with (bloody) diarrhea
compared to PT groups II + III (Fisher exact test, P = 0.006). One patient categorized in PT group I
developed HC (serotype O26:H11), and family members of another patient categorized in PT group I
(serotype O26:H11) also had gastrointestinal complaints. Interestingly, symptoms reported by
patients that are not associated with acute disease, such as persistent diarrhea and/or abdominal
complaints without loose stools, were absent in patients categorized in PT group I (0%) but present in
patients in PT groups II, III, and IV (33%, 29%, and 25%, respectively). The age distribution of patients
did not differ between PT groups (PT group I: mean age = 27 years; 95% CI = 10 to 44 years; PT group
II: mean age = 36 years; 95% CI = 23 to 48 years; PT group III: mean age = 41 years; 95% CI = 32 to 50
years) (ANOVA, P = 0.33; F = 1.1), although the median age of patients in PT group I was considerably
lower (15 years) compared to PT group II (33 years) and PT group III (39 years).
Chapter 2
44
Table 3. Pathotype distribution of virulence factors and stx subtypes
Total no. (%)
No. (%)a
of isolates or enriched BGB broths for PT:
Statistical comparison of PT I + PT II vs PT III
b
Virulence genotype I II III Pc
OR (95% CI)
Enriched BGB broths (n=65)
stx1 (all) 39 (60) 6 (55) 8 (42) 25 (71) 0.021 0.3 (0.08 – 0.9) stx1a 31 (48) 6 (55) 7 (37) 18 (51) stx1c 6 (9) 0 1 (5) 5 (14) stx2 (all) 35 (54) 8 (73) 14 (74) 13 (37) 0.006 4.7 (1.4 – 15.6) stx2a 7 (11) 3 (27) 4 (21) 0 0.003 ∞(1,7 – inf) stx2b 9 (14) 2 (18) 0 7 (20) stx2c 8 (12) 4 (36) 2 (11) 2 (6) 4.1 (0.7 – 32.7) stx2d 2 (3) 1 (9) 0 1 (3) stx2e 2 (3) 1 (9) 0 1 (3) stx2f 9 (14) 0 9 (47) 0 < 0.0001 ∞ (2.5 – inf) stx2a or stx2c 14 (22) 7 (64) 5 (26) 2 (6) 0.002 11.0 (2.0 – 80.5)
Isolates (n=26)
astA 7 (27) 3 (43) 3 (50) 1 (8) EHEC-hlyA 20 (77) 7 (100) 4 (67) 9 (69) toxB 7 (27) 6 (86) 1 (17) 0 0.005 ∞ (1.9 – inf) mch cluster 9 (35) 0 0 9 (69) < 0.0001 < 0.0001 (0 – 0.3)
ireA 8 (31) 0 0 8 (62) 0.002 < 0.0001 (0 – 0.4)
eae 13 (50) 7 (100) 6 (100) 0 < 0.0001 ∞ (18.4 – inf) efa 7 (27) 6 (86) 1 (17) 0 0.005 ∞ (1.9 – inf) iha 20 (77) 7 (100) 1 (17) 12 (92) lpfA 16 (62) 3 (43) 3 (50) 8 (62) iss 15 (58) 5 (71) 0 10 (77) cif 9 (35) 6 (86) 3 (50) 0 < 0.0001 ∞ (3.5 – inf) espA 17 (65) 6 (86) 6 (100) 5 (38) 0.011 19.2 (1.5 – 537.4)
tccP 12 (46) 7 (100) 5 (83) 0 < 0.0001 ∞ (10.4 – inf) eaaA 10 (39) 0 0 10 (77) < 0.0001 < 0.0001 (0 – 0.2)
espP 11 (42) 5 (71) 4 (67) 2 (15) 0.015 12.4 (1.4 – 142.1)
nleA/B 12 (46) 7 (100) 5 (83) 0 < 0.0001 ∞ (10.4 – inf) etpD 4 (15) 2 (29) 2 (33) 0 katP 7 (27) 5 (71) 1 (17) 1 (8) tir cluster 10 (39) 6 (86) 4 (67) 0 < 0.0001 ∞ (4.8 – inf)
a Total no. of enriched BGB broths: PT I, 11; PT II, 19; PT III, 35. Total no. of isolates: PT I, 7; PT II, 6; PT III, 13.
b PT I and II combined (associated with high risk for diarrhea and high/moderate risk for severe disease) are
compared to PT III (lower risk for diarrhea and severe disease). ∞, infinite. c P values (from the Fisher exact test) are shown only if the P value was <0.05.
Diagnostic Algorithm for STEC Risk Assessment
45
DISCUSSION
We here present the first prospective study that uses a diagnostic algorithm directly applied on stool
samples of patients presenting with gastrointestinal complaints to assess the public health risk of
STEC. Although the disease severity and incidence of STEC is not solely based on the pathogenic
potential of the organism but also on host-associated and environmental factors, enough
Figu
re 3
. C
lust
er a
nal
ysis
of
po
ten
tial
vir
ule
nce
gen
es
in c
ult
ure
d s
trai
ns.
Th
e es
cV-n
egat
ive
clu
ster
in
clu
ded
all
PT
gro
up
III
iso
late
s an
d a
n s
tx-n
egat
ive
iso
late
, EA
EC O
10
4:H
4 (
PT
gro
up
IV
). T
he
escV
-po
siti
ve c
lust
er
incl
ud
ed a
ll P
T gr
ou
ps
I_II
iso
late
s
and
tw
o s
tx-n
ega
tive
EP
EC i
sola
tes
(O8
8:H
25
an
d O
NT:
H3
1)
that
clu
ste
red
in
a d
isti
nct
bra
nch
wit
h t
he
two
stx
2f-
po
siti
ve
STEC
iso
late
s. S
po
ts d
epic
ted
in g
ray
rep
rese
nt
amb
igu
ou
s re
sult
s th
at w
ere
rega
rded
as
neg
ativ
e.
Chapter 2
46
information has accumulated that the presence of virulence factors (VF) carried additional to the stx
genes varies considerably between STEC strains, and could therefore be used to categorize the
potential risk of STEC (5, 17, 33-36).
The detection frequency of the stx genes observed in this study (1.8%) was comparable with previous
studies performed in The Netherlands (30, 37). The diagnostic algorithm enabled categorization of
STEC infections into 4 pathotype (PT) groups. The majority of the initial stx-PCR positive samples
(48%) were categorized in PT group III, while 15% and 26% of stx-PCR positive samples were
categorized in PT group I and PT group II, respectively, both having a high risk for diarrhea and
moderate to high risk for severe disease. The presence of stx genes could not be confirmed after
enrichment in 11% of samples and these were categorized in PT group IV. Stx subtyping and genetic
characterization was performed in order to confirm the validity of the proposed categorization of
STEC infections.
Previous studies have indicated that the subtype of shiga toxin produced may influence the clinical
outcome of STEC infections (23, 24). STEC harboring stx2a or stx2c are associated with HUS and bloody
diarrhea, while strains carrying stx1c or stx2b have often been isolated from patients with milder
infections (38). Although STEC carrying stx2d usually predict a milder disease, strains that produce
elastase-activatable Stx2d may predict a severe clinical outcome of the infection (39). Other variants,
such as stx2e and stx2f, have been associated with animals and are rarely isolated from humans (24,
40).
In our study, there was a strong association between presence of stx2, in particular stx2a or stx2c, and
samples categorized in PT group I + II (LEE-positive), while the presence of the stx1 gene was
associated with samples categorized in PT group III. Similar to a previous study performed in Belgium,
stx1a was the most detected subtype (41). Furthermore, the detection frequency of the stx2f gene in
our study (12.3%) was comparable with previous studies (41, 42). Stx2f was, together with stx2b, the
first-most detected stx2 subtype among samples that were serogroup O157 PCR negative in this
study. Similar to previous studies, all stx2f PCR positive samples also contained the escV gene (LEE-
positive) (41-43). With exception of stx2b, the detection frequencies of subtypes stx1c (9%) and stx2e
(3%) that are associated with milder disease or asymptomatic carriage were similar to the incidence
detected in Belgium (41).
Furthermore, cluster analysis of VF clearly showed a separation into an escV-negative (PT group III)
and escV-positive cluster (PT group I + II) with a significant difference in the number of “accessory”
virulence factors (VF) present between these PT groups. Furthermore, VF that play an important role
in toxin production, and attachment to host cells, were highly associated with PT group I + II or PT
group I alone, while other VF were associated with PT group III. Previous studies also reported that
Diagnostic Algorithm for STEC Risk Assessment
47
the number of VF present in STEC isolates increases the pathogenic potential of STEC and the strong
association of certain “accessory” VF and severe illness and outbreaks (12, 17, 22, 27, 33, 34, 36, 44).
Interestingly, the accessory virulence gene content of both the stx2f STEC positive isolates, that
clustered in a distinct branch with two stx-negative EPEC isolates, was lower compared to the other
STEC isolates categorized in PT group II. Others have also reported that stx2f STEC form a distinct
group within STEC with regard to virulence genes and their association with a relatively mild disease
(41, 42).
Our findings with respect to the main clinical features of STEC infection are consistent with those of
others (9, 40). Patients with STEC infections categorized in PT group I presented significantly more
often with (bloody) diarrhea, suggesting that the pathogenic potential of STEC in this group is higher
compared to STEC categorized in PT group II + III, as was confirmed by stx subtyping and genetic
characterization. Although there was no clear association between patient age and PT groups in our
study, the age distribution of patients in PT group I was considerably lower in comparison to patients
categorized in PT group II and PT group III. Others did also report a close relation between patient’s
young age and infection with more virulent (LEE-positive) STEC strains (40). Furthermore, this study
revealed a high number (45%) of other enteric pathogens detected in individual stx-PCR positive
samples (data not shown). However, the clinical relevance of these mixed infections was beyond the
scope of this study.
Although stx subtyping and genetic characterization confirmed the validity of the PT classification,
categorization with this molecular-based PT approach should be regarded as presumptive. Additional
subtyping of stx genes, genetic characterization and O:H-serotyping of STEC isolates will provide a
clearer assessment of the potential public health risk. Hence, a high culture yield remains important
for facilitating these laboratory procedures.
An important step in the diagnostic algorithm is the use of an enrichment step, which was performed
on the initial stx PCR positive stool samples. Performing this step has several advantages. First,
confirmation by performing PCR on the enrichment broth increases the positive predictive value for
detection of STEC; 89% of the initial stx PCR positive results could be confirmed. In the majority of
the samples (85%) the stx Ct values were lower after enrichment, which suggests the presence of
viable STEC. In a part of the samples (11%) no stx genes could be detected after enrichment (PT
group IV). The stx Ct values for direct qPCR were relatively high in all these samples, suggestive for
presence of low loads of non-viable STEC or free stx DNA. Another possibility would be detection of
free stx phages in the stool of these patients, which has been described previously in stool of healthy
individuals (45).
Chapter 2
48
Second, stx subtyping was performed directly from the DNA isolate obtained from the enriched
broth. To our knowledge, subtyping of stx genes is only being performed on STEC isolates, which will
take additional time for obtaining final subtyping results. Third, although not statistically proven,
culture yield will improve using enrichment; in the majority of the initial stx PCR positive samples the
“stx gene” load increased after enrichment, suggestive for the presence of viable STEC. Higher
amounts of STEC bacteria in the background of intestinal flora will increase the odds of isolation by
culture. The culture yield in our study (38%) was lower compared to other studies (24, 41). However,
the amount of colonies screened with PCR (maximum of 10) in our study was considerably lower
compared to those studies. Hence, increasing the total amount of colonies screened, and routinely
screening DNA isolated from a loopful of bacteria growth from the first streaking area of culture
plates (as was performed for two samples in this study) would increase the probability of obtaining
an isolate or at least confirm the growth of STEC.
Furthermore, due to more easily identification of suspicious colonies the CHROMagar STEC medium
proved to be an effective supplemental medium for isolation of especially more virulent (LEE-
positive) STEC serotypes as described previously by others (46-48). The medium also supported the
growth of EAEC (O104:H4) and EPEC (O88:H25, and ONT:H31), suggesting that it could also be an
useful tool for support of EAEC and EPEC isolation as described previously (49).
Unfortunately, our diagnostic algorithm only includes direct molecular screening for the stx genes,
rendering the detection of shiga toxin lost (STL) EHEC impossible (25, 26, 50). Another limitation of
this study was the concise amount of clinical information that was available on request forms that
may have influenced clinical associations. Furthermore, the number of STEC isolates that were
characterized was limited.
In conclusion, the proposed diagnostic algorithm for risk categorization of STEC infection offers a
rapid testing format that could be easily implemented in laboratories that already perform qPCR-
based detection of STEC. It enables stx-PCR positive stool samples to be categorized for the potential
risk to public health. This risk assessment may provide valuable information to aid community health
services in estimating the level of action required (with regard to source/contact tracing, and
intervention measures to minimize secondary transmission) to address the potential threat, as well
as a useful tool for public health surveillance. However, the proposed risk categorization should be
regarded as presumptive and interpreted with care as infections with STEC serotypes categorized in
PT group III, such as O117:H7, can still pose a public health concern as has been shown recently (51).
Currently, a multicenter prospective cohort study is being conducted that will verify the performance
of the proposed molecular-based pathotyping approach on a larger scale, in order to justify its
Diagnostic Algorithm for STEC Risk Assessment
49
application in case of STEC infections for determining if swift action by community health services is
warranted or not.
ACKNOWLEDGEMENTS
The authors would like to thank Alex W. Friedrich (Department of Medical Microbiology, University
of Groningen, University Medical Center Groningen, Groningen, the Netherlands) for critically
reviewing this manuscript and his helpful suggestions. We also thank Evert van Zanten (Certe
Laboratory for Infectious Disease, Department of Research & Development, Groningen, The
Netherlands) for help with construction of dendrograms for cluster analysis of VF.
This study formed part of a larger multidisciplinary research initiative called STEC-ID-net.
REFERENCES
1. Paton JC, Paton AW. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli
infections. Clin Microbiol Rev 11:450-79.
2. Griffin PM, Tauxe RV. 1991. The epidemiology of infections caused by Escherichia coli O157:H7,
other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 13:60-
98.
3. Gyles CL. 2007. Shiga toxin-producing Escherichia coli: an overview. J Anim Sci 85:E45-62.
4. Kaper JB, Nataro JP, Mobley HL. 2004. Pathogenic Escherichia coli. Nat Rev Microbiol 2:123-140.
5. Karmali MA, Mascarenhas M, Shen S, Ziebell K, Johnson S, Reid-Smith R, Isaac-Renton J,
Clark C, Rahn K, Kaper JB. 2003. Association of genomic O island 122 of Escherichia coli EDL 933
with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious
disease. J Clin Microbiol 41:4930-4940
6. Karmali MA. 1989. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev 2:15-
38.
7. Rangel JM, Sparling PH, Crowe C, Griffin PM, Swerdlow DL. 2005. Epidemiology of Escherichia
coli O157:H7 outbreaks, United States, 1982-2002. Emerg Infect Dis 11:603-9.
8. (BIOHAZ), S. O. o. t. P. o. B. H. 2007. Monitoring of verotoxigenic Escherichia coli (VTEC) and
identification of human pathogenic VTEC types. EFSA Journal 579:1-61.
9. (BIOHAZ), E. P. o. B. H. 2013. Scientific Opinion on VTEC-seropathotype and scientific criteria
regarding pathogenicity assessment. EFSA Journal 11:3138.
10. Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth, A, Bauwens A, Peters G, Karch H. 2011.
Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic
syndrome in Germany, 2011: A microbiological study. Lancet Infect Dis 11:671–676.
11. Piérard D, De Greve H, Haesebrouck F, Mainil J. 2012. O157:H7 and O104:H4 Vero/Shiga toxin-
producing Escherichia coli outbreaks: respective role of cattle and humans. Vet Res 43:13.
Chapter 2
50
12. Prager R, Annemüller S, Tschäpe H. 2005. Diversity of virulence patterns among shiga toxin-
producing Escherichia coli from human clinical cases-need for more detailed diagnostics. Int J Med
Microbiol 295:29-38.
13. Schmidt MA. 2010. LEEways: tales of EPEC, ATEC and EHEC. Cell. Microbiol. 12:1544-1552.
14. Karch H, Schubert S, Zhang D, Zhang W, Schmidt H, Olschlager T, Hacker J. 1999. A genomic
island, termed high-pathogenicity island, is present in certain non-O157 Shiga toxin-producing
Escherichia coli clonal lineages. Infect Immun 67:5994-6001.
15. Makino S, Tobe T, Asakura H, Watarai M, Ikeda T, Takeshi K, Sasakawa C. 2003. Distribution of
the secondary type III secretion system locus found in enterohemorrhagic Escherichia coli O157:H7
isolates among Shiga toxin-producing E. coli strains. J Clin Microbiol 41:2341-7.
16. Brunder W, Schmidt H, Frosch M, Karch H. 1999. The large plasmids of Shiga-toxin-producing
Escherichia coli (STEC) are highly variable genetic elements. Microbiology 145 ( Pt 5):1005-14.
17. Bugarel M, Martin A, Fach P, Beutin L. 2011. Virulence gene profiling of enterohemorrhagic
(EHEC) and enteropathogenic (EPEC) Escherichia coli strains: a basis for molecular risk assessment of
typical and atypical EPEC strains. BMC Microbiol 11:142.
18. Newton HJ, Sloan J, Bulach DM, Seemann T, Allison CC, Tauschek M, Robins-Browne RM,
Paton JC, Whittam TS, Paton AW, Hartland EL. 2009. Shiga toxin-producing Escherichia coli
strains negative for locus of enterocyte effacement. Emerg Infect Dis 15:372-80.
19. Iyoda S, Tamura K, Itoh K, Izumiya H, Ueno N, Nagata K, Togo M, Terajima J, Watanabe H. 2000. Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia
coli O86:HNM isolated from patients. FEMS Microbiol Lett 191:7-10.
20. Morabito S, Karch H, Mariani-Kurkdjian P, Schmidt H, Minelli F, Bingen E, Caprioli A. 1998.
Enteroaggregative, Shiga toxin-producing Escherichia coli O111:H2 associated with an outbreak of
hemolytic-uremic syndrome. J Clin Microbiol 36:840-2.
21. Prager R, Lang C, Aurass P, Fruth A, Tietze E, Flieger A. 2014. Two Novel EHEC/EAEC Hybrid
Strains Isolated from Human Infections. PLoS One 9:e95379.
22. Boerlin P, McEwen SA, Boerlin-Petzold F, Wilson JB, Johnson RP, Gyles CL. 1999. Associations
between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J Clin
Microbiol 37:497-503.
23. Persson S, Olsen KE, Ethelberg S, Scheutz F. 2007. Subtyping method for Escherichia coli shiga
toxin (verocytotoxin) 2 variants and correlations to clinical manifestations. J Clin Microbiol 45:2020-4.
24. Friedrich AW, Bielaszewska M, Zhang WL, Pulz M, Kuczius T, Ammon A, Karch H. 2002.
Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical
symptoms. J Infect Dis 185:74-84.
25. Bielaszewska M, Köck R, Friedrich AW, von Eiff C, Zimmerhackl LB, Karch H, Mellmann A.
2007. Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm? PLoS
One 2:e1024
26. Friedrich AW, Zhang W, Bielaszewska M, Mellmann A, Köck R, Fruth A, Tschäpe H, Karch H.
2007. Prevalence, virulence profiles, and clinical significance of Shiga toxin-negative variants of
enterohemorrhagic Escherichia coli O157 infection in humans. Clin Infect Dis 45:39-45.
27. Haugum K, Brandal LT, Lindstedt BA, Wester AL, Bergh K, Afset JE. 2014. PCR-based detection
and molecular characterization of shiga toxin-producing Escherichia coli strains in a routine
microbiology laboratory over 16 years. J Clin Microbiol 52:3156-63.
28. Todd WT, Dundas S. 2001. The management of VTEC O157 infection. Int J Food Microbiol 66:103-
10.
29. Vallieres E, Saint-Jean M, Rallu F. 2013. Comparison of three different methods for detection of
Shiga toxin-producing Escherichia coli in a tertiary pediatric care center. J Clin Microbiol 51:481-6.
30. de Boer RF, Ott A, Kesztyus B, Kooistra-Smid A M. 2010. Improved detection of five major
gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol 48:4140-6.
31. Lede IO, Kraaij-Dirkzwager MM, van den Kerkhof JHTC, Notermans DW. 2012. Lack of
uniformity with notifications of Shiga-toxin producing Escherichia coli and Shigella towards and by
community health services. Infectieziekten Bulletin 23:116-118.(In dutch).
32. Scheutz F, Teel LD, Beutin L, Pierard D, Buvens G, Karch H, Mellmann A, Caprioli A, Tozzoli R,
Morabito S, Strockbine NA, Melton-Celsa AR, Sanchez M, Persson S, O'Brien AD. 2012.
Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx
nomenclature. J Clin Microbiol 50:2951-2963.
Diagnostic Algorithm for STEC Risk Assessment
51
33. Brandt SM, King N, Cornelius AJ, Premaratne A, Besser TE, On SL. 2011. Molecular risk
assessment and epidemiological typing of Shiga toxin-producing Escherichia coli by using a novel PCR
binary typing system. Appl Environ Microbiol 77:2458-70.
34. Girardeau JP, Dalmasso A, Bertin Y, Ducrot C, Bord S, Livrelli V, Vernozy-Rozand C, Martin C. 2005. Association of virulence genotype with phylogenetic background in comparison to different
seropathotypes of Shiga toxin-producing Escherichia coli isolates. J Clin Microbiol 43:6098-107.
35. Toma C, Martinez Espinosa E, Song T, Miliwebsky E, Chinen I, Iyoda S, Iwanaga M, Rivas M. 2004. Distribution of putative adhesins in different seropathotypes of Shiga toxin-producing
Escherichia coli. J Clin Microbiol 42:4937-46.
36. Wickham ME, Lupp C, Mascarenhas M, Vazquez A, Coombes BK, Brown NF, Coburn BA, Deng
W, Puente JL, Karmali MA, Finlay BB. 2006. Bacterial genetic determinants of non-O157 STEC
outbreaks and hemolytic-uremic syndrome after infection. J Infect Dis 194:819-27.
37. van Duynhoven YT, Friesema IH, Schuurman T, Roovers A, van Zwet AA, Sabbe LJ, van der
Zwaluw WK, Notermans DW, Mulder B, van Hannen EJ, Heilmann FG, Buiting A, Jansen R,
Kooistra-Smid AM. 2008. Prevalence, characterisation and clinical profiles of Shiga toxin-producing
Escherichia coli in The Netherlands. Clin Microbiol Infect
14:437-45.
38. Friedrich AW, Borell J, Bielaszewska M, Fruth A, Tschape H, Karch H. 2003. Shiga toxin 1c-
producing Escherichia coli strains: phenotypic and genetic characterization and association with human
disease. J Clin Microbiol 41:2448-53.
39. Bielaszewska M, Friedrich AW, Aldick T, Schurk-Bulgrin R, Karch H. 2006. Shiga toxin
activatable by intestinal mucus in Escherichia coli isolated from humans: predictor for a severe clinical
outcome. Clin Infect Dis 43:1160-7.
40. Beutin L, Krause G, Zimmermann S, Kaulfuss S, Gleier K. 2004. Characterization of Shiga toxin-
producing Escherichia coli strains isolated from human patients in Germany over a 3-year period. J Clin
Microbiol 42:1099-108.
41. Buvens G, De Gheldre Y, Dediste A, de Moreau AI, Mascart G, Simon A, Allemeersch D, Scheutz
F, Lauwers S, Pierard D. 2012. Incidence and virulence determinants of verocytotoxin-producing
Escherichia coli infections in the Brussels-Capital Region, Belgium, in 2008-2010. J Clin Microbiol
50:1336-45.
42. Friesema I, van der Zwaluw K, Schuurman T, Kooistra-Smid M, Franz E, van Duynhoven Y, van
Pelt W. 2014. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing
E. coli (STEC) infections in the Netherlands, January 2008 to December 2011. Euro Surveill 19:26-32.
43. Prager R, Fruth A, Siewert U, Strutz U, Tschape H. 2009. Escherichia coli encoding Shiga toxin 2f
as an emerging human pathogen. Int J Med Microbiol 299:343-53.
44. Bosilevac JM, Koohmaraie M. 2011. Prevalence and characterization of non-O157 shiga toxin-
producing Escherichia coli isolates from commercial ground beef in the United States. Appl Environ
Microbiol 77:2103-12.
45. Martinez-Castillo A, Quiros P, Navarro F, Miro E, Muniesa M. 2013. Shiga toxin 2-encoding
bacteriophages in human fecal samples from healthy individuals. Appl Environ Microbiol 79:4862-8.
46. Hirvonen JJ, Siitonen A, Kaukoranta SS. 2012. Usability and performance of CHROMagar STEC
medium in detection of Shiga toxin-producing Escherichia coli strains. J Clin Microbiol 50:3586-90.
47. Wylie JL, Van Caeseele P, Gilmour MW, Sitter D, Guttek C, Giercke S. 2013. Evaluation of a new
chromogenic agar medium for detection of Shiga toxin-producing Escherichia coli (STEC) and relative
prevalences of O157 and non-O157 STEC in Manitoba, Canada. J Clin Microbiol 51:466-71.
48. Tzschoppe M, Martin A, Beutin L. 2012. A rapid procedure for the detection and isolation of
enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, O145 and
O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables. Int J Food
Microbiol 152:19-30.
49. Hauswaldt SI, Rodloff AC, Solbach W, Knobloch JKM. 2012. Improving diagnostics of
diarrheagenic Escherichia coli by use of a new chromogenic medium. ECCMID:P1760.
50. Schmidt H, Scheef J, Huppertz HI, Frosch M, Karch H. 1999. Escherichia coli O157:H7 and
O157:H(-) strains that do not produce Shiga toxin: phenotypic and genetic characterization of isolates
associated with diarrhea and hemolytic-uremic syndrome. J Clin Microbiol 37:3491-6.
51. Simms I, Gilbart VL, Byrne L, Jenkins C, Adak GK, Hughes G, Crook PD. 2014. Identification of
verocytotoxin-producing Escherichia coli O117:H7 in men who have sex with men, England,
November 2013 to August 2014. Eurosurveillance 19:pii=20946.
Chapter 2
52
Supplementary Materials
Supplementary Table S1. Primer and probe sequences used for five real-time (multiplex) qPCR
reactions.
Target Multiplex reaction
Sequence name Sequence (5’ – 3’)a, b
Tm (°C)
Conc (nM)
Reference
stx1 A stx1F934_mod TGG CAT TAA TAC TGA ATT GTC ATC ATC 59.2 300
(1, 2)
stx1F934F_mod1d TGG CAT TAA TAT TAA ATT GCC ATC AT 58.7 300 stx1R1042_G GCG TAA TCC CAC GGA CTC TTC 59.6 300 stx1R1042_modC GCG TAA TCC CAC GCA CTC TT 58.8 300 stx1R1042_mod1d GAG TAA TCC CAC GCC CAC TTC 59.4 300 stx1P990_mod_MGB FAM-TTC CTT CTA TGT GTC CGG CAG-
NFQMGB 69.0 100
stx1P990_mod1c_MGB FAM-CCT TCT ATG TGC CCG GTA G-NFQMGB
69.0 100
stx1P990_mod1d_MGB FAM-TCC TTC TAT GTG CCC GAC AG-NFQMGB
69.0 100
stx2 A stx2F_LvI CCG GAA TGC AAA TCA GTC GT 59.5 300
(2) stx2R_G_LvI ACC ACT GAA CTC CAT TAA CGC C 59.0 300 stx2R_A_LvI TAC CAC TAA ACT CCA TTA ACG CCA 58.7 300 stx2P_LvI_MGB VIC-ACT CAC TGG TTT CAT CAT A-
NFQMGB 68.9 100
stx2f A stx2F_mod2f_LvI GGA ACG TAC AGG GAT GCA GAT T 59.0 300
(1) stx2R_mod2f_LvI CGT CCT CTG AAC TCC ATT AAA TCC 59.0 300 stx2P_mod2f_LvI VIC-ATG AAC CAA CCA GTG AAT-
NFQMGB 69.0 100
escV A escV_F GCG TCA TTY TGA CCG CTT TAG 56.4 300
(3) escV_R1 TCC TGA AAA GAG AGC ACG GG 59.0 300 escV_R2 TCC TGA AAA GAA AGC ACA GGG 58.0 300 escV-TM CY5- ACT GAC GGG AAC GAA CCT TCA
ATC ATT TTC -BBQ 68.9 200
wzxO103 B Ec_wzxO103_F CGT TGT TAT CTA TGG TGG GCT TAG T 58.6 300
This study Ec_wzxO103_R CAC CTG CAA CCG CAT TAT TTA A 58.7 300 Ec_wzxO103_PTM FAM-TTG GCC TCA AAG GCG CAT TAG
TGT CT-BHQ1 68.2 75
wzxO121 B Ec_wzxO121_F CAT GGC GGG ACA ATG ACA 58.4 400
(4) Ec_wzxO121_R CGA TAG TGA AGA ACA AAA TAT GAA
GAG TTC 59.2 400
Ec_wzxO121_PMGB VIC-TGC TGG ACT ACA GAA AA-NFQMGB 69.0 100 rfbEO157 B Ec_rfbEO157_F CGA TGA GTT TAT CTG CAA GGT GAT 58.3 600
(5) Ec_rfbEO157_R TTT CAC ACT TAT TGG ATG GTC TCA A 58.6 600 Ec_rfbEO157_PTM CY5 -CCT TAA TTC CTC TCT TTC CTC TGC
GGT CCT -BBQ 68.5 150
wzxO26 C Ec_wzxO26_F CGC GAC GGC AGA GAA AAT T 59.9 400
(5) adapted
Ec_wzxO26_R AGC AGG CTT TTA TAT TCT CCA ACT TT 58.2 400 Ec_wzxO26_PMGB VIC-CCG TTA AAT CAA TAC TAT TTC ACG
A-NFQMGB 68.0 100
ihp1O145 C Ec_ihp1O145_F CGA TAA TAT TTA CCC CAC CAG TAC AG 58.0 400
(6) adapted
Ec_ihp1O145_R GCC GCC GCA ATG CTT 59.0 400 Ec_ihp1O145_PMGB FAM-CGA TAT TGT GTG CAT TCT-
NFQMGB 68.0 100
Diagnostic Algorithm for STEC Risk Assessment
53
Target Multiplex reaction
Sequence name Sequence (5’ – 3’)a, b
Tm (°C)
Conc (nM)
Reference
wbdIO111 C Ec_wbdIO111_F CGA GGC AAC ACA TTA TAT AGT GCT TT 58.8 400
(5) Ec_wbdIO111_R TTT TTG AAT AGT TAT GAA CAT CTT GTT
TAG C 58.8 400
Ec_wbdIO111_PTM CY5-TTG AAT CTC CCA GAT GAT CAA CAT CGT GAA-BBQ
68.7 150
wzxO104 D Ec_wzxO104_F TGT CGC GCA AAG AAT TTC AAC 59.8 400
(7) adapted
Ec_wzxO104_R ATC CTT TAA ACT ATA CGC CCT AGA AAC C
59.6 400
Ec_wzxO104_PMGB VIC-TTT GTA TTA GCA ATA AGT GGT GTC-NFQMGB
68.0 100
aggR D aggR_F CAA TAA GGA AAA GRC TTG AGT CAG A 59.4 300
(3) aggR_R1 TCA AGC AAC AGC AAT GCT GC 59.7 300 aggR_R2 TTA TCA AGC AAT AGC AAT GCT GCT 59.1 300 aggR_P FAM-CCT TAT GCA ATC AAG AAT-
NFQMGB 69.0 50
aat D aat_F GGG CAG TAT ATA AAC AAC AAT CAA
TGG 59.8 300
(3)
aat_R1 GGG CAG TAT ATA AAC AAC AAC CAG TG 58.9 300 aat_R2 GCT TCA TAA GCC GAT AGA AGA TTA
TAG G 59.2 300
aat_PMGB1 FAM-TCT CAT CTA TTA CAG ACA GCC-NFQMGB
69.0 25
aat_PMGB2 FAM-CTC ATC TAT TAC AGA CAG CAA T-NFQMGB
69.0 25
bfpA D bfpA_F1 ATC ACA CCT GCG GTA ACG G 58.0 600
(3) bfpA_F2 TCA CAC CGG CGG TAA CG 58.6 600 bfpA_R CGA RAA AGG TCT GTC TTT GAT TGA 60.7 600 bfpA_PTM CY5-CAG CAA GCG CAA GCA CCA TTG C-
BBQ 68.7 200
PhHV (internal control)
All PhHV-267s GGG CGA ATC ACA GAT TGA ATC 58.1 300
(2, 8) PhHV-337as GCG GTT CCA AAC GTA CCA A 58.1 300
PhHV-1-MGB NED-CGC CAC CAT CTG GAT-NFQMGB 70.0 100
a Fluorescent dyes used are represented in bold. FAM, 6-carboxyfluorescein; VIC, 6-carboxyrhodamine; BHQ1, black hole
quencher-1; NFQ-MGB, non-fluorescent quencher minor groove binder; Cy5, reactive water-soluble fluorescent dye of the
cyanine dye family; BBQ, black berry quencher; NED, 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-
carboxyfluorescein. b Y, C or T; R, A or G.
REFERENCES for Table S1
1. de Boer RF, Ott A, Kesztyus B, Kooistra-Smid AM. 2010. Improved detection of five major
gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol 48:4140-6.
2. Schuurman T, Roovers A, van der Zwaluw WK, van Zwet AA, Sabbe LJ, Kooistra-Smid AM, van
Duynhoven YT. 2007. Evaluation of 5'-nuclease and hybridization probe assays for the detection of
shiga toxin-producing Escherichia coli in human stools. J Microbiol Methods 70:406-15.
Chapter 2
54
3. Friesema IH, de Boer RF, Duizer E, Kortbeek LM, Notermans DW, Norbruis OF,Bezemer DD,
van Heerbeek H, van Andel RN, van Enk JG, Fraaij PL, Koopmans MP, Kooistra-Smid AM, van
Duynhoven YT. 2012. Etiology of acute gastroenteritis in children requiring hospitalization in the
Netherlands. Eur J Clin Microbiol Infect Dis 31:405-15.
4. Tzschoppe M, Martin A, Beutin L. 2012. A rapid procedure for the detection and isolation of
enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, O145 and
O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables. Int J Food
Microbiol 152:19-30.
5. Perelle S, Dilasser F, Grout J, Fach P. 2004. Detection by 5'-nuclease PCR of Shiga-toxin producing
Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's
most frequent clinical cases. Mol Cell Probes 18:185-92.
6. Perelle S, Dilasser F, Grout J, Fach P. 2003. Development of a 5'-nuclease PCR assay for detecting
Shiga toxin-producing Escherichia coli O145 based on the identification of an 'O-island 29' homologue.
J Appl Microbiol 94:587-94.
7. Bugarel M, Beutin L, Martin A, Gill A, Fach P. 2010. Micro-array for the identification of Shiga
toxin-producing Escherichia coli (STEC) seropathotypes associated with Hemorrhagic Colitis and
Hemolytic Uremic Syndrome in humans. Int J Food Microbiol 142:318-29.
8. van Doornum GJ, Guldemeester J, Osterhaus AD, Niesters HG. 2003. Diagnosing herpesvirus
infections by real-time amplification and rapid culture. J Clin Microbiol 41:576-80.
55
CHAPTER 3
Molecular Characterization and Phylogeny of Shiga Toxin-
Producing Escherichia coli Isolates Obtained from Two
Dutch Regions Using Whole Genome Sequencing
Mithila Ferdous1, Alexander W. Friedrich1, Hajo Grundmann1, Richard F. de Boer2, Peter D. Croughs3, Md.A. Islam4, Marjolein F.Q. Kluytmans-van den Bergh5,6, Anaa M.D. Kooistra-Smid1,2, John W.A. Rossen1
1Department of Medical Microbiology, University of Groningen, University Medical Center
Groningen, Groningen, the Netherlands. 2Department of Medical Microbiology, Certe Laboratory for Infectious Diseases, Groningen, the
Netherlands. 3Star-MDC, Rotterdam, the Netherlands. 4Department of Psychiatry, Rob Giel Research Center, University of Groningen, University Medical
Center Groningen, Groningen, the Netherlands. 5Julius Center for Health Sciences and Primary Care, UMC Utrecht, Utrecht, the Netherlands. 6Amphia Academy Infectious Disease Foundation, Amphia Hospital, Breda, the Netherlands.
Keywords
Shiga Toxin-Producing Escherichia coli (STEC), Whole Genome Sequencing (WGS), Multilocus
sequence typing (MLST), Core genome MLST, Disease outcome, Phylogenetic analysis, Genetic
diversity
Clinical Microbiology and Infection 22 (2016) 642.e1-642.e9
Chapter 3
56
ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) is one of the major causes of human gastrointestinal
disease and has been implicated in sporadic cases and outbreaks of diarrhoea, haemorrhagic colitis
and haemolytic uremic syndrome worldwide. In this study we determined the molecular
characteristics and phylogenetic relationship of STEC isolates, and their genetic diversity was
compared with that of other E. coli populations. Whole genome sequencing (WGS) was performed
on 132 clinical STEC isolates obtained from faeces of 129 Dutch patients with gastrointestinal
complaints. STEC isolates of this study belonged to 44 different sequence types (STs), 42
serogenotypes and 14 stx subtype combinations. Antibiotic resistance genes were more frequently
present in stx1 positive isolates compared to stx2 and stx1+stx2 positive isolates. The iha, mchB,
mchC, mchF, subA, ireA, senB, saa, sigA genes were significantly more frequently present in eae
negative than in eae positive STEC isolates. Presence of virulence genes encoding type III secretion
proteins and adhesins was associated with isolates obtained from patients with bloody diarrhoea.
Core genome phylogenetic analysis showed that isolates clustered according to their ST or
serogenotypes irrespective of stx subtypes. Isolates obtained from patients with bloody diarrhoea
were from diverse phylogenetic background. Some STEC isolates shared common ancestors with
non-STEC isolates. WGS is a powerful tool for clinical microbiology allowing high resolution molecular
typing, population structure analysis and detailed molecular characterization of strains. STEC isolates
of a substantial genetic diversity and of distinct phylogenetic groups were observed in this study.
Characterization of STEC Using Whole Genome Sequencing
57
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) is a pathogen of significant public health concern
associated with both outbreaks and sporadic cases of human gastrointestinal illness worldwide (1).
Enterohaemorrhagic E. coli (EHEC), a subpopulation of STEC, can cause bloody diarrhoea in humans
and some of them can cause haemolytic-uremic syndrome (HUS) (2). The ability of STEC to cause
disease is associated with the production of Shiga-like toxins (Stx), which are classified into two major
types Stx1 and Stx2 (encoded by stx1 and stx2 genes). Stx1 and Stx2 are further categorised into
several subtypes; according to the new classification proposed by Scheutz et al, Stx1 consists of three
variants, Stx1a, Stx1c and Stx1d, whereas Stx2 is a diverse group composed of seven distinct variants,
namely Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f and Stx2g (3). The production of Stx might not be
solely responsible for pathogenesis of STEC (4); several mobile genetic elements, e.g., plasmids,
transposons, phages and pathogenicity islands also play a role in disease outcome (4,5). One of the
major virulence factors is the outer membrane protein intimin, encoded by eae gene, which is part of
a pathogenicity island named the locus of enterocyte effacement (LEE) (6). Intimin is thought to be
the genetic determinant of the formation of attaching and effacing (A/E) lesions, which is
characterized by the intimate attachment of the bacteria to the enterocyte membrane and by the
effacement of the microvilli of the enterocyte (6).
More than 100 O-serotypes (based on the somatic antigen) of STEC have been associated with
human disease (7), STEC O157:H7 being the predominant serotype implicated in food-borne
infections world-wide and the cause of outbreaks in many countries like Canada, Japan, the United
Kingdom and the United States (8). However, several non-O157 STEC types have also been associated
with sporadic cases and outbreaks. Six O-serotypes (O26, O111, O103, O121, O45, and O145) have
been reported by the CDC as the cause of 71% of non-O157 STEC infection and these serotypes are
considered as the “top (or big) 6 STEC” (7). Moreover, in Europe infections caused by non-O157 STEC
strains are more common than those by O157:H7 strains (8).
Different methods have been used to classify STEC. Karmali et al introduced seropathotypes to assess
the pathogenic potential of STEC based on their reported frequencies in human illness. Subsequently,
the classification was modified by the European Food Safety Authority (EFSA) based on the health
outcome of reported confirmed human VTEC cases (9). For epidemiological purposes various genetic
fingerprinting methods have been developed to identify, trace and prevent dissemination of STEC
(10). Among the sequence based methods, multilocus sequence typing (MLST) is a reliable method to
determine genetic relatedness of epidemiologically-unrelated isolates, but has limited discriminatory
power (11). Currently whole genome sequencing (WGS) of bacterial genomes is an accessible and
Chapter 3
58
affordable method (12,13). An obvious application for WGS is epidemiological typing to detect and
support outbreak investigations, to define transmission pathways of pathogens, and to reveal
laboratory cross-contamination (14). Due to the public health importance of STEC infections,
epidemiological and molecular surveillance systems are essential for early outbreak detection and to
differentiate STEC strains based on their potential to cause severe illness in human (15,16). This study
was performed to determine the molecular characteristics, phylogenetic relationship and diversity of
STEC isolates from faeces of patients obtained from two regions in the Netherlands, and to reveal the
relation between molecular determinants and disease outcome.
MATERIALS and METHODS
Collection of Isolates
A multicentre prospective study, STEC-ID-net, was performed during the period from April 2013 to
March 2014 in the Dutch regions Groningen and Rotterdam. Stool samples from patients with
suspected infectious gastroenteritis were screened using qPCR targeting the stx1/stx2/escV genes
and positive samples were further processed to obtain pure stx-positive isolates (17).
Phenotypic antibiotic resistance pattern
Antibiotic resistance patterns of the STEC isolates were determined using VITEK2 (bioMerieux, Marcy
l’Etoile, France) following European Committee on Antimicrobial Susceptibility Testing guidelines.
Whole genome sequencing
To perform the whole genome sequencing of stx positive isolates, DNA was extracted using the
UltraClean® microbial DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, US) according to the
manufacturer’s protocol. A DNA library was prepared using the Nextera XT kit (Illumina, San Diego,
CA, US) according to the manufacturer’s instructions and then ran on a Miseq (Illumina) for
generating paired-end 250-bp reads aiming at a coverage of at least 60 fold.
Data analysis and molecular characterization.
De novo assembly was performed using CLC Genomics Workbench v7.0.3 (CLC bio A/S, Aarhus,
Denmark) after quality trimming (Qs ≥ 28) with optimal word sizes based on the maximum N50 value
(18). The average N50 value of the sequenced 132 STEC isolates was 144166 (range 23341 to 387919)
and the average number of contigs was 178 (range 59 to 497). Gene annotation was performed by
uploading the assembled genome on RAST server version 2.0 (19). The sequence types (STs) and O-
Characterization of STEC Using Whole Genome Sequencing
59
and H- serogenotypes were identified by uploading the assembled genomes to the MLST finder 1.7
(20) and SerotypeFinder 1.1 tool (21) respectively of Center for Genomic Epidemiology (CGE)
website. The virulence genes and stx subtypes were determined using CGE VirulenceFinder 1.2 (22),
and antibiotic resistance genes were determined by CGE ResFinder 2.1 (23). For the CGE server the
threshold of ID was set to 85% and the percentage of minimum overlapping gene length to 60%. The
sequences assigned unknown sequence type by CGE MLST finder were submitted to the EnteroBase
database of the University of Warwick.
This whole-genome shotgun project has been deposited in NCBI under BioProject PRJNA285020. The
GenBank accession numbers are listed in Supplementary Table S1.
Statistical analysis
To determine the association of virulence genes with different patient groups and to compare the
presence of virulence genes in eae-positive and -negative STEC isolates, the Pearson chi-square test
was used. To observe the effect of the virulence genes on patient groups, univariate binary logistic
regression was performed. All analyses were done using two-tailed tests at a 5% significance level.
The statistical analyses were performed using IBM SPSS Statistics 22 (IBM SPSS, Chicago, IL, USA).
Phylogenetic analysis of STEC isolates
To determine the phylogenetic relationship of the isolates, a gene-by-gene approach was performed
using SeqSphere+ v3.0 (Ridom, Münster, Germany). Briefly, an ad hoc core genome MLST (cgMLST)
scheme was developed using the genome of E. coli O157:H7 strain Sakai (accession no. NC_002695)
as reference genome and additional ten E. coli as query genomes (Supplementary File S2) to extract
open reading frames (ORFs) from the genome of each isolate using MLST+ Target Definer v2.1.0 of
SeqSphere+. Only the ORFs without premature stop codon and ambiguous nucleotides from contigs
of assembled genomes were included. The genes shared by all isolates analysed were defined as the
core genome for phylogenetic analysis [18]. A Neighbour Joining (NJ) tree was constructed based on
a distance matrix among the isolates depending on the core genome of all isolates. To compare the
isolates with previously reported ones, five additional genomes of STEC (E. coli strain Sakai, strain
EDL933, strain 11368, strain 2011c-3493 and strain 12009) available on NCBI were included in the
phylogenetic analysis. To see the allele differences among the isolates a minimum spanning tree
(MST) was constructed by SeqSphere+ based on the numerical allele type for each isolates according
to the sequence identity of each gene (24). An additional NJ tree based on the accessory genome of
the isolates was also constructed.
Chapter 3
60
Phylogenetic comparison of STEC isolates with the Diarrhoeagenic E. coli (DEC) reference collection
The phylogenetic relationship of the STEC isolates of this study with isolates of the DEC reference
collection (n=76) (25) was determined by cgMLST. The DEC consists of predominant clones of
diarrhoeagenic E. coli, including 27 STEC, 25 Enteropathogenic E. coli (EPEC) and 24 non STEC/EPEC E.
coli.
Genetic Diversity of STEC isolates
To reveal the genetic diversity of the STEC isolates of this study, median pairwise distance (MPD) was
calculated (26) and the MPD was compared with that of two other independent strain collections,
i.e.,76 isolates of the DEC collection and 131 isolates that were randomly selected from a collection
of ESBL (extended-spectrum beta-lactamase) producing Enterobacteriaceae (ESBL-E) from a multi-
centre study on the epidemiology of ESBL-E in Dutch hospitalised patients (SoM study, unpublished
data). As neither of the underlying population followed normal distribution pattern, the median of
the pair-wise distances and interquartile ranges (IQR) were calculated in all three populations. To
identify the group differences on MPD of three E. coli isolates, the Kruskal-Wallis test was used.
Additionally, to compare the genetic diversity within the STEC isolates with that of other E. coli
populations, the Mann-Whitney U-test was performed and the statistical significance level was
corrected by Bonferroni correction for multiple testing.
RESULTS
STEC isolates and patient groups
From 425 stx positive faecal samples, 132 STEC isolates (Groningen, n= 70 and Rotterdam, n= 62)
from 129 patients were obtained. From three patients two different types of STEC were isolated.
Characteristics of the isolates are summarized in Supplementary Table S1. Based on clinical outcomes
(available for 110 patients), patients were classified into groups with bloody diarrhoea (including one
HUS patient) (n=26), non-bloody diarrhoea (n=64) and no diarrhoea (n=20) but having other clinical
symptoms as abdominal pain, nausea, malaise and so on (Supplementary Table S1).
Serotypes, sequence types and stx subtypes
Forty-two different serogenotypes and 44 different STs were found among the 132 isolates. The most
predominant serogenotypes were O91:H14 (n=19), O157:H7 (n=17) and O26:H11 (n=15) (Figure 1a).
The most predominant STs were ST33 (n=19), ST11 (n=17) and ST21 (n=17) (Figure 1b). For six of the
isolates we found new STs assigned by EnteroBase database (Table S1). Fourteen different stx
Characterization of STEC Using Whole Genome Sequencing
61
subtypes (combinations) were found among the isolates with stx1a being most predominant (n=55,
41.6%) (Figure 1c). Almost all O91:H14 isolates contained only the stx1a gene except two, of which
one (STEC 2620) contained the stx1a+stx2b and the other (STEC 2110-1) only the stx2a gene (Table
S1). One stx2 positive isolate (STEC 2826) had both stx2d (encoding Stx A subunit) and stx2a
(encoding Stx B subunit) gene.
Presence of virulence genes
Among the eae positive isolates (n=71), 33 (46%) contained stx1, 21 (30%) contained stx2, 16 (23%)
contained stx1+stx2 and one isolate was found without stx gene. Among the eae negative isolates
(n=61), the prevalence of stx1, stx2 and stx1+stx2 positive isolates was 49% (n=30), 26% (n=16) and
24.5% (n=15) respectively. In eae positive isolates, the virulence genes tir, espA, espB, espF, espJ,
espP, nleA, nleB, nleC, etpD, katP, toxB, efa1 and cif were significantly more often present, whereas
iha, mchB, mchC, mchF, subA, ireA, sepA, senB, saa and sigA genes were significantly more often
found in eae negative isolates (Figure 2). The associations and the effects (odds ratios and their
confidence interval) of the presence of virulence genes with bloody diarrhoea and diarrhoea are
presented in Table 1.
Antibiotic resistance patterns and presence of resistance genes
Phenotypic antibiotic resistance patterns of the isolates are shown in Supplementary Table S3. In
thirty-three (25%) STEC isolates at least one antibiotic resistance gene was found. Although not
significant, presence of resistance genes was higher in only stx1 positive isolates (35.5%) compared
to stx2 (16.5%) (P=0.063) and stx1+stx2 (15%) (P=0.054) positive isolates. Antibiotic resistance genes
found in STEC isolates are shown in Table 2. Two isolates (STEC 381-1 and STEC 1255) of serotype
O104:H4 and O5:H9 were ESBL producers and contained the blaCTX-M-15 and blaCTX-M-1 genes,
respectively.
Chapter 3
62
Figure 1. Distribution of different serogenotypes (a), sequence types (STs) (b), and stx subtype combinations
(c) among STEC isolates.
*Other serogenotypes and sequence types of all isolates are summarized in supplementary Table S1.
Figure 2. Comparison of virulence genes in eae-positive and eae-negative STEC isolates. Blue and red bars
represent the frequency of virulence genes in eae-positive and eae-negative isolates, respectively.
*P < 0.05, **P < 0.001
Characterization of STEC Using Whole Genome Sequencing
63
Table 1. Distribution of virulence genes (other than stx) among isolates obtained from three
patient groups
Virulence gene
category
Virulence genes
Patient groups n (%)
Pa
ORb (95% CI)
BD (N=26)
NBD (N=64) ND (N=20) BD vs NBD BD vs ND
Adhesin
eae 21(80) 34(53) 7(35) 0.006* 3.7 (1.24-11.04) 7.8(2.04-29.7)
tir 21(80) 34(53) 7(35) 0.006* 3.7 (1.2-11.04) 7.8(2.04-29.7)
espB 18(69) 24(37.5) 6(30) 0.009* 3.7(1.4-9.9) 5.2(1.4-18.6)
iha 14(54) 36(56) 15(75) 0.272 0.9(.36-2.2) 0.39(0.11-1.38)
efa1 11(42) 18(28) 4(20) 0.231 1.87(0.72-4.8) 2.93(0.76-11.2)
saa 1(4) 2(3) 1(5) 0.924 1.24(0.10-14.3) 0.76(0.04-12.9)
Toxin
ehxA 24(92) 41(64) 13(65) 0.023* 6.7(1.4-31) 6.4(1.1-35.7)
toxB 12(46) 15(23) 4(20) 0.063 2.8(1.06-7.3) 3.4(0.89-13)
astA 17(65) 26(41) 7(35) 0.059 2.7(1.06-7.13) 3.5(1.03-11.9)
subA 2(8) 10(15.6) 5(25) 0.273 0.45(0.92-2.21) 0.25(0.04-1.45)
cnf1 0(0) 1(1.6) 0(0) 0.696 Undefined Undefined
cdtB 0(0) 1(1.6) 1(5) 0.441 Undefined Undefined
senB 1(4) 5(8) 2(10) 0.704 0.47(0.05-4.2) 0.36(0.03-4.28)
Secretion system
espA 21(80) 33(51) 7(35) 0.005* 3.9(1.3-11.7) 7.8(2.04-29.7)
espC 1(3.8) 3(4.7) 0(0) 0.619 0.81(0.08-8.19) Undefined
espF 19(73) 27(42) 5(25) 0.003* 3.7(1.3-10) 8.1(2.1-30.8)
espI 3(11.5) 8(12.5) 2(10) 0.954 0.91(0.22-3.75) 1.17(0.18-7.8)
espJ 21(80) 33(51) 6(30) 0.002* 3.9(1.3-11.7) 9.8(2.5-38.4)
nleA 20(77) 27(42) 6(30) 0.002* 4.5(1.6-12.9) 7.7(2.07-29.1)
nleB 19(73) 30(47) 6(30) 0.011* 3.07(1.1-8.3) 6.3(1.7-23)
nleC 16(61.5) 22(34) 6(30) 0.035* 3.05(1.1-7.8) 3.7(1.08-12.9)
etpD 12(46) 15(43) 1(5) 0.005* 2.8(1.06-7.3) 16.2(1.8-140)
cif 12(46) 26(40.6) 6(30) 0.534 1.25(0.50-3.13) 2.00(0.58-6.83)
tccP 2(8) 13(20) 1(5) 0.125 0.33(0.06-1.56) 1.58(0.13-18.8)
SPATE
espP 16(61.5) 23(36) 6(30) 0.045* 2.8(1.1-7.3) 3.7(1.08-12.9)
pic 1(4) 4(6) 0(0) 0.494 0.60(0.06-5.63) Undefined
sepA 1(4) 2(3) 0(0) 0.69 1.24(0.10-14.2) Undefined
sigA 1(4) 5(8) 3(15) 0.387 0.47(0.05-4.24) 0.22(0.02-2.36)
Colicin cma
3(11.5) 3(4.7) 1(5) 0.465 2.65(0.49-14) 2.47(0.23-25.8)
cba 7(27) 12(19) 5(25) 0.648 1.6(0.54-4.65) 1.10(0.29-4.19)
celb 6(23) 17(26.6) 5(25) 0.941 0.83(0.28-2.41) 0.9(0.23-3.51)
Microcin
mchB 4(15) 13(20) 8(40) 0.110 0.71(0.21-2.43) 0.27(0.06-1.1)
mchC 4(15) 13(20) 8(40) 0.110 0.71(0.21-2.43) 0.27(0.06-1.1)
mchF 6(23) 17(26) 8(40) 0.407 0.83(0.28-2.41) 0.45(0.12-1.61)
mcmA 1(4) 1(1.6) 1(5) 0.657 2.5(0.15-41.8) 0.76(0.04-12.9)
Fimbriae lpfA 20(77) 44(69) 17(85) 0.323 1.51(0.53-4.34) 0.59(0.12-2.71)
Other ireA 3(11.5) 14(22) 8(40) 0.071 0.46(0.12-1.78) 0.19(.04-.87)
katP 10(38.5) 22(34) 4(20) 0.379 1.12(0.46-3.06) 2.5(0.64-9.6)
BD, Bloody diarrhoea; NBD, Non-bloody diarrhea; ND, No diarrhea; CI, confidence interval; OR, odds ratio. *Statistically significant p values. aP values were obtained from Chi-square test comparing three groups
bOR obtained from binary logistic regression. OR was also calculated for group D vs ND but was found to be not significant,
therefore is not mentioned in this table.
Chapter 3
64
Table 2. Presence of antibiotic resistance genes in STEC isolates.
Isolate Id Serogenotype stx subtype Presence of antibiotic resistance genes
STEC 168 O91:H14 stx1a dfrA1, strA-B, sul2
STEC 169 O6:H10 stx1c aadA1, blaTEM-1B, dfrA1, mphB, strA-B, sul1-2, tetA
STEC 196 O91:H14 stx1a aadA1, sul1, tetA STEC 200 O174:H21 stx2c strA-B, sul2, tetB STEC 299 O5:H9 stx1a strA-B, sul2 STEC 329 O91:H14 stx1a aadA5, catB3, dfrA1, sul1, tetA STEC 338 O104:H4 stx2a blaTEM-1B, dfrA7, sul1 STEC 370 O111:H8 stx1a aadA1, aph(3')-Ia, catA, dfrA1, mphB, strA-B, sul1, tetA, tetM
STEC 381-1 O104:H4 stx2a bla CTX-M-15, blaTEM-1B, dfrA7, sul1 STEC 381-4 O104:H4 stx2a blaTEM-1B, dfrA7, sul1 STEC 479 O26:H11 stx1a aadA1, blaTEM-1B, dfrA1, mphB, strA-B, sul1-2, tetA
STEC 487 O26:H11 stx1a aadA1, blaTEM-1B, dfrA1, mphB, strA-B, sul1-2, tetA
STEC 690 O69:H11 stx1a aac(3)-IIa, aadA1, aph(3')-Ic, blaTEM-1A, catA1, dfrA1, mphB, strA-B, sul1-2, tetA-B
STEC 691 O69:H11 stx1a aac(3)-IIa, aadA1, aph(3')-Ic, blaTEM-1A, catA1, dfrA1, mphB, strA-B, sul1-2, tetA-B
STEC 757 O69:H11 stx1a aac(3)-IIa, aadA1, aph(3')-Ic, blaTEM-1A, catA1, dfrA1, mphB, strA-B, sul1-2, tetA-B
STEC 1255 O5:H9 stx1a+ stx2a blaCTX-M-1, mphA STEC 1500 O76:H19 stx1c strA-B STEC 1585 O91:H14 stx1a aadA1, sul1, tetA STEC 2193 O103:H2 stx1a blaTEM-1C, strA-B, sul2 STEC 2236 O113:H4 stx2d strA-B, sul2, tetB STEC 2359 O55:H12 stx1a blaTEM-1B, strA-B, sul2, tetA STEC 2441 O5:H9 stx1a strA-B, sul2 STEC 2564 O117:H7 stx1a dfrA14, strA-B, sul2, tetA STEC 2573 O112:H19 stx1a+ stx2d blaTEM-1B, catA, floR, strA-B,sul2, tetA STEC 2633 O146:H10 stx1a aadA1, sul1, tetA STEC 2743 O103:H2 stx1a aadA1, blaTEM-1B, dfrA1, strA-B, sul1-2, tetA STEC 2770 O157:H7 stx1a+ stx2c blaTEM-1B, strA-B, sul2, tetA STEC 2797 O100:H30 stx2e aadA1, blaTEM-1B, dfrA1, strA-B, sul1-2, tetA STEC 2820 O157:H7 stx1a+ stx2c aadA1, aph(3')-Ia , blaTEM-1B, dfrA8, strA-B, sul2, tetA STEC 2821 O157:H7 stx1a+ stx2c blaTEM-1B, strA-B, sul2, tetA STEC 3084 O91:H14 stx1a aadA5, catB, dfrA1, sul1, tetA STEC 3087 O91:H14 stx1a aadA5, catB, dfrA1, sul1, tetA STEC 3106 O91:H14 stx1a tetA
Phylogenetic Analysis
The core genome phylogenetic tree was constructed including those 2069 genes (defined as core
genome) shared by all 137 isolates including 5 reference STEC isolates (Figure 3). To describe the
isolates from the tree, the NJ tree in Figure 3 is arbitrarily divided into 8 groups (Group 1-Group 8).
Isolates of the same serogenotype or ST clustered together irrespective of their stx subtypes and
isolation region (Groningen or Rotterdam). Group 2, 4, 5 and 7 contained isolates of serogenotypes
O157:H7, O26:H11, O91:H14 and O103:H2, respectively, which were the most prevalent
serogenotypes in this study. In Group 7 isolates of serotype O128:H2 formed a subcluster. Group 3
and Group 6 contained heterogeneous isolates of different STs and serogenotypes. All but one (STEC
Characterization of STEC Using Whole Genome Sequencing
65
309, in Group 7) of the stx 2f positive isolates clustered together (in Group 1) and belonged to
O63:H6 and O113:H6 serogenotypes (Figure 3).
Several subclusters were observed in a single ST cluster. In Group 4, isolates of serogenotype
O26:H11 formed different subclusters within ST21; notably, one of the subclusters contained three
O69:H11 isolates which had a 290 minimum allele difference (data not shown) from their closest
O26:H11 isolate STEC 380. In some cases, isolates from the same O serogroups e.g., O5:H9 and
O5:H19 were found to be scattered at distinct positions in the tree. On the other hand most of the
cases, isolates of the same H type irrespective of their O serotypes shared a common ancestor, e.g.,
serogroups O103:H2 and O128:H2, serogroups O174:H21, O146:H21 and O91:H21, and serogroups
O113:H6 and O63:H6.
To reveal the genetic relatedness of the isolates based on their virulence genes and other mobile
genetic elements an additional NJ tree was constructed. This tree was based on the accessory
genome containing 2586 genes that were present in at least one isolate but not in all of the 137
isolates (Figure S4). The tree was almost identical to the core genome phylogenetic tree and no
clusters based on disease severity were found.
Distribution of isolates with clinical manifestation, geographical location and epidemiological link
The isolates obtained from the patients with bloody diarrhoea belonged to different serogenotypes
and STs (Figure 3). No significant geographical distribution was observed among the STEC isolates in
relation to their genotypes. Two exceptions should be mentioned within Group 6, in which all 4
isolates belonging to ST442 were obtained from patients in the Rotterdam region. In addition, in
Group 1 seven out of nine stx2f isolates were obtained from patients in the Groningen region. All
other closely related subclusters contained isolates from both regions. Isolates of two clusters
containing three (STEC 338, STEC 381-1 and STEC 381-4 in Group 6) and two (STEC 690 and STEC 757
in Group 4) epidemiologically related isolates were also closely genetically related. In figure 3, we
also highlighted isolates having almost similar core genomes; e.g. STEC 563 and STEC 709 (3-allele
difference), STEC 479 and STEC 487 (no allele difference), STEC 2174 and STEC 2363 (1-allelle
difference), STEC 384 and STEC 464 (no allele difference), STEC 299 and STEC 2441 (2-allele
difference); for those isolates no epidemiological data were available. The number of allele
differences was obtained from MST analyses (data not shown).
Chapter 3
66
Phylogenetic comparison of STEC isolates with the DEC reference collection
Figure 4 shows a NJ tree representing all the STEC isolates from this study and E. coli isolates from
the DEC collection. STEC isolates of this study represented a diverse collection and did not form a
separate cluster but were interspersed among the DEC isolates. The phylogenetic tree shows that
STEC serogroups O157:H7 evolved from E. coli O55:H7 isolates (DEC5), and O26:H11 clustered with
DEC9 and DEC10 isolates in a separate lineage. In both cases clusters contained only STEC isolates. In
contrast, STEC O103:H2 shared a common ancestor with EPEC O111:H2 (DEC12) and O128:H2
(DEC11A-D). STEC O63:H6 and O113:H6, both belonging to the stx2f subtype, shared a common
ancestor with EPEC O55:H6 (DEC1 and DEC2). Among the non-STEC/EPEC isolates, DEC13, DEC14 and
DEC15 isolates shared a common ancestor with several of our STEC isolates of heterogeneous
serotypes and DEC6 (O111:H21) isolates clustered together with our STEC ST10 isolates.
Genetic diversity of STEC isolates compared to DEC isolates and ESBL-producing E. coli isolates
The MPD of the STEC, DEC and ESBL-producing E. coli isolates was 0.86 (IQR 0.24), 0.93 (IQR 0.24)
and 0.97 (IQR 0.10), respectively. The result from the Kruskal-Wallis test indicated that the MPD of
the three populations (STEC, DEC and ESBL) was significantly different (p<0.001). Additionally, a
Mann-Whitney U test showed that there was a highly significant difference (p<0.001) between the
MPD of STEC and ESBL, and DEC and ESBL (p<0.001) but not between the MPD of STEC and DEC
(p=0.137).
Characterization of STEC Using Whole Genome Sequencing
67
Figure 3. Neighbour Joining (NJ) Phylogenetic tree of STEC isolates based on 2069 genes (defined as core
genome) shared by 137 STEC isolates (including 5 reference STEC). The whole tree is arbitrarily divided into 8
groups indicated by circles (Group 1-Group 8). Predominant serogenotypes are mentioned in the circles. Each
isolate Id is followed by isolation region (G for Groningen and R for Rotterdam), serogenotype, sequence type
and stx subtype. Closely related isolates with an allele difference <5 (obtained from the minimum spanning tree
analysis) are highlighted with red lines behind them. New sequence types were not updated in Seqsphere
server during analysis therefore were left as ‘?’ as an unknown sequence type. STEC, Shiga toxin-producing
Escherichia coli.
Chapter 3
68
Figure 4. Neighbour Joining (NJ) Phylogenetic tree of STEC isolates and DEC reference isolates based on 1231
genes (defined as core genome) shared by 208 isolates. The non- STEC isolates are marked with a red asterisk.
For each isolates, the serogenotypes and sequence types are mentioned behind the isolate name. DEC,
diarrhoeagenic E. coli; STEC, Shiga toxin-producing E. coli.
DISCUSSION
In this study, STEC isolates were collected from faecal samples of patients with gastrointestinal
complaints from two regions of the Netherlands. Molecular characterization and high resolution
typing of the isolates was performed using WGS. Serogenotyping, MLST and subtyping of stx genes
revealed a diverse group of STEC in the study population. Twenty five per cent of the isolates carried
one or more antibiotic resistance gene(s), including ESBL genes. Resistance genes were mostly found
in stx1 positive isolates belonging to O-serotypes other than the big six known to be most frequently
involved in severe human infection. These isolates often originate from food-producing animals
which are regularly treated with antibiotics that may lead to STEC becoming resistant. In addition, as
Characterization of STEC Using Whole Genome Sequencing
69
stx1 positive bacteria often cause only mild symptoms without bloody diarrhea and patients may
receive antibiotic therapy if no detailed diagnostics is performed (27-29). Recently, several reports
including the one on the 2011 German E. coli O104:H4 outbreak, have described the association of
STEC isolates with ESBL genes (30). Antibiotic resistance genes are mainly carried on mobile genetic
elements that can be transferred from one bacterium to the other while subjected to selective
pressure, e.g., by exposure to antibiotics. Transferring of resistance genes into clinically significant
bacteria for human could make their treatment option complicated (31).
The presence of several virulence genes (iha, mchB, mchC, mchF, subA, ireA, senB, saa, sigA) was
significantly higher in eae-negative isolates compared to eae-positive ones similar as reported by
previous studies in which these genes were described as additional virulence factors in eae negative
strains (32,33). The presence of virulence genes (eae, tir, espA, espF, espJ) associated with the LEE
pathogenicity island and non-LEE-encoded effector (nle) that encode translocated substrates of the
type III secretion system was more frequent in isolates obtained from patients with bloody
diarrhoea. These genetic determinants were also described to be associated with highly pathogenic
STEC and therefore with severe disease (15,34) although a wide number of STEC LEE-negative strains
also have been associated with sporadic cases and outbreaks (4). There was no correlation between
the serogenotype or STs and the disease outcome. Thus, isolates obtained from bloody diarrhoea did
not belong to a specific phylogenetic cluster but were scattered throughout the phylogenetic tree.
This finding supports the idea that STEC from different phylogenetic backgrounds could be
responsible for severe disease outcome in human by acquiring virulence factors contributing to their
pathogenicity (15). Furthermore, clustering of isolates according to their STs and serogenotype
pattern irrespective of stx subtypes suggests that Stx converting phages are carried by a genetically
diverse group of E. coli (35). In addition, the phylogenetic tree based on the accessory genome of the
isolates also showed no correlation with disease severity. Clearly, disease outcome is multifactorial
and does not only depends on the genetic contents or virulence factors of the isolates but also on
host susceptibility factors and several Stx phage related factors (36).
MLST provides an adequate tool for producing genetic profiles for a vast number of isolates
especially in non-epidemic circumstances, i.e., for national reference services or when comparing
large international strain collections, but has a low discriminatory power. We found several
subclusters within the same ST and serogenotype clusters. In addition, isolates highly similar in their
core genome were also identified using cgMLST. Five historical isolates obtained from outbreaks or
sporadic cases were included in the phylogeny and we found that two of our O157:H7 isolates
clustered with two O157:H7 isolates associated with a previous outbreak (18) and that three
Chapter 3
70
epidemiologically related O104:H4 isolates clustered with the 2011 E. coli O104:H4 outbreak isolate
2011c-3493 (24). Therefore, the detailed gene-by-gene phylogenetic approach using cgMLST enabled
us to discriminate among isolates within same STs and helped us to identify potentially more virulent
clones thereby improving risk assessment and outbreak management. Isolates belonging to different
serogroups clustered with each other, suggesting that using just serogroups may cause misleading
conclusions about the phylogenetic relatedness between STEC strains and their health risks (37).
However, the two regions (Groningen and Rotterdam) from which the isolates were obtained are
only approximately 250 km away and probably both regions share many food sources. This may be
an explanation that no geographical distribution was observed among the STEC isolates of the
different regions. In some cases, isolates of the same O-serogroup were located in different
phylogenetic clusters. On the other hand isolates of the same H type, irrespective of their O
serotype, shared a common ancestor. This finding is in concordance with previous findings where H-
serogroups were described as monophyletic, whereas O-serogroups were described as polyphyletic
(37,38).
Comparing STEC isolates of this study with DEC isolates revealed that the STEC isolates represent a
heterogeneous group. Some of the serogroups formed distinct branches containing only STEC
isolates. However, some serogroups shared a common ancestor with EPEC and other stx/eae
negative DEC isolates. This supports the hypothesis that Stx converting bacteriophages can integrate
into different E. coli pathogroups thereby converting them into a more pathogenic variant (33).
Moreover, STEC isolates of this study had a similar diversity pattern compared to DEC isolates but
were less diverse than ESBL-producing E. coli isolates. This result supports the idea that Stx
converting bacteriophages may have a preference in host selection (36).
Our study has several limitations. Firstly, it was not possible to link all the isolate characteristics with
patient disease outcome and epidemiology due to lacking of patient information. Therefore, an
unknown epidemiological linkage may exist among isolates that might have influenced the reported
diversity. Moreover, as some of the faecal samples were also positive for parasites, and other
bacteria causing gastro-enteritis and all samples were not tested for gastroenteric viruses, it was not
possible to reveal the exact aetiology of the disease outcome.
In conclusion, STEC isolates of a substantial genetic diversity and of distinct phylogenetic groups were
observed in two regions of the Netherlands. WGS serves perfectly well for detailed characterization
of STEC strains compared to serotyping and MLST that have less discriminatory power and do not
provide any information on virulence and resistance genes. WGS of STEC could be very useful for
outbreak tracing within a clinical outbreak, but as STEC strains are very diverse it may not always be
Characterization of STEC Using Whole Genome Sequencing
71
suitable for comparing different outbreaks. WGS may not always be useful to find common ancestors
of STEC, because of its great heterogeneity and incorporation of mobile genetic elements, but so far
it is the best available method. There was no clear correlation between serogenotypes, stx subtype
or STs and the outcome of the disease, as it is also influenced by several factors in addition to
virulence factors or a specific pathotype.
ACKNOWLEDGEMENTS
We would like to thank all the project members of STEC-ID-net study. This work was done in
collaboration with the ESCMID Study Group on Molecular Diagnostics (ESGMD), Basel, Switzerland.
REFERENCES
1. Paton JC, Paton AW. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli
infections. Clin Microbiol Rev 11:450-79.
2. Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A, Middendorf B, Harmsen D, Schmidt
MA, Karch H. 2008. Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic
Escherichia coli. Emerg Infect Dis 14:1287-90.
3. Scheutz F, Teel LD, Beutin L, Pierard D, Buvens G, Karch H, Mellmann A, Caprioli A, Tozzoli R,
Morabito S, Strockbine NA, Melton-Celsa AR, Sanchez M, Persson S, O'Brien AD. 2012. Multicenter
evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J
Clin Microbiol 50:2951-2963.
4. Farfan MJ, Torres AG. 2012. Molecular mechanisms that mediate colonization of Shiga toxin-producing
Escherichia coli strains. Infect Immun 80:903-913.
5. Torres AG, Vazquez-Juarez RC, Tutt CB, Garcia-Gallegos JG. 2005. Pathoadaptive mutation that
mediates adherence of shiga toxin-producing Escherichia coli O111. Infect Immun 73:4766-76.
6. Elliott SJ, Sperandio V, Giron JA, Shin S, Mellies JL, Wainwright L, Hutcheson SW, McDaniel TK,
Kaper JB. 2000. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both
LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli.
Infect Immun 68:6115-6126.
7. Brooks JT, Sowers EG, Wells JG, Greene KD, Griffin PM, Hoekstra RM, Strockbine NA. 2005. Non-
O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002. J Infect Dis.
192:1422-9.
8. Blanco JE, Blanco M, Alonso MP, Mora A, Dahbi G, Coira MA, Blanco J. 2004. Serotypes, virulence
genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients:
prevalence in Lugo, Spain, from 1992 through 1999. J Clin Microbiol 42:311-9.
9. Karmali MA, Mascarenhas M, Shen S, Ziebell K, Johnson S, Reid-Smith R, Isaac-Renton J, Clark C,
Rahn K, Kaper JB. 2003. Association of genomic O island 122 of Escherichia coli EDL 933 with
verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease.
J Clin Microbiol 41:4930-4940
10. Heir E, Lindstedt BA, Vardund T, Wasteson Y, Kapperud G. 2000. Genomic fingerprinting of
shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis
(PFGE) and fluorescent amplified-fragment-length polymorphism (FAFLP). Epidemiol Infect 125:537-548.
Chapter 3
72
11. European Food Safety Authority. 2011. Urgent advice on the public health risk of Shiga-toxin producing
Escherichia coli in fresh vegetables EFSA Journal. 9:2274.
12. Fricke WF, Rasko DA. 2014. Bacterial genome sequencing in the clinic: bioinformatics challenges and
solutions. Nat Rev Genet 15:49-55.
13. Kwong JC, McCallum N, Sintchenko V, Howden BP. 2015. Whole genome sequencing in clinical and
public health microbiology. Pathology 47:199-210.
14. Koser CU, Ellington MJ, Cartwright EJ, Gillespie SH, Brown NM, Farrington M, Holden MT,
Dougan G, Bentley SD, Parkhill J, Peacock SJ. 2012. Routine use of microbial whole genome sequencing
in diagnostic and public health microbiology. PLoS Pathog 8:e1002824.
15. Haugum K, Johansen J, Gabrielsen C, Brandal LT, Bergh K, Ussery DW, Drabløs F, Afset JE. 2014.
Comparative genomics to delineate pathogenic potential in non-O157 Shiga toxin-producing Escherichia
coli (STEC) from patients with and without haemolytic uremic syndrome (HUS) in Norway. PLoS One
9:e111788.
16. Berenger BM, Berry C, Peterson T, Fach P, Delannoy S, Li V, Tschetter L, Nadon C, Honish L, Louie
M, Chui L. 2015. The utility of multiple molecular methods including whole genome sequencing as tools to
differentiate Escherichia coli O157:H7 outbreaks. Euro Surveill 20.
17. de Boer RF, Ferdous M, Ott A, Scheper HR, Wisselink GJ, Heck ME, Rossen JW, Kooistra-Smid
AM. 2015. Assessing the public health risk of Shiga toxin-producing Escherichia coli by use of a rapid
diagnostic screening algorithm. J Clin Microbiol 53:1588–1598.
18. Ferdous M, Zhou K, Mellmann A, Morabito S, Croughs PD, de Boer RF, Kooistra-Smid AM, Rossen
JW, Friedrich AW. 2015. Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or
Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome
Sequencing. J Clin Microbiol 53:3530-3538.
19. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM,
Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D,
Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O.
2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75.
20. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Ponten
T, Ussery DW, Aarestrup FM, Lund O. 2012. Multilocus sequence typing of total-genome-sequenced
bacteria. J Clin Microbiol 50:1355-1361.
21. Joensen KG, Tetzschner AM, Iguchi A, Aarestrup FM, Scheutz F. 2015. Rapid and Easy In Silico
Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data. J Clin Microbiol
53:2410-2426.
22. Joensen, KG, Scheutz, F, Lund, O, Hasman, H, Kaas, RS, Nielsen, EM, Aarestrup, FM. 2014. Real-
time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic
Escherichia coli. J. Clin. Microbiol. 52:1501-1510.
23. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen
MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640-
2644.
24. Ferdous M, Zhou K, de Boer RF, Friedrich AW, Kooistra-Smid AMD, Rossen JWA. 2015.
Comprehensive Characterization of Escherichia coli O104:H4 Isolated from Patients in the Netherlands.
Front Microbiol 6:1348.
Characterization of STEC Using Whole Genome Sequencing
73
25. Hazen TH, Sahl JW, Redman JC, Morris CR, Daugherty SC, Chibucos MC, Sengamalay NA, Fraser-
Liggett CM, Steinsland H, Whittam TS, Whittam B, Manning SD, Rasko DA. 2012. Draft genome
sequences of the diarrheagenic Escherichia coli collection. J Bacteriol 194:3026-7.
26. Tsirogiannis C, Sandel B. 2014. Computing the skewness of the phylogenetic mean pairwise distance in
linear time. Algorithms Mol Biol 9:15.
27. Iweriebor BC, Iwu CJ, Obi LC, Nwodo UU, Okoh AI. 2015. Multiple antibiotic resistances among Shiga
toxin producing Escherichia coli O157 in feces of dairy cattle farms in Eastern Cape of South Africa. BMC
Microbiol 15:213.
28. Park HJ, Yoon JW, Heo EJ, Ko EK, Kim KY, Kim YJ, Yoon HJ, Wee SH, Park YH, Moon JS. 2015.
Antibiotic Resistance and Virulence Potentials of Shiga Toxin-Producing Escherichia coli Isolates from
Raw Meats of Slaughterhouses and Retail Markets in Korea. J Microbiol Biotechnol 25:1460-6.
29. Ishii Y, Kimura S, Alba J, Shiroto K, Otsuka M, Hashizume N, Tamura K, Yamaguchi K. 2005.
Extended-spectrum beta-lactamase-producing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a
new concern. J Clin Microbiol 43:1072-5.
30. Mandakini R, Dutta TK, Chingtham S, Roychoudhury P, Samanta I, Joardar SN, Pachauau AR,
Chandra R. 2015. ESBL-producing Shiga-toxigenic E. coli (STEC) associated with piglet diarrhoea in
India. Trop Anim Health Prod. 47:377-81.
31. Bennett PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in
bacteria. 2008. Br J Pharmacol 153 (Suppl 1):S347-57.
32. Franz E, van Hoek AH, Wuite M, van der Wal FJ, de Boer AG, Bouw EI, Aarts HJ. 2015. Molecular
hazard identification of non-O157 Shiga toxin-producing Escherichia coli (STEC). PLoS One 10:e0120353.
33. Steyert SR, Sahl JW, Fraser CM, Teel LD, Scheutz F, Rasko DA. 2012. Comparative genomics and stx
phage characterization of LEE-negative Shiga toxin-producing Escherichia coli. Front Cell Infect Microbiol
2:133.
34. Delannoy S, Beutin L, Fach P. 2013. Discrimination of enterohemorrhagic Escherichia coli (EHEC) from
non-EHEC strains based on detection of various combinations of type III effector genes. J Clin Microbiol
51:3257-62.
35. Tozzoli R, Grande L, Michelacci V, Ranieri P, Maugliani A, Caprioli A, Morabito S. 2014. Shiga
toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion. Front
Cell Infect Microbiol 4:80.
36. Gamage SD, Patton AK, Hanson JF, Weiss AA. 2004. Diversity and host range of Shiga toxin encoding
phage. Infect Immun 72:7131-9.
37. Ju W, Cao G, Rump L, Strain E, Luo Y, Timme R, Allard M, Zhao S, Brown E, Meng J. 2012.
Phylogenetic analysis of non-O157 Shiga toxin-producing Escherichia coli strains by whole-genome
sequencing. J Clin Microbiol 50:4123-7.
38. Yan X, Fratamico PM, Bono JL, Baranzoni GM, Chen CY. 2015. Genome sequencing and comparative
genomics provides insights on the evolutionary dynamics and pathogenic potential of different H-serotypes
of Shiga toxin-producing Escherichia coli O104. BMC Microbiol 15:83.
Chapter 3
74
Supplementary Materials Supplementary Table S1. Information of the patients and STEC isolates used in this study.
Isolate ID Collection date
Collection Region
Host disease Host age
Host sex
Serogenotype
stx subtyp
e
Sequence type
Bioproject ID NCBI accession
number
STEC 29 Apr-13 Groningen Abdominal discomfort,
Nausea 6 F O91:H14 stx1a 33 PRJNA285020 LNFU00000000
STEC 66 May-13 Groningen Diarrhoea 4 M O165:H25 stx1a+s
tx2a 119 PRJNA285020 LNFT00000000
STEC 168 Jun-13 Groningen Diarrhoea 81 F O91:H14 stx1a 33 PRJNA285020 LNFV00000000
STEC 169 Jun-13 Groningen Unknown 14 F O6:H10 stx1c 43 PRJNA285020 LNZJ00000000
STEC 196 Jun-13 Groningen Abdominal complain
1 M O91:H14 stx1a 33 PRJNA285020 LNZK00000000
STEC 200 Jun-13 Groningen Malaise 80 F O174:H21 stx2c 677 PRJNA285020 LNZL00000000
STEC 299 Jul-13 Groningen Bloody Diarrhoea 27 F O5:H9 stx1a 342 PRJNA285020 LOCR00000000
STEC 309 Jul-13 Groningen Abdominal pain 13 F O128:H2 stx2f 20 PRJNA285020 LOCS00000000
STEC 329 Jul-13 Groningen Diarrhoea 11 M O91:H14 stx1a 33 PRJNA285020 LOCT00000000
STEC 338 Jul-13 Groningen HUS 23 F O104:H4 stx2a 678 PRJNA285020 JRJF00000000
STEC 343 Jul-13 Groningen Diarrhoea 27 F O157:H7 stx2c 11 PRJNA285020 LDOZ00000000
STEC 370 Jul-13 Groningen Unknown 67 F O111:H8 stx1a 16 PRJNA285020 LOCU00000000
STEC 380 Jul-13 Groningen Diarrhoea 69 F O26:H11 stx1a 21 PRJNA285020 LOCV00000000
STEC 381-1
Jul-13 Groningen Diarrhoea 22 F O104:H4 stx2a 678 PRJNA285020 JRKD00000000
STEC 381-4
Jul-13 Groningen Diarrhoea 22 F O104:H4 stx2a 678 PRJNA285020 JRLD00000000
STEC 384 Jul-13 Groningen Diarrhoea 62 M O103:H2 stx1a 17 PRJNA285020 LOCW00000000
STEC 464 Aug-13 Groningen Bloody Diarrhoea 43 F O103:H2 stx1a 17 PRJNA285020 LOCX00000000
STEC 477 Aug-13 Groningen Diarrhoea 1 F O26:H11 stx1a 21 PRJNA285020 LOCY00000000
STEC 479 Aug-13 Groningen Bloody Diarrhoea 2 F O26:H11 stx1a 21 PRJNA285020 LOCZ00000000
STEC 487 Aug-13 Groningen Diarrhoea 82 F O26:H11 stx1a 21 PRJNA285020 LODA00000000
STEC 545 Aug-13 Groningen Diarrhoea 60 F O9:H9 stx2e 10 PRJNA285020 LODB00000000
STEC 559 Aug-13 Groningen Diarrhoea 1 M O182:H25 stx1a 300 PRJNA285020 LODC00000000
STEC 563 Aug-13 Groningen Diarrhoea 19 F O26:H11 stx1a+s
tx2a 21 PRJNA285020 LODD00000000
STEC 565 Aug-13 Groningen Bloody Diarrhoea 63 F O121:H19 stx2a 655 PRJNA285020 LODE00000000
STEC 605 Aug-13 Groningen Unknown 52 M O157:H7 stx2c 11 PRJNA285020 LFUA00000000
STEC 623 Aug-13 Groningen Bloody Diarrhoea 25 M O157:H7 stx1a+s
tx2c 11 PRJNA285020 LFUB00000000
STEC 627 Aug-13 Groningen Nausea 15 F O5:H9 stx1a 342 PRJNA285020 LODF00000000
STEC 645 Aug-13 Groningen Diarrhoea 31 F O91:H14 stx1a 33 PRJNA285020 LODG00000000
STEC 690 Sep-13 Groningen Diarrhoea 1 F O69:H11 stx1a 21 PRJNA285020 LOFJ00000000
STEC 691 Sep-13 Groningen Abdominal pain 43 M O69:H11 stx1a 21 PRJNA285020 LOFK00000000
STEC 707 Sep-13 Groningen Diarrhoea 27 M O103:H2 stx1a 17 PRJNA285020 LOFL00000000
STEC 709 Sep-13 Groningen Bloody Diarrhoea 55 F O26:H11 stx1a+s
tx2a 21 PRJNA285020 LOFM00000000
STEC 731 Sep-13 Groningen Diarrhoea 1 F O145:Hnt stx2a 32 PRJNA285020 LOFN00000000
STEC 757 Sep-13 Groningen Abdominal discomfort
3 F O69:H11 stx1a 21 PRJNA285020 LOFO00000000
STEC 764 Sep-13 Groningen Diarrhoea 19 F O26:H11 stx1a 21 PRJNA285020 LOFP00000000
STEC 771 Aug-13 Groningen Diarrhoea 30 F O157:H7 stx1a+s
tx2c 11 PRJNA285020 LGAZ00000000
STEC 793 Sep-13 Groningen Bloody Diarrhoea 23 F O63:H6 stx2f 6044b,c
PRJNA285020 LOFQ00000000
STEC 886 Oct-13 Groningen Diarrhoea 16 F O91:H14 stx1a 33 PRJNA285020 LOFR00000000
Characterization of STEC Using Whole Genome Sequencing
75
Isolate ID Collection date
Collection Region
Host disease Host age
Host sex
Serogenotype
stx subtyp
e
Sequence type
Bioproject ID NCBI accession
number
STEC 915 Oct-13 Groningen Bloody Diarrhoea 8 M O157:H7 stx1a+s
tx2c 11 PRJNA285020 LFUH00000000
STEC 931 Oct-13 Groningen Bloody Diarrhoea 34 F O26:H11 stx2a 29 PRJNA285020 LOFS00000000
STEC 940 Oct-13 Groningen Diarrhoea 5 M O63:H6 stx2f 6045b,d
PRJNA285020 LOFT00000000
STEC 989 Oct-13 Groningen Bloody Diarrhoea 58 M O157:H7 stx1a+s
tx2c 11 PRJNA285020 LGBA00000000
STEC 994 Oct-13 Groningen Bloody Diarrhoea 43 M O157:H7 stx2c 11 PRJNA285020 LGBB00000000
STEC 1109 Oct-13 Groningen Abdominal pain,
Nausea 78 M O157:H7 stx2c 11 PRJNA285020 LGBC00000000
STEC 1117 Nov-13 Groningen Unknown 2 F O26:H11 stx1a 21 PRJNA285020 LOFU00000000
STEC 1161 Nov-13 Groningen Diarrhoea 29 F O84:H2 stx1a 6042b,e
PRJNA285020 LOFV00000000
STEC 1178 Nov-13 Groningen Diarrhoea 4 F O113:H6 stx2f 121 PRJNA285020 LOFW00000000
STEC 1188 Nov-13 Groningen Abdominal discomfort
5 F O91:H14 stx1a 33 PRJNA285020 LOFX00000000
STEC 1198 Nov-13 Groningen Unknown unknown
unknown
O63:H6 stx2f 583 PRJNA285020 LOFY00000000
STEC 1201 Nov-13 Groningen Bloody Diarrhoea 24 F O103:H2 stx1a 17 PRJNA285020 LOFZ00000000
STEC 1225 Nov-13 Groningen Bloody Diarrhoea 11 F O182:H25 stx1a 300 PRJNA285020 LOGA00000000
STEC 1236 Nov-13 Groningen Diarrhoea 51 F O26:H11 stx1a 21 PRJNA285020 LOGB00000000
STEC 1255 Nov-13 Groningen Bloody Diarrhoea 8 F O5:H9 stx1a+s
tx2a 342 PRJNA285020 LOGC00000000
STEC 1270 Nov-13 Groningen Abdominal complain
3 F O113:H6 stx2f 121 PRJNA285020 LOGD00000000
STEC1284 Nov-13 Groningen Diarrhoea 0 F O103:H2 stx1a 17 PRJNA285020 LOGE00000000
STEC 1293 Dec-13 Groningen Bloody Diarrhoea 42 M O26:H11 stx1a+s
tx2a 21 PRJNA285020 LOGF00000000
STEC 1299 Dec-13 Groningen Diarrhoea 5 M O128:H2 stx1c+s
tx2b 25 PRJNA285020 LOGG00000000
STEC 1303 Dec-13 Groningen Diarrhoea 4 F O113:H6 stx2f 121 PRJNA285020 LOGH00000000
STEC 1363 Dec-13 Groningen Abdominal pain 40 M O128:H2 stx2b 25 PRJNA285020 LOGI00000000
STEC 1375 Dec-13 Groningen Diarrhoea 87 F O185:H7 stx2c 2387 PRJNA285020 LOGJ00000000
STEC 1442 Jan-14 Groningen Bloody Diarrhoea 11 M O145:Hnt stx2a 32 PRJNA285020 LOGK00000000
STEC 1465 Jan-14 Groningen Unknown 31 M O128:H2 stx1c 25 PRJNA285020 LOGL00000000
STEC 1473 Jan-14 Groningen Diarrhoea 75 F O113:H6 stx2f 121 PRJNA285020 LOGM00000000
STEC 1500 Jan-14 Groningen Diarrhoea 21 F O76:H19 stx1c 675 PRJNA285020 LOGN00000000
STEC 1506 Jan-14 Groningen Abdominal pain 12 F O5:H19 stx1c+s
tx2b 447 PRJNA285020 LPWV00000000
STEC 1513 Jan-14 Groningen Diarrhoea 14 F O103:H2 stx1a 17 PRJNA285020 LOGO00000000
STEC1528 Feb-14 Groningen Diarrhoea 57 M O75:H7 stx2b 80 PRJNA285020 LOGP00000000
STEC1532 Feb-14 Groningen Diarrhoea 55 M O103:H2 stx1a 17 PRJNA285020 LOGQ00000000
STEC 1585 Feb-14 Groningen Abdominal complain
83 M O91:H14 stx1a 33 PRJNA285020 LOGR00000000
STEC 1634 Apr-14 Groningen Diarrhoea 46 F Ont:H8 stx2b 26 PRJNA285020 LOGS00000000
STEC 1686 Apr-14 Groningen Unknown 50 M O27:H30 stx1a+s
tx2b 753 PRJNA285020 LOGT00000000
STEC 2064 May-13 Rotterdam Diarrhoea 83 M O91:H14 stx1a 33 PRJNA285020 LOJC00000000
STEC 2074 May-13 Rotterdam Abdominal pain,
Nausea 29 M O146:H21 stx1c 442 PRJNA285020 LOJD00000000
STEC 2075 May-13 Rotterdam Diarrhoea 1 M O157:H7 stx2c 11 PRJNA285020 LGBD00000000
STEC 2110.1
Jun-13 Rotterdam Abdominal pain 9 F O91:H14 stx2a 33 PRJNA285020 LPWW0000000
0
STEC 2110.3
Jun-13 Rotterdam Abdominal pain 9 F O26:H11 stx lost 21 PRJNA285020 LOJE00000000
STEC 2112 Jun-13 Rotterdam Diarrhoea 10 F O157:H7 stx1a+s
tx2a 11 PRJNA285020 LGBE00000000
STEC 2144 Jun-13 Rotterdam Diarrhoea 21 F O26:H11 stx1a 21 PRJNA285020 LOGU00000000
Chapter 3
76
Isolate ID Collection date
Collection Region
Host disease Host age
Host sex
Serogenotype
stx subtyp
e
Sequence type
Bioproject ID NCBI accession
number
STEC 2174 Jun-13 Rotterdam Abdominal pain,
Nausea 20 M O91:H14 stx1a 33 PRJNA285020 LOGV00000000
STEC 2193 Jun-13 Rotterdam Bloody Diarrhoea 14 F O103:H2 stx1a 17 PRJNA285020 LOGW00000000
STEC 2211 Jul-13 Rotterdam Bloody Diarrhoea 51 F O108:H25 stx1a 300 PRJNA285020 LOGX00000000
STEC 2236 Jul-13 Rotterdam Diarrhoea 62 M O113:H4 stx2d 10 PRJNA285020 LOGY00000000
STEC 2257 Jul-13 Rotterdam Unknown 4 F O157:H7 stx1a+s
tx2c 11 PRJNA285020 LGBF00000000
STEC 2270 Jul-13 Rotterdam Diarrhoea 1 F O111:H8 stx1a 16 PRJNA285020 LPWX00000000
STEC 2334 Aug-13 Rotterdam Diarrhoea 1 F O63:H6 stx2f 583 PRJNA285020 LOGZ00000000
STEC 2346 Aug-13 Rotterdam Diarrhoea 0 F O26:H11 stx1a 21 PRJNA285020 LOHA00000000
STEC 2359 Aug-13 Rotterdam Diarrhoea 41 M O55:H12 stx1a 101 PRJNA285020 LOHB00000000
STEC 2363 Aug-13 Rotterdam Unknown 67 F O91:H14 stx1a 33 PRJNA285020 LPWY00000000
STEC 2410 Aug-13 Rotterdam Diarrhoea 14 F O157:H7 stx2c 11 PRJNA285020 LGBG00000000
STEC 2419 Sep-13 Rotterdam Diarrhoea 38 F O103:H2 stx1a 17 PRJNA285020 LPWZ00000000
STEC 2441 Sep-13 Rotterdam Diarrhoea 79 F O5:H9 stx1a 342 PRJNA285020 LOHC00000000
STEC 2450 Sep-13 Rotterdam Diarrhoea 82 F O146:H21 stx1c+s
tx2b 442 PRJNA285020 LPXA00000000
STEC 2499 Sep-13 Rotterdam Abdominal pain 10 M O181:H49 stx1a+s
tx2d 4043
b,f PRJNA285020 LOIE00000000
STEC 2505 Sep-13 Rotterdam Diarrhoea 59 M O128:H2 stx1c+s
tx2b 25 PRJNA285020 LPXB00000000
STEC 2539 Sep-13 Rotterdam Unknown 79 F O63:H6 stx2f 583 PRJNA285020 LOIF00000000
STEC 2564 Sep-13 Rotterdam Abdominal complain
70 F O117:H7 stx1a 504 PRJNA285020 LOIG00000000
STEC 2573 Sep-13 Rotterdam Bloody Diarrhoea 29 M O112:H19 stx1a+s
tx2d 201 PRJNA285020 LOIH00000000
STEC 2591 Sep-13 Rotterdam Diarrhoea 68 F O174:H2 stx1a+s
tx2d 661 PRJNA285020 LOII00000000
STEC 2595 Sep-13 Rotterdam Diarrhoea 2 M O17/O77/O44:H18
stx2d 69 PRJNA285020 LOIJ00000000
STEC 2620 Sep-13 Rotterdam Bloody Diarrhoea 25 F O91:H14 stx1a+s
tx2b 33 PRJNA285020 LPXC00000000
STEC 2633 Oct-13 Rotterdam Unknown unknown
unknown
O146:H10 stx1a 745 PRJNA285020 LOIK00000000
STEC 2667 Oct-13 Rotterdam Unknown 4 F O157:H7 stx1a+s
tx2c 11 PRJNA285020 LGBH00000000
STEC 2708 Oct-13 Rotterdam Diarrhoea 52 M O110:H9 stx1c 10 PRJNA285020 LOIL00000000
STEC 2743 Oct-13 Rotterdam Diarrhoea 3 M O103:H2 stx1a 1786 PRJNA285020 LOIM00000000
STEC 2746 Oct-13 Rotterdam Unknown 31 F O128:H2 stx1c+s
tx2b 25 PRJNA285020 LPXD00000000
STEC 2764 Nov-13 Rotterdam Diarrhoea 39 M O174:H8 stx1c 13 PRJNA285020 LOIN00000000
STEC 2770 Nov-13 Rotterdam Unknown unknown
unknown
O157:H7 stx1a+s
tx2c 11 PRJNA285020 LPXF00000000
STEC 2788 Nov-13 Rotterdam Bloody Diarrhoea 19 F O91:H14 stx1a 33 PRJNA285020 LOIO00000000
STEC 2797 Nov-13 Rotterdam Unknown 55 F O100:H30 stx2e 993 PRJNA285020 LOIP00000000
STEC 2820 Nov-13 Rotterdam Unknown 3
month
unknown
O157:H7 stx1a+s
tx2c 11 PRJNA285020 LGBQ00000000
STEC 2821 Nov-13 Rotterdam Bloody Diarrhoea 72 F O157:H7 stx1a+s
tx2c 11 PRJNA285022 LGBI00000000
STEC 2826 Nov-13 Rotterdam Diarrhoea 19 F O91:H21 stx2a+s
tx2da
442 PRJNA285020 LOJA00000000
STEC 2839 Nov-13 Rotterdam Unknown 41 M O76:H19 stx1c 675 PRJNA285020 LOJB00000000
STEC 2841 Nov-13 Rotterdam Diarrhoea 50 M Ont:H20 stx1c 6060b PRJNA285020 LOIQ00000000
STEC 2861 Nov-13 Rotterdam Unknown 57 F Ont:H20 stx2d 724 PRJNA285020 LOIR00000000
Characterization of STEC Using Whole Genome Sequencing
77
Isolate ID Collection date
Collection Region
Host disease Host age
Host sex
Serogenotype
stx subtyp
e
Sequence type
Bioproject ID NCBI accession
number
STEC 2868 Nov-13 Rotterdam Bloody Diarrhoea 14 F O157:H7 stx1a+s
tx2a 11 PRJNA285020 LGBJ00000000
STEC 2894.1
Dec-13 Rotterdam Diarrhoea 19 F Ont:H20 stx1a 6041b,g
PRJNA285020 LOIS00000000
STEC 2894.2
Dec-13 Rotterdam Diarrhoea 19 F O177:H25 stx2c 659 PRJNA285020 LOIT00000000
STEC 2920 Dec-13 Rotterdam Bloody Diarrhoea 79 F O26:H11 stx1a 21 PRJNA285020 LOIU00000000
STEC 2938 Dec-13 Rotterdam Diarrhoea 76 F O182:H25 stx1a 300 PRJNA285020 LOIV00000000
STEC 2953 Dec-13 Rotterdam Unknown 15 M O113:H4 stx1c+s
tx2b 10 PRJNA285020 LOIW00000000
STEC 2954 Dec-13 Rotterdam Diarrhoea 1 F O174:H8 stx1c+s
tx2b 13 PRJNA285020 LPXE00000000
STEC 2962 Dec-13 Rotterdam Diarrhoea 20 F O91:H14 stx1a 33 PRJNA285020 LOIX00000000
STEC 2980 Jan-14 Rotterdam Diarrhoea 11
month
F O128:H2 stx1c+s
tx2b 25 PRJNA285020 LOIY00000000
STEC 3031 Jan-14 Rotterdam Bloody Diarrhoea 17 F O38:H26 stx1c+s
tx2b 548 PRJNA285020 LOIZ00000000
STEC 3039 Feb-14 Rotterdam Abdominal complain
36 M O146:H21 stx1c+s
tx2b 442 PRJNA285020 LPUH00000000
STEC 3055 Feb-14 Rotterdam Diarrhoea 10 F O91:H14 stx1a 33 PRJNA285020 LPUI00000000
STEC 3084 Mar-14 Rotterdam Unknown 0 M O91:H14 stx1a 33 PRJNA285020 LPUJ00000000
STEC 3087 Mar-14 Rotterdam Diarrhoea 51 M O91:H14 stx1a 33 PRJNA285020 LPUK00000000
STEC 3094 Mar-14 Rotterdam Diarrhoea 7 M O103:H2 stx1a 17 PRJNA285020 LPUL00000000
STEC 3098 Mar-14 Rotterdam Unknown 60 F O174:H21 stx2c 677 PRJNA285020 LPUM00000000
STEC 3106 Mar-14 Rotterdam Unknown 72 M O91:H14 stx1a 33 PRJNA285020 LPUN00000000
a
The A subunit of the stx gene was of subtype stx2d and the B subunit was of subtype stx2a b
These new STs were assigned by EnteroBase, but was not updated in Seqsphere, therefore in Figure 3 they are shown as unknown ST. c ST6044 has a single nucleotide change in purA gene of ST583,
d ST6045 has a single nucleotide change in recA gene of ST583
e ST6042 has a single nucleotide change in fumC gene of ST306
f ST6043 has a single nucleotide change in recA gene of ST173
g ST6041 has a single nucleotide change in fumC gene of ST724
Reference STEC isolates from NCBI
Strain name
Year of isolation
Country of isolation
Disease outcome Serogenot
ype stx subtype
Sequence type
NCBI accession number
EDL 933 1982 Michigan, USA Isolated from ground beef
O157:H7 stx1a+stx2a 11 CP008957
Sakai 1996 Japan HUS O157:H7 stx1a+stx2a 11 NC_002695
2011c-3493 2011 USA HUS O104:H4 stx2a 678 NC_018658
11368 2001 Japan Diarrhoea(outbre
ak) O26:H11 stx1a 21 NC_013361
12009 2001 Japan Bloody diarrhoea O103:H2 stx1a 17 NC_013353
Chapter 3
78
Supplementary File S2
Resulting Targets:
2952 targets were defined for MLST+ (2884626 bases)
1699 targets were used as Accessory targets (1431581 bases)
553 targets were discarded
Reference Genome:
* GenBank entry NC_002695.1, 5498450 bases, 5204 genes (Escherichia coli O157:H7 str. Sakai chromosome,
complete genome.)
Query Genomes (10):
* GenBank entry NC_017626.1, 5241977 bases, 4793 genes (Escherichia coli 042, complete genome.)
* GenBank entry NC_013364.1, 5371077 bases, 4968 genes (Escherichia coli O111:H- str. 11128, complete
genome.)
* GenBank entry NC_011601.1, 4965553 bases, 4548 genes (Escherichia coli O127:H6 str. E2348/69
chromosome, complete genome.)
* GenBank entry NC_011353.1, 5572075 bases, 5315 genes (Escherichia coli O157:H7 str. EC4115
chromosome, complete genome.)
* GenBank entry NC_002655.2, 5528445 bases, 5286 genes (Escherichia coli O157:H7 str. EDL933
chromosome, complete genome.)
* GenBank entry NC_013008.1, 5528136 bases, 5253 genes (Escherichia coli O157:H7 str. TW14359
chromosome, complete genome.)
* GenBank entry NC_013361.1, 5697240 bases, 5360 genes (Escherichia coli O26:H11 str. 11368 chromosome,
complete genome.)
* GenBank entry NC_013941.1, 5386352 bases, 5010 genes (Escherichia coli O55:H7 str. CB9615 chromosome,
complete genome.)
* GenBank entry NC_017656.1, 5263980 bases, 4912 genes (Escherichia coli O55:H7 str. RM12579
chromosome, complete genome.)
* GenBank entry NC_017646.1, 5313531 bases, 5009 genes (Escherichia coli O7:K1 str. CE10 chromosome,
complete genome)
Reference Genome Filters:
* Minimum length filter
* Start Codon Filter
* Stop Codon Filter
* Homologous Gene Filter
* Gene overlap filter
* Excluded Sequences Filter
Query Genomes Filters:
* Stop Codon Percentage Filter
Characterization of STEC Using Whole Genome Sequencing
79
Supplementary Table S3. Antibiotic resistance profiles of STEC isolates of this study.
Iso
late
Id
Am
pic
illin
Am
oxi
cilli
n+
Cla
vula
nic
aci
d
Pip
erac
illin
/Taz
ob
actu
m
Cef
uro
xim
e
Cef
oxi
tin
Cef
ota
xim
e
Cef
tazi
dim
e
Cef
epim
e
Imip
enem
Mer
op
enem
Gen
tam
icin
Tob
ram
ycin
Cip
rofl
oxa
cin
No
rofl
oxa
cin
Nit
rofu
ran
toin
Co
listi
n
Trim
eth
op
rim
Trim
eth
op
rim
/Su
lfa
met
ho
xazo
le
Tetr
acyc
lin
Ph
eno
typ
e
STEC 168 R R
STEC 169 R R R R
STEC 196 R
STEC 200 I R
STEC 299 I
STEC 329 R R R
STEC 338 R R
STEC 370 R R R R
STEC 381-1 R R R R R R ESBL
STEC 381-4 R R
STEC 479 R R R R R
STEC 487 R R R R R
STEC 690 R R R R R R
STEC 691 R R R R R R
STEC 757 R R R R R R R
STEC 1255 R R R R R ESBL
STEC 1500 I
STEC 1585 R
STEC2193 R
STEC 2236 R
STEC 2359 R R
STEC 2441 I
STEC 2564 I R
STEC 2573 R R
STEC 2633 I R
STEC 2743 R R R R R
STEC 2770 R R
STEC 2797 R R R R
STEC 2820 I R R R
STEC 2821 R R
STEC 3084 R R R
STEC 3087 R R R
STEC 3106 R R R=resistant, I= intermediate
Chapter 3
80
Supplementary Figure S4. Neighbour Joining (NJ) Phylogenetic tree of STEC isolates based on 2586 genes
(defined as accessory genome) which were present in at least one isolate but not in all of the 137 isolates
(including 5 reference STEC genomes). Each isolate Id is followed by isolation region (G for Groningen and R for
Rotterdam) and sequence type. New sequence types were not updated in Seqsphere server during analysis
therefore were left as ‘?’ as an unknown ST.
81
CHAPTER 4
Comprehensive Characterization of Escherichia coli
O104:H4 Isolated from Patients in the Netherlands
Mithila Ferdous1*, Kai Zhou1*, Richard F. de Boer 2, Alexander W. Friedrich1, Anna M.D.
Kooistra-Smid 1,2 , John W.A. Rossen1
1 Department of Medical Microbiology, University of Groningen, University Medical Center
Groningen, Groningen, the Netherlands 2 Certe - Laboratory for Infectious Diseases, Groningen, the Netherlands
*These authors equally contributed to this work
Key words
Shiga toxin-producing E. coli –STEC, Enterohemorrhagic E. coli –EHEC, Hemolytic uremic syndrome,
Outbreak, Antimicrobial resistance, Whole genome sequencing, Phylogenetic analysis
Front. Microbiol (2015) 6:1348.
Chapter 4
82
ABSTRACT
In 2011, a Shiga toxin-producing Enteroaggregative Escherichia coli (EAEC Stx2a+) O104:H4 strain
caused a serious outbreak of acute gastroenteritis and hemolytic-uremic syndrome (HUS) in
Germany. In 2013, E. coli O104:H4 isolates were obtained from a patient with HUS and her friend
showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these
isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and
compared. Whole-genome sequencing (WGS) was performed for detailed characterization and to
determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4
isolates of 2009 and 2011 available on NCBI were included in the virulence and phylogenetic analysis.
All E. coli O104:H4 isolates tested were positive for stx2a, aatA and terD but were negative for escV.
All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC
Stx2a+ O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and
showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-
genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the
isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one-allele
difference was found between the isolate of the HUS-patient and that of her friend. Our study shows
that EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are
still circulating necessitating proper surveillance to prevent further outbreaks with these potentially
pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but
its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of
2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection
control strategies.
Comparing Escherichia coli O104:H4 isolates
83
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) are a pathogenic group of Escherichia coli (E. coli)
producing the potent cytotoxin Shiga-toxin (Stx) similar to the one produced by Shigella dysenteriae
serotype 1 (1). STEC may cause a broad spectrum of illness, ranging from diarrhea to the potentially
fatal hemolytic uremic syndrome (HUS) (2). A subset of STEC can cause bloody diarrhea in humans
and they are known as enterohemorrhagic E. coli (EHEC) while a subset of EHEC can cause HUS and
are known as hemolytic uremic syndrome-associated E coli HUSEC (3). In addition to Shiga toxin-
converting bacteriophages, STEC may contain several other mobile genetic elements encoding
virulence factors as pathogenicity islands (PAI), and a large, approximately 90 kb plasmid (pO157) (4).
Stx production is common to all HUS-associated E. coli isolates regardless of their serotype. When the
toxin enters the blood stream it binds to receptors on endothelial cells abundantly present in kidneys
and brain, leading to neurological sequel and/or to microvascular disease that may result in HUS (5).
In 2011, a large outbreak was reported in Germany caused by an Enteroaggregative E. coli (EAEC)
O104:H4 strain lysogenized with the Stx2a bacteriophage and thereby becoming an EAEC/STEC
hybrid strain (6, 7). Besides the stx2a gene, this unusual strain had virulence properties of EAEC
including plasmid pAA carrying the aggregative adherence fimbriae (AAF) variant I encoded by the
aggA gene whose expression is regulated by the aggR gene. In addition, it contained a protein-coat
secretion system (Aat), dispersin (Aap), a putative type VI secretion system (Aai), and a rare
combination of serine protease autotransporters of Enterobacteriaceae (SPATEs) genes, i.e., sepA,
sigA and pic (8, 9). It also contained a terD gene (tellurite resistance gene as a marker for the ter
cluster) and a plasmid-borne extended spectrum beta-lactamase (ESBL) gene blaCTX-M-15 resulting in
resistance to several antibiotics (7, 8). However, the outbreak clone did not possess the escV gene
encoding the predicted outer membrane protein and a marker for the locus of enterocyte
effacement (LEE) PAI (9, 10).
In clinical microbiology, Whole Genome Sequencing (WGS) has already shown its value in outbreak
investigations and epidemiological typing due to its high-resolution discriminatory power and
detailed virulence profiling, thereby becoming more and more important in routine diagnostics (11-
13). In this study, we characterized EAEC Stx2a+ O104:H4 strains isolated from a HUS patient and her
friend who travelled together to Turkey in 2013 prior to diagnosing the patient with HUS. For this, a
WGS approach in parallel with routine phenotypic and genotypic laboratory methods was used.
Analyses were performed to get more insight into the antibiotic resistance and virulence profiles of
the isolates and to reveal their genetic relationship with the 2011 German outbreak EAEC Stx2a+
O104:H4 isolates.
Chapter 4
84
MATERIALS AND METHODS
E. coli isolates used in this study
In July 2013, four E. coli isolates were obtained from a HUS patient (isolate 338) and her friend
(isolates 381-1, 381-3 and 381-4). They were compared to three EAEC Stx2a+ O104:H4 strains named
7N, 8G and LB227103 which were a kind gift of Dr. Alexander Mellmann (Institute of Hygiene,
University of Muenster, Muenster, Germany) and were isolated during the 2011 German outbreak
period from stool samples of patients, submitted to the National Consulting Laboratory for Hemolytic
Uremic Syndrome in Münster, Germany, between May 23 and June 2, 2011 (6, 14). In addition,
publically available genomes of five previously reported strains (TY-2482, 2011C-3494, 2009EL-2050,
2009EL-2071 and 55989) were included in virulence and phylogenetic analyses. Detailed information
on the isolates used in this study is shown in Table 1.
Diagnostic procedures
Fecal samples from the HUS patient (patient 338) and her friend (patient 381) were collected for
diagnostic purposes at Certe Laboratory for Infectious Diseases as described previously (18). Shortly,
fecal samples were screened for the presence of the virulence genes stx1, stx2 and escV by real-time
PCR (qPCR) and stx-positive samples were subsequently enriched in Brilliant green bile (BGB) broth.
After DNA isolation from the enriched broth, qPCR was performed for detection of the EAEC pAA
plasmid encoding genes aggR and aatA and, O-serogroup encoding genes (O26, O91, O103, O104,
O111, O121, O145 and O157). In parallel, part of the BGB broth was plated on CHROMagar STEC and
Sorbitol MacConkey agar plates (Mediaproducts BV, Groningen, the Netherlands). To obtain a pure
isolate from the selective agar plates, more than 30 colonies of each patient were analysed for the
presence of stx genes by qPCR and for ESBL production by using CHROM ID ESBL agar (bioMérieux,
Marcy I'Etoile, France). In addition, sorbitol fermentation and tellurite resistance of the isolates were
determined by observing their ability to grow on CT-SMAC plates (Sorbitol MacConkey agar with
Cefixime and Tellurite). In addition, conventional PCRs for stx-subtypes 2a, 2b, 2c, 2d, 2e, 2f and 2g
were performed to determine the subtype of the stx2 gene as described previously (19). Finally, a
multiplex PCR targeting typical molecular features of the 2011 outbreak strain, i.e. the presence of
the wzyO104 (O antigen polymerase as a marker for the E. coli O104 gene cluster), fliCH4 (encoding the
H4 specific flagellin), stx2, and terD genes was performed (6, 20).
Comparing Escherichia coli O104:H4 isolates
85
Antimicrobial Resistance
Antibiotic resistance patterns of the four isolates of 2013 (338, 381-1, 381-3 and 381-4) and three
isolates of 2011 (7N, 8G and LB227103) were analyzed using VITEK2 (bioMerieux, Marcy l'Etoile,
France) and E-Test (bioMérieux, Marcy l'Etoile, France) following EUCAST guidelines. Presence of
ESBL, carbapenemases and AmpCs genes was confirmed using the Check-MDR CT103 assay according
to the manufacturer’s protocol (Check-Points BV Wageningen, the Netherlands).
DNA isolation
For repetitive sequence based PCR fingerprinting and whole genome sequencing, genomic DNA was
extracted by the UltraClean® microbial DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, US)
according to the manufacturer’s protocol. For other molecular assays DNA was extracted using a
DNeasy blood and tissue kit (QIAGEN, GmbH, Hilden, Germany) following the manufacturer’s
protocol.
Multilocus sequence typing (MLST)
MLST was performed using a 3130xl Genetic Analyzer (Applied Biosystems) to sequence internal
fragments of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) according to the
protocol described in the E. coli MLST databases
(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html). The alleles and sequence
types (ST) were assigned in accordance with the same database.
Virulence properties
To assess the presence of known E. coli virulence genes, a DNA microarray analysis was performed
using the E. coli genotyping combined assay kit according to the manufacturer’s protocol (Clondiag,
Alere Technologies, GmbH, Jena, Germany) (21).
Repetitive sequence based PCR (rep-PCR)
Isolated DNA was amplified using the DiversiLab Escherichia kit for repetitive sequence based PCR
fingerprinting following the manufacturer’s instructions (bioMérieux, Marcy-l’Etoile, France). The
rep-PCR products were detected and analysed using lab-on-a-chip microfluidics technology on an
Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). Further analysis was performed with the web-
based DiversiLab software (version 3.4) using the Pearson correlation coefficient to calculate pairwise
Chapter 4
86
similarities among all samples. In this study, we used 95% similarity thresholds for the data analysis
(22).
Whole genome sequencing and analysis
A DNA library was prepared using the Nextera XT kit (Illumina, San Diego, CA, US) according to the
manufacturer’s instructions and then run on a Miseq (Illumina) for generating paired-end 250-bp
reads. De novo assembly was performed using default settings of CLC Genomics Workbench v6.0.5
(CLC bio A/S, Denmark) as described previously (23). The MLST sequence type was also confirmed
from the whole genome sequence by uploading the assembled genomes to the CGE MLST server
(version 1.7) (24); the configuration for MLST was set to E. coli scheme 1. The resistome was
generated by uploading assembled genomes to the CGE Resfinder server 2.0 (25). The default
settings for Resfinder were used except for the threshold of ID that was set to 85%. The
serogenotype of the isolates was identified using CGE SerotypeFinder 1.0 (26). The virulence genes
were identified by mapping reads to a pseudomolecule generated by concatenating a set of E. coli
virulence genes described before (6, 27). In addition to the isolates of 2013 and 2011, the genomes
of two isolates of 2009 (2009EL-2050, 2009EL-2071) were included for comparison of the virulence
genes. The genome sequence of 2011C-3493 (GenBank accession no. NC_018658.1) was used as
reference for extracting open reading frames (ORFs) from contigs of each strain by SeqSphere v1.0
(Ridom GmbH, Münster, Germany). Only the ORFs without premature stop codons and ambiguous
nucleotides were extracted, and the sequences of those ORFs shared by all samples analyzed here
were defined as the core genome for subsequent phylogenetic analysis (28). According to the
sequence identity of each ORF, a numerical allele type was assigned by SeqSphere. SeqSphere used
the allelic profile formed by the combination of all alleles in each genome for constructing the
minimum-spanning tree. This Whole Genome Shotgun project has been deposited at
DDBJ/EMBL/GenBank under the accession number JRJF01000000 (E. coli 338), JRKD01000000 (E. coli
381-1), JRLM01000000 (E. coli 381-3), JRLD01000000 (E. coli 381-4), JRKE01000000 (E. coli 7N),
JRLN00000000 (E. coli 8G), JRKF01000000 (E. coli LB227103). The version described in this paper is
the first version.
RESULTS
Demographic and clinical characteristics of patients
Strain 338 was isolated from the fecal sample of a 22 year-old female patient (338) admitted to the
Intensive Care Unit of the University Medical Center Groningen (UMCG) in July 2013, after returning
Comparing Escherichia coli O104:H4 isolates
87
from a holiday in Turkey. She presented with abdominal pain, bloody diarrhea, vomiting and
eventually developed HUS. Patient 381, a 24-year-old female travelling together with patient 338,
showed symptoms of diarrhea, abdominal cramps and abdominal pain but was not hospitalized.
Table 1. Characteristics of isolates analyzed in this study.
a NA = Not applicable as these isolates are stx negative
Primary screening results
Direct screening of feces by qPCR revealed the presence of stx1, stx2 and escV genes (patient 338)
and stx2 gene (patient 381). Additional molecular screening of DNA isolated from enriched BGB broth
for other virulence genes and O-serogroups resulted in detection of aggR, aatA and wzyO104 in both
patients. Subsequently, as a result of colony screening for the presence of the stx gene and/or for
ESBL production one stx2-positive non ESBL-producing isolate (isolate 338) was obtained from
patient 338, whereas two different stx2-positive isolates were recovered from patient 381: an ESBL
producing isolate (381-1) and a non ESBL producing isolate (381-4). In addition, one stx-negative but
ESBL-producing isolate (381-3) was obtained from this patient. All stx-positive isolates contained the
aatA and terD but not the escV gene, similar to the 2011 German outbreak isolates. No stx1-positive
Isolate ID Date of isolation
Country of Isolation
Clinical manifestations
stx subtype
Serotype ESBL MLST Source
338 July 2013 Netherlands HUS 2a O104:H4 negative ST-678 this study
381-1 July 2013 Netherlands Diarrhea 2a O104:H4 positive ST-678 this study
381-3 July 2013 Netherlands Diarrhea NAa O126:H2 positive ST-10 this study
381-4 July 2013 Netherlands Diarrhea 2a O104:H4 negative ST-678 this study
7N 2011 Germany Unknown 2a O104:H4 positive ST-678 this study
8G 2011 Germany Unknown 2a O104:H4 positive ST-678 this study
LB227103 2011 Germany HUS 2a O104:H4 positive ST-678 (6, 14)
TY-2482 2011 Germany HUS 2a O104:H4 positive ST-678 (12)
2011C-3493
2011 U.S.A. HUS 2a O104:H4 positive ST-678 (15)
2009EL–2050
2009 Republic of Georgia
Bloody diarrhea
2a O104:H4 negative ST-678 (15)
2009EL–2071
2009 Republic of Georgia
Bloody diarrhea
2a O104:H4 negative ST-678 (15)
55989 1995 Central African Republic
Diarrhea NAa O104:H4 negative ST-678
(16, 17)
Chapter 4
88
isolates were obtained. The aggR gene was detected in the 2013 and two of the three 2011 isolates
(7N and 8G) but unexpectedly not in isolate LB227103. All stx-positive isolates were subtyped as
stx2a and shared the same serotype (O104:H4) as the 2011 German outbreak isolates, whereas the
stx negative ESBL producing isolate was serotype O126:H2. All isolates were sorbitol fermenting and
tellurite resistant. MLST analysis showed that all 2013 EAEC Stx2a+ O104:H4 isolates belonged to
ST678 as the 2011 outbreak strains, whereas the stx-negative isolate (381-3) was assigned to ST10.
The screening results are summarized in Table 1.
Phenotypic and genotypic resistance profiles
Isolates 338 and 381-4 (both ESBL negative) were resistant to ampicillin and trimethoprim. Isolate
381-1 (ESBL positive) was resistant to ampicillin, cefotaxime, ceftazidime, cefepime, cefuroxime, and
trimethoprim and this resistance pattern was almost identical to that of the stx-negative/ESBL
positive isolate 381-3 from the same patient except that the latter was susceptible to trimethoprim.
The resistance pattern of isolate 381-1 differed from that of three of the 2011 outbreak isolates
which were resistant to amoxicillin-clavulanic acid, tetracycline and trimethoprim/sulfamethoxazole
(Table 2).
In agreement with the results of the phenotypic resistance pattern, the resistome of the three 2013
EAEC Stx2a+ O104:H4 isolates differed from that of the 2011 isolates: the strA, strB (streptomycin
resistance), sul2 (sulfonamide resistance) and tetA (tetracycline resistance) genes were only found in
the 2011 outbreak isolates (Table 2). A blaCTX-M-15 gene was detected in isolate 381-1 (stx-positive) and
381-3 (stx-negative) as well as in the 2011 isolates. In addition, WGS showed the presence of the
dfrA7 gene in all isolates except 381-3 resulting in the latter’s trimethoprim-sensitive phenotype. A
truncated sul1 gene resulting from an insertion of IS26 was found in all EAEC Stx2a+ O104:H4
isolates.
Virulence Profile
DNA microarray results showed that the virulence profile of the 2013 isolates was similar to that of
the EAEC Stx2a+ O104:H4 isolates of 2011 and 2009 used in this study. The only difference was the
presence of the microcin H47 biosynthesis gene mchB and mchC, which were identified in the 2009
and 2011 strains, but were absent in the EAEC Stx2a+ O104:H4 isolates of 2013.
The results obtained by WGS were identical to that of the DNA microarray and confirmed the
absence of the aggR gene in the outbreak strain LB227103. WGS revealed the presence of several
additional virulence genes including aap, aaiC, aggA, lpfAO113, lpfAO26, fyuA, irp2 and set1 (not
detected by PCR or microarray) in our isolates (Table 3).
Comparing Escherichia coli O104:H4 isolates
89
Table 2. Phenotypic antibiotic resistance patterns and presence of corresponding antibiotic
resistance genes among the isolates.
2011 isolates
2013 isolates
Name of Antibiotics
7N 8G LB227103 338 381-1 381-4 381-3
Ampicillin R/blaTEM-1 R/blaTEM-1 R/blaTEM-1 R/blaTEM-1 R/blaTE -1 R/blaTEM-1 R/blaTEM-1
Amoxicillin+ Clav acid
R/blaTEM-1 R/blaTEM-1 R/blaTEM-1 S I S S
Cefuroxime R/blaCTX-M-15 R/blaCTX-M-15 R/blaCTX-M-15 S/N R/blaCTX-M-15 S/N R/blaCTX-M-15
Cefotaxime R/blaCTX-M-15 R/blaCTX-M-15 R/blaCTX-M-15 S/N R/blaCTX-M-15 S/N R/blaCTX-M-15
Ceftazidime R/ blaCTX-M-15 R/ blaCTX-M-15 R/ blaCTX-M-15 S/N R/ blaCTX-M-15 S/N R/blaCTX-M-15
Cefepime R/blaCTX-M-15 R/blaCTX-M-15 R/blaCTX-M-15 S/N R/blaCTX-M-15 S/N R/blaCTX-M-15
Co-trimoxazole R/sul1 & 2 R/sul1 & 2 R/sul1 & 2 S/sul1a S/sul1
a S/sul1
a S/N
Tetracycline R/tetA R/tetA R/tetA S/N S/N S/N S/N
Trimethoprime R/dfrA7 R/dfrA7 R/dfrA7 R/dfrA7 R/dfrA7 R/dfrA7 S/N
Streptomycin ND/strA-B ND/strA-B ND/strA-B ND/N ND/N ND/N ND/N
a sul1 gene was deactivated by an IS26 insertion
(R represents drug resistance, S represents drug sensitivity; I represents intermediate; N represents not found; ND represents not determined)
Chapter 4
90
Table 3. Virulence genes detected by qPCR, microarray and whole genome sequencing (WGS)
analysis.
Gene name Function 7N 8G LB227103
338 381-1 381-4 2009EL-2050
2009EL-2071
55989
aapa synthesis of anti-aggregation protein
dispersin + + + + + + + + +
aatA necessary for translocation of dispersin (Aap)
+ + + + + + + + +
aaiCa aggR-activated island C + + + + + + + + +
aggA a
(AAF/I)
Pilin subunit of aggregative adherence fimbriae I
+ + + + + + + + -
aafAa Pilin subunit of aggregative adherence
fimbriae II - - - - - - - - -
agg3A a
(AAF/III)
Pilin subunit of aggregative adherence fimbriae III
- - - - - - - - +
agg4A a
Pilin subunit of aggregative adherence fimbriae IV
- - - - - - - - -
Agg5A a
Pilin subunit of aggregative adherence fimbriae V
- - - - - - - - -
aggR Transcriptional regulator AggR + + - + + + + + +
astA (EAST1) heat-stable enterotoxin 1 - - - - - - - - +
bfpA Bundle-forming pili - - - - - - - - -
cdt (I-V) Cytolethal distending toxin - - - - - - - - -
eae Intimin - - - - - - - - -
EHEC-hlyA EHEC haemolysin - - - - - - - - -
elt Heat-labile enterotoxin (LT) - - - - - - - - -
espP Serine protease - - - - - - - - -
est1a Heat-stable enterotoxin (STIa) - - - - - - - - -
est1b Heat-stable enterotoxin (STIb) - - - - - - - - -
fyuA a
Component of iron uptake system on HPI + + + + + + + + +
ial a
Invasive plasmid (pInv) - - - - - - - - -
iha Iron-regulated gene + + + + + + + + +
irp2 a
Component of iron uptake system on HPI + + + + + + + + +
lpfAO113a
Structural subunit of LPF of STEC O113 + + + + + + + + +
lpfAO157-OI141
a
Structural subunit of LPF of STEC O157:H7 - - - - - - - - -
lpfAO157-OI154
a
Structural subunit of LPF of STEC O157:H7 - - - - - - - - -
lpfAO26 a
Structural subunit of long polar fimbriae (LPF) of STEC O26
+ + + + + + + + +
mchB synthesis of Microcin + + + - - - + + -
mchC synthesis of Microcin + + + - - - + + -
mchF synthesis of Microcin + + + + + + + + -
pet plasmid-encoded toxin - - - - - - - - -
pic gene encoding protein for intestinal colonization
+ + + + + + + + +
saa STEC autoagglutinating adhesin - - - - - - - - -
sat Secreted autotransporter toxin - - - - - - - - -
set1a Shigella enterotoxin 1 + + + + + + + + +
sepA Shigella extracellular protease + + + + + + + + -
Comparing Escherichia coli O104:H4 isolates
91
Gene name Function 7N 8G LB227103
338 381-1 381-4 2009EL-2050
2009EL-2071
55989
sfpA plasmid-borne gene encoding the pilin subunit
- - - - - - - - -
sigA Shigella IgA-like protease homologue + + + + + + + + +
stx1 Shiga toxin 1 - - - - - - - - -
stx2 Shiga toxin 2 + + + + + + + + -
subA Subtilase cytotoxin - - - - - - - - -
ter cluster Tellurite resistance + + + + + + + + -
aData derived only from WGS
Phylogenetic analyses
Rep-PCR analysis was used to investigate the genetic relationship between the different isolates.
EAEC Stx2a+ O104:H4 isolates of 2011 and 2013 clustered into two different groups sharing a
similarity of less than 95% whereas the two non O104:H4 isolates (E. coli ATCC 25922 and E. coli
O126:H2) separated from the O104:H4 clusters with less than 80% similarity (Figure 1). To type the
different isolates with an even higher resolution, a core-genome phylogenetic analysis based on WGS
results was performed. In total 3764 ORFs were shared by all isolates analyzed and these were
defined as the core genome for further phylogenetic analysis in this study. The minimum-spanning
tree shows that the three 2013 EAEC Stx2a+ O104:H4 isolates clustered together with one to two-
allele difference among them. One-allele difference of isolate 381-1 from isolates 338 and 381-4 was
found in the gene encoding a carboxyterminal protease and was caused by a non-synonymous point
mutation (1481T>A) resulting in a premature protein (Leu494Stop) in isolate 381-1. In addition, one-
allele difference of isolate 381-4 from isolates 338 and 381-1 was found in a gene encoding a
putative cation:proton antiport protein caused by a non-synonymous point mutation (1465A>C)
resulting in an amino-acid change in the protein (Ile489Leu) in isolate 381-4. No allele differences
were found between the three 2011 outbreak isolates (7N, 8G and LB227103), and they clustered
together with two other reported outbreak strains (TY-2482 and 2011C-3493) with two to three-
allele difference among them (Figure 2). The 2009 isolates (2009El-2071 and 2009El-2050) were
separated from each other with a five-allele difference. The cluster of 2013 O104:H4 isolates were
separated from the 2009 and 2011 O104:H4 clusters with a minimum of 20 and 27 different alleles,
respectively (Figure 2 and Table S1). There were more than 193 different alleles between the typical
EAEC strain 55989 (O104:H4 stx-negative) and the 2013 EAEC stx2a+ O104:H4 isolates, whereas
isolate 381-3 (O126:H2, stx-negative) was far distinct from any of the O104:H4 strains analyzed, with
more than 3291-allele differences (Figure 2).
Chapter 4
92
Figure 2. Core-genome phylogenetic analysis of E. coli strains. The minimum spanning tree was generated by
SeqSphere and based on allelic profiles comparing 3764 alleles present in all analyzed strains and defined as
their core genome. Numbers in the lines indicate the number of allele differences between isolates. Black lines
indicate minimum distances and red lines connect isolates with more than minimum distances. Different colors
represent isolates of different time periods except isolate 381-3, which was isolated in 2013 but marked in a
different color as it is stx negative. EAEC 55989 was used as the hypothetical EAEC Stx2a+ O104:H4 progenitor.
Figure 1. Dendrogram of E. coli
isolates based on rep-PCR results.
Stx2a+ O104:H4 isolates of 2011 and
2013 are separated into two clusters
with a similarity of less than 95%
whereas the non O104:H4 E. coli
isolates are clearly separated with a
similarity of less than 80%.
Comparing Escherichia coli O104:H4 isolates
93
DISCUSSION
In this study, EAEC Stx2a+ O104:H4 isolates obtained from stool samples of a HUS patient and her
friend, travelling together to Turkey in 2013, were characterized and compared with the 2011
German EAEC Stx2a+ O104:H4 outbreak isolates and with two isolates from cases of bloody diarrhea
that occurred in the Republic of Georgia in 2009. The phylogenetic relationship of these isolates was
also compared with EAEC 55989, the hypothetical EAEC Stx2a+ O104:H4 progenitor strain isolated in
central Africa in 1995. Besides using established molecular typing methods routinely used in our
laboratory, WGS was used. Initially, the three EAEC Stx2a+ O104:H4 isolates obtained in 2013 were
suspected to be similar to the 2011 German outbreak strains based on PCR results. The 2013 isolates
could not be distinguished from the 2011 outbreak strains using a multiplex screening PCR targeting
O104wzy, fliCH4, stx2, and terD genes as characteristic features of the 2011 outbreak strains (6, 20).
Our microarray analyses revealed more detailed information but did not allow us to definitely
discriminate the 2013 EAEC Stx2a+ O104:H4 isolates from the 2011 ones. Differences found between
the 2011 and 2013 EAEC Stx2a+ O104:H4 isolates included two virulence genes (mchB and mchC) and
three drug-resistant genes (tetA, strA-B and sul2). However, these genes are known to be located in
multiple genomic islands and could therefore be easily lost or obtained (29, 30). Although rep-PCR
separated the 2011 and 2013 EAEC Stx2a+ O104:H4 isolates into two clusters, this technique is not
suitable for large-scale/global outbreak typing, as the inter-laboratory reproducibility is not sufficient
for this. In addition, also the intra-laboratory reproducibility may vary making it necessary for an
accurate typing to include all the isolates that need to be compared within the same run (22). The
high resolution of WGS enabled us to discriminate the 2013 isolates from the 2011 German outbreak
in the most accurate way.
In this study, a gene-by-gene typing approach also known as whole genome MLST (wgMLST),
extended MLST, MLST+ or core genome MLST (cgMLST) was used (31). The EAEC Stx2a+ O104:H4
isolates of 2013 appear to differ from those of 2011 and 2009 by a minimum of 27 and 20 alleles,
respectively. This suggests that 2013 isolates were relatively closer related to the 2009 clone of
southeast Europe than to the 2011 outbreak clone. Unfortunately, there is no established threshold
for the MLST+ approach to address intra- and inter-cluster differences yet. In addition, only the core
genome was used for constructing the phylogenetic tree minimizing the chance of including mobile
genetic elements (MGEs) in the analysis as there is no consensus on how MGEs should be taken into
account in bacterial phylogeny (32, 33). As the estimated mutation rate of E. coli is reported to be 1.1
nucleotide per genome per year (34), the 27-allele difference between the 2013 and 2011 isolates
suggests that the EAEC Stx2a+ O104:H4 isolates from the different time periods belong to different
Chapter 4
94
clones. Moreover, only a one-allele difference was found between the isolate of the HUS patient and
that of her friend, strongly supporting the hypothesis that either both patients obtained STEC from
the same source or that one transmitted STEC to the other.
In addition to high resolution typing, WGS provided us with more detailed molecular characteristics
of the isolates. This helped us to explain in more detail the phenotypic results of the isolates. For
example, an insertion within the sul1 gene resulting in protein truncation was found in all 2013 EAEC
Stx2a+ O104:H4 isolates, which could explain why the isolates were susceptible to
trimethoprim/sulfamethoxazole in spite of the presence of the sul1 gene detected by the microarray.
On the other hand, a sul2 gene existing in the 2011 but not the 2013 isolates may confer resistance
to trimethoprim/sulfamethoxazole in spite of the fact that also the 2011 isolates carried the
truncated sul1.
Notably, one of the 2013 EAEC Stx2a+ O104:H4 isolates (381-1) was blaCTX-M-15 positive as the 2011
outbreak isolates. As isolate 381-3 (stx-negative/O126:H2/blaCTX-M-15) was also obtained from the
same host, it is likely that 381-1 acquired this ESBL gene from isolate 381-3 or the other way around.
Our study did, however, not address a detailed analysis of the MGEs and plasmids as the aim of the
study was to show the value of WGS technique in routine clinical diagnostics to identify and compare
potentially pathogenic strains.
Recently, two similar isolates of EAEC Stx2a+ O104:H4 causing bloody diarrhea and HUS were
reported in Belgium. Patients of both cases travelled to Tunisia and Turkey respectively, and the
infection may have been acquired in those regions (35). This is similar to our and other studies
describing EAEC Stx2a+ O104:H4 infections associated with travelling to Turkey, Tunisia, Egypt and
North Africa (30, 36).
Our study shows that WGS has the potential to be integrated in current routine laboratories to
relatively rapidly obtain reproducible and detailed information in one single step. This facilitates
hospitals and public health organizations to make appropriate strategies for infection control and
public health measures in real time (37). Surely, some challenges have to be overcome before
applying WSG to routine diagnostics, like the availability of user-friendly bioinformatics tools,
automation of the workflow and availability of genomic databases (38).
In conclusion, the detailed characterization of EAEC Stx2a+ O104:H4 isolates obtained in 2013
including the high resolution typing approach helped us to distinguish them from the 2011 German
outbreak strains. It further supported the general idea that, although the 2011 outbreak is stopped,
EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak strain in their core genome still
Comparing Escherichia coli O104:H4 isolates
95
circulate. They still pose a serious risk for public health being a potential source for causing a new
EAEC Stx2a+ O104:H4 outbreak if not monitored carefully.
ACKNOWLEDEEMENTS
We thank Dr. Alexander Mellmann for providing us with E. coli O104:H4 strains of the German
outbreak and Brigitte Dijkhuizen for technical assistance.
This study was partly supported by the Interreg IVa-funded projects EurSafety Heath-net (III-1-02=73)
and SafeGuard (III-2-03=025) and by a University Medical Center Groningen Healthy Ageing Pilots
grant.
REFERENCES
1. Gyles CL. 2007. Shiga toxin-producing Escherichia coli: an overview. J Anim Sci 85.
2. Farrokh C, Jordan K, Auvray F, Glass K, Oppegaard H, Raynaud S, Thevenot D, Condron R, De Reu K,
Govaris A, Heggum K, Heyndrickx M, Hummerjohann J, Lindsay. 2013. Review of Shiga-toxin-producing
Escherichia coli (STEC) and their significance in dairy production. Int J Food Microbiol 162:190–212.
3. Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A, Middendorf B, Harmsen D, Schmidt MA,
Karch H. 2008. Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia
coli. Emerg Infect Dis 14:1287-90.
4. Karch H. 2001. The role of virulence factors in enterohemorrhagic Escherichia coli (EHEC)-associated
hemolytic-uremic syndrome. Semin. Thromb Hemost 27:207–213.
5. Welinder-Olsson C, Kaijser B. 2005. Enterohemorrhagic Escherichia coli (EHEC). Scand J Infect Dis 37:405–
416.
6. Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth, A, Bauwens A, Peters G, Karch H. 2011.
Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in
Germany, 2011: A microbiological study. Lancet Infect Dis 11:671–676.
7. Brzuszkiewicz E, Thürmer A, Schuldes J, Leimbach A, Liesegang H, Meyer FD, Boelter J, Petersen H,
Gottschalk G, Daniel R. 2011. Genome sequence analyses of two isolates from the recent Escherichia coli
outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia
coli (EAHEC). Arch Microbiol 193:883–891.
8. Rasko DA, Webster DR, Sahl JW, Bashir A, Boisen N, Scheutz F, Paxinos EE, Sebra R, Chin CS,
Iliopoulos D, Klammer A, Peluso P, Lee L, Kislyuk AO, Bullard J, Kasarskis A, Wang S, Eid J, Rank D,
Redman JC, Steyert SR, Frimodt-Møller J, Struve C, Petersen AM, Krogfelt KA, Nataro JP, Schadt EE,
Waldor MK. 2011. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany.
N Engl J Med 365:709–717.
9. Scheutz F, Nielsen EM, Frimodt-Møller J, Boisen N, Morabito S, Tozzoli R, Nataro JP, Caprioli A. 2011.
Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain
causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. Euro Surveill 16.
Chapter 4
96
10. Müller D, Benz I, Liebchen A, Gallitz I, Karch H, Schmidt, MA. 2009. Comparative analysis of the locus of
enterocyte effacement and its flanking regions. Infect Immun 77:3501–3513.
11. Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y,
Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM, Moore RL,
Guenther S, Rothberg JM, Karch H. 2011. Prospective genomic characterization of the German
enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS
One 6:e22751.
12. Rohde H, Qin J, Cui Y, Li D, Loman NJ, Hentschke M, Chen W, Pu F, Peng Y, Li J, Xi F, Li S, Li Y,
Zhang Z, Yang X, Zhao M, Wang P, Guan Y, Cen Z, Zhao X, Christner M, Kobbe R, Loos S, Oh J, Yang
L, Danchin A, Gao GF, Song Y, Li Y, Yang H, Wang J, Xu J, Pallen MJ, Wang J, Aepfelbacher M, Yang
R, E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium. 2011. Open-source genomic analysis of
Shiga-toxin-producing E. coli O104:H4. N Engl J Med 365:718-724.
13. Grad YH, Lipsitch M, Feldgarden M, Arachchi HM, Cerqueira GC, FitzGerald M, Godfrey P, Haas BJ,
Murphy CI, Russ C, Sykes S, Walker BJ, Wortman JR, Young S, Zeng Q, Abouelleil A, Bochicchio J,
Chauvin S, Desmet T, Gujja S, McCowan C, Montmayeur A, Steelman S, Frimodt-Møller J, Petersen
AM, Struve C, Krogfelt KA, Bingen E, Weill FX, Lander ES, Nusbaum C, Birren BW, Hung DT, Hanage
WP. 2012. Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011. Proc Natl Acad
Sci 109:3065–3070.
14. Pritchard L, Holden NJ, Bielaszewska M, Karch H, Toth IK. 2012. Alignment-free design of highly
discriminatory diagnostic primer sets for Escherichia coli O104:H4 outbreak strains. PLoS One 7:e34498.
15. Ahmed SA, Awosika J, Baldwin C, Bishop-Lilly K. a, Biswas B, Broomall S, Chain PS, Chertkov O,
Chokoshvili O, Coyne S, Davenport K, Detter JC, Dorman W, Erkkila TH, Folster JP, Frey KG, George
M, Gleasner C, Henry M, Hill KK, Hubbard K, Insalaco J, Johnson S, Kitzmiller A, Krepps M, Lo CC,
Luu T, McNew LA, Minogue T, Munk CA, Osborne B, Patel M, Reitenga KG, Rosenzweig CN, Shea A,
Shen X, Strockbine N, Tarr C, Teshima H, van Gieson E, Verratti K, Wolcott M, Xie G, Sozhamannan S,
Gibbons HS; Threat Characterization Consortium. 2012. Genomic Comparison of Escherichia coli O104:H4
Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding
Phage stx2. PLoS One 7.
16. Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C,
Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C, Karoui ME,
Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguénec C, Lescat M, Mangenot S, Martinez-
Jéhanne V, Matic I, Nassif X, Oztas S, Petit MA, Pichon C, Rouy Z, Ruf CS, Schneider D, Tourret J,
Vacherie B, Vallenet D, Médigue C, Rocha EP, Denamur E. 2009. Organised genome dynamics in the
Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 5.
17. Miko A, Delannoy S, Fach P, Strockbine N. a, Lindstedt BA, Mariani-Kurkdjian P, Reetz J, Beutin L. 2013. Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from
different origins and sources. Int J Med Microbiol 303:410–421.
18. de Boer RF, Ferdous M, Ott A, Scheper HR, Wisselink GJ, Heck ME, Rossen JW, Kooistra-Smid AM.
2015. Assessing the public health risk of Shiga toxin-producing Escherichia coli by use of a rapid diagnostic
screening algorithm. J Clin Microbiol 53:1588–1598.
19. Scheutz F, Teel LD, Beutin L, Pierard D, Buvens G, Karch H, Mellmann A, Caprioli A, Tozzoli R,
Morabito S, Strockbine NA, Melton-Celsa AR, Sanchez M, Persson S, O'Brien AD. 2012. Multicenter
evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin
Microbiol 50:2951-2963.
20. Zhang W, Bielaszewska M, Bauwens A, Fruth A, Mellmann A, Karch H. 2012. Real-time multiplex PCR for
detecting Shiga toxin 2-producing Escherichia coli O104:H4 in human stools. J Clin Microbiol 50:1752–1754.
Comparing Escherichia coli O104:H4 isolates
97
21. Geue L, Schares S, Mintel B, Conraths FJ, Müller E, Ehricht R. 2010. Rapid microarray-based genotyping
of enterohemorrhagic Escherichia coli serotype O156:H25/H-/Hnt isolates from cattle and clonal relationship
analysis. Appl Environ Microbiol 76:5510–5519.
22. Voets GM, Leverstein-Van Hall Ma, Kolbe-Busch S, Van Der Zanden A, Church D, Kaase M, Grisold A,
Upton M, Cloutman-Green E, Cantón R, Friedrich AW, Fluit AC; DiversiLab Study Group. 2013.
International multicenter evaluation of the diversilab bacterial typing system for escherichia coli and klebsiella
spp. J Clin Microbiol 51:3944–3949.
23. Ferdous M, Zhou K, Mellmann A, Morabito S, Croughs PD, de Boer RF, Kooistra-Smid AM, Rossen JW,
Friedrich AW. 2015. Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or
Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing. J
Clin Microbiol 53:3530-3538.
24. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Ponten T,
Ussery DW, Aarestrup FM, Lund O. 2012. Multilocus sequence typing of total-genome-sequenced bacteria. J
Clin Microbiol 50:1355-1361.
25. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV.
2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640-2644.
26. Joensen KG, Tetzschner AM, Iguchi A, Aarestrup FM, Scheutz F. 2015. Rapid and Easy In Silico
Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data. J Clin Microbiol 53:2410-
2426.
27. Lima IF, Boisen N, Quetz Jda S, Havt A, de Carvalho EB, Soares AM, Lima NL, Mota RM, Nataro JP,
Guerrant RL, Lima AÂ. 2013. Prevalence of enteroaggregative Escherichia coli and its virulence-related genes
in a case-control study among children from north-eastern Brazil. J Med Microbiol 62:683-93.
28. Leopold SR, Goering RV, Witten A, Harmsen D, Mellmann A. 2014. Bacterial whole-genome sequencing
revisited: Portable, scalable, and standardized analysis for typing and detection of virulence and antibiotic
resistance genes. J Clin Microbiol 52:2365–2370.
29. Guy L, Jernberg C, Arvén Norling J, Ivarsson S., Hedenström I, Melefors Ö, Liljedahl U, Engstrand L,
Andersson SG. 2013. Adaptive Mutations and Replacements of Virulence Traits in the Escherichia coli
O104:H4 Outbreak Population. PLoS One 8: 1–13.
30. Grad YH, Godfrey P, Cerquiera GC, Mariani-Kurkdjian P, Gouali M, Bingen E, Shea TP, Haas BJ,
Griggs A, Young S, Zeng Q, Lipsitch M, Waldor MK, Weill FX, Wortman JR, Hanage WP. 2013.
Comparative genomics of recent Shiga toxin-producing Escherichia coli O104:H4: short-term evolution of an
emerging pathogen. MBio 4, 1–10.
31. Maiden MCJ, Jansen van Rensburg MJ, Bray JE, Earle SG, Ford Sa, Jolley Ka, McCarthy ND. 2013.
MLST revisited: the gene-by-gene approach to bacterial genomics. Nat. Rev. Microbiol 11:728–36.
32. Daubin V, Gouy M, Perrière G. 2002. A phylogenomic approach to bacterial phylogeny: evidence of a core of
genes sharing a common history. Genome Res 12:1080-90.
33. Frost LS, Leplae R, Summers AO, Toussaint A. 2005. Mobile genetic elements: the agents of open source
evolution. Nat Rev Microbiol 3:722-32.
34. Reeves PR, Liu B, Zhou Z, Li D, Guo D, Ren Y, Clabots C, Lan R, Johnson JR, Wang L. 2011. Rates of
mutation and host transmission for an escherichia coli clone over 3 years. PLoS One 6.
35. De Rauw K, Vincken S, Garabedian L, Levtchenko E, Hubloue I, Verhaegen J, Craeghs J, Glupczynski Y,
Mossong J, Piérard D. 2014. Enteroaggregative Shiga toxin-producing Escherichia coli of serotype O104:H4 in
Belgium and Luxembourg. New Microbes New Infect 2:138-43.
Chapter 4
98
36. Ecdc/Efsa 2011. STEC/VTEC in humans, food and animals in the EU/EEA. ECDC/EFSA joint Technical report
23.
37. Reuter S, Ellington MJ, Cartwright EJP, Köser CU, Török, ME, Gouliouris T, Harris SR, Brown NM,
Holden MT, Quail M, Parkhill J, Smith GP, Bentley SD, Peacock SJ. 2013. Rapid bacterial whole-genome
sequencing to enhance diagnostic and public health microbiology. JAMA Intern Med 173:1397–404.
38. Sherry NL, Porter JL, Seemann T, Watkins A, Stinear TP, Howden BP. 2013. Outbreak investigation using
high-throughput genome sequencing within a diagnostic microbiology laboratory. J. Clin.Microbiol 51,1396–
1401.
99
CHAPTER 5
The Mosaic Genome Structure and Phylogeny of Shiga
Toxin-Producing Escherichia coli O104:H4 is Driven by
Short-term Adaptation
Kai Zhou1, Mithila Ferdous1, Richard F. de Boer2, Anna M. D. Kooistra-Smid1,2, Hajo
Grundmann1, Alexander W. Friedrich1, John W.A. Rossen1
1Department of Medical Microbiology, University of Groningen, University Medical Center
Groningen, Groningen, the Netherlands 2Certe - Laboratory for Infectious Diseases, Groningen, the Netherlands
Keywords
Shiga Toxin-Producing Escherichia coli, Escherichia coli O104:H4, Stx2-encoding prophage, Next-
generation sequencing, Comparative Genomics, Genomic Structural Variation, Genomic islands,
Prophages, Single Nucleotide Polymorphism, Antibiotic Resistance
Clinical Microbiology and Infection (2015); 21: 468.e7–468.e18
Chapter 5
100
ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) O104:H4 emerged as an important pathogen when it
caused a large outbreak in Germany in 2011. Little is known about the evolutionary history and
genomic diversity of the bacterium. The current communication describes a comprehensive analysis
of STEC O104:H4 genomes from the 2011 outbreak and other non-outbreak related isolates.
Outbreak-related isolates formed a tight cluster which shared a monophyletic relation with two non-
outbreak clusters, suggesting that all three clusters originated from a common ancestor. Eight single
nucleotide polymorphisms, seven of which were non-synonymous, distinguished outbreak from non-
outbreak isolates. Lineage-specific markers indicated that recent partitions were driven by selective
pressures associated with niche adaptation. Based on the results, an evolutionary model for STEC
O104:H4 is proposed. Our analysis provides the evolutionary context at population level and
describes the emergency of clones with novel properties, which is necessary for developing
comprehensive approaches to early warning and control.
The plastic genome structure of STEC O104:H4
101
INTRODUCTION
From early May to July 2011, nearly 4000 clinical infections were ascertained by health authorities in
Germany accounting for the largest STEC outbreak on record. Over 900 patients developed hemolytic
uremic syndrome (HUS) of which 54 were fatal (1). Two features set this outbreak apart from
previous ones caused by STEC O157:H7, including the high incidence of HUS (>20%) and a rare
serotype O104:H4 (1). Isolates associated with the outbreak had an unusual combination of virulence
factors not only attributed to STEC but also to enteroaggregative E. coli (EAEC) harboring the Stx2-
encoding prophage and pAA-like plasmid (2), which may have contributed to the high rate of HUS (3).
Moreover, outbreak isolates contained an extended spectrum β-lactamase (ESBL) gene, which is
rather rare in STEC (4).
Using the power of Next-Generation Sequencing (NGS) technology, the first available draft sequence
of an outbreak strain (TY-2482) isolated from a 16-year-old girl became available while the outbreak
was still ongoing. It revealed a high degree of genome plasticity with numerous mobile genetic
elements (MGEs) and three plasmids (2). Further analysis showed that outbreak strains shared the
same sequence type (ST) known as ST678 with a historical STEC O104:H4 strain 01-09591, which was
isolated from a child presenting with HUS in Germany in 2001. Genomic comparisons revealed a
genetic relationship of 99.8% nucleotide similarity with an AggR positive EAEC O104:H4 strain 55989
isolated in Central Africa in the late 1990s (2). This was further supported by a study that included
additional EAEC O104:H4 strains into the phylogenetic analysis. It was therefore suggested that EAEC
O104:H4 strain 55989 represented a clade at the root of the emerging clone of STEC O104:H4 that
rapidly expanded in 2011 (5). The limited number of single nucleotide polymorphisms (SNPs) among
all sequenced outbreak isolates suggested their clonality (6,7). However, it remains to be elucidated
how this clone evolved and attained its repertoire of virulence factors. In this study, we attempt to
shed light on the evolution of STEC O104:H4 by describing the genome structure and population
structure of outbreak and available non-outbreak isolates obtained from sporadic infections reported
before and after the outbreak.
MATERIALS AND METHODS
Strains analyzed in this study
In all, 23 E. coli isolates have been used in this study (Table 1). Seven isolates were sequenced as part
of our previous study (Ferdous et al., unpublished). Briefly, DNA libraries were prepared using the
Nextera XT v2 kit (Illumina, San Diego, CA, US) according to the manufacturer’s instructions and then
run on a Miseq (Illumina, San Diego, CA, US) for generating paired-end 250-bp reads. De novo
assembly was performed using CLC Genomics Workbench v6.0.5 (CLC bio A/S, Denmark) after quality
Chapter 5
102
trimming (Qs ≥ 28) with optimal word sizes based on the maximum N50 value. Four of the seven
isolates were obtained from a HUS patient (338) and her travel partner (381-1, 381-3 and 381-4), and
the other three (7N, 8G and 9Z) were isolated during the 2011 outbreak in Germany. Apart from
isolate 381-3, which is non-O104:H4, stx negative and ST10, the other six isolates belong to STEC
O104:H4 / ST678. Therefore 381-3 was only used for plasmid comparison in this study. In addition, 17
genomes were obtained from publically available databases: genome sequences of 55989, 2009EL-
2050, 2009EL-2071, 2011C-3493 and E112/10 were downloaded from the NCBI database, and the
others were downloaded from
http://www.broadinstitute.org/annotation/genome/Ecoli_O104_H4/Downloads.html. Detailed
information of all the isolates analyzed in this study is listed in Table 1. The GenBank accession
number of all isolates analyzed in this study: NC_011748 (55989), NC_018650 (2009EL-2050),
NC_018661 (2009EL-2071), NC_018658 (2011C-3493), NZ_AHAV00000000 (E112/10), AFVR00000000
(TY-2482), AFUX01000000 (Ec11-4404), AFVA01000000 (Ec11-4632.1), AIPQ01000000 (Ec12-0465),
AIPR01000000 (Ec12-0466), AGWF01000000 (Ec11-9450), AGWH01000000 (Ec11-9941),
AGWG01000000 (Ec11-9990), AFRL01000000 (04-8351), AFRK01000000 (09-7901), AFPS00000000
(HUSEC041), JRJF00000000 (338), JRKD00000000 (381-1), JRLM00000000 (381-3), JRLD00000000
(381-4), JRKE00000000 (7N), JRLN00000000 (8G), JRKF00000000 (9Z).
Annotation
To annotate the genomes, contigs were first oriented and ordered using ABACAS (8) against the
reference TY-2482 chromosome and plasmids with the following settings: using sensitive mapping in
Numer, a minimum percent identity of 40, a minimum percent contig coverage of 20, minimum
contig converage difference set to 0, and reference sequence is circular. The start coordinate of all
genomes has been reset according to the first nucleotide of TY-2482 (GATGTTGCTCCCCCAAG).
Contigs were concatenated following this order as a pseudomolecule with appending the unmapped
contigs at the end. Each ordered genome was manually curated after performing automatic
annotation on the RAST server (9).
Mapping and SNP analysis
Reads were mapped to the chromosome of TY-2482 by CLC Genomics Workbench v6.05 with default
settings. To acquire reliable SNPs, the regions of MGEs (prophages and genomic islands) and repeats
were masked during mapping. Candidate SNPs were called by the algorithm Quality-based variant
detection of CLC Genomics Workbench. SNPs were filtered out if one of the following occurred: (i)
their quality score was below 30; (ii) the neighborhood quality was below 30; (iii) the minimum
The plastic genome structure of STEC O104:H4
103
variant frequency was below 35%; (iv) the minimum coverage was below 10; (v) only detected on a
single strand. SNPs called from assembly genomes were identified by Mauve (10).
Genome analysis: genomic islands, prophages and plasmids
Fragments larger than 5 kb that were absent in at least one genome were detected by BLAST and
were defined as genomic islands (GEIs) in this study. The prophages were predicted on the web
server PHAST (11) followed by manual curations. Only “intact” prophages detected by PHAST were
included in the further analysis, and those were grouped according to the sequence similarity aligned
by Mauve. The plasmid analysis was mainly dependent on BLASTn. The contigs of each sample were
blasted against the reference plasmid and plotted by BLAST Ring Image Generator (BRIG) (12). The
reference plasmid was artificially generated by concatenating sequences of a set of plasmids,
including pTY1, pTY2, pTY3 (2), p55989 (13), pHUSEC41-1, pHUSEC41-2, pHUSEC41-3, pHUSEC41-4
(14), and p09EL50 (15).
Core-genome phylogenetic analysis
The whole genomes were aligned by Mauve. Fragments (≥500 bp) shared by all genomes were
collected and then concatenated. The resulting pseudomolecules were defined as the core genome,
which was used for the phylogenetic analysis. SNPs were collected from the core genomes by in-
house scripts. A maximum likelihood phylogeny was estimated by RAxML v7.2.8 (16) with 1000
bootstrap replications under the general time-reversible model with Gamma correction (GTR+G).
RESULTS
Core-genome phylogeny of STEC O104:H4
To reveal the evolutionary relationship of STEC O104:H4 analyzed in this study, a core-genome
phylogenetic analysis based on single nucleotide polymorphisms (SNPs) was performed. A maximum-
likelihood (ML) phylogenetic tree was constructed based on 3659 SNPs detected from the alignments
of the 4.5 Mbp core genome (Figure 1). The phylogeny showed that the sequenced German outbreak
isolates 7N, 8G, and 9Z from our previous study [Ferdous et al., submitted] shared a monophyletic
relationship (outbreak clade; highlighted in red in Figure 1) with two other German outbreak isolates
(TY-2482 and 2011C-3493) and two French outbreak isolates (Ec11-4404 and Ec11-4632.1). Three
isolates from 2013 (338, 381-1 and 381-4) clustered in a separated clade (non-outbreak clade A,
shortly clade A; highlighted in green in Figure 1) together with four other non-outbreak isolates
(E112/10, Ec11- 9941, Ec11-9990 and Ec12-0466). E112/10 was isolated in 2010 from a Swedish
patient, and Ec11- 9941, Ec11-9990 and Ec12-0466 were isolated after the outbreak in France 2011.
Chapter 5
104
Notably, Ec12-0466 formed a separated branch within this clade. Two additional 2011 isolates from
France (Ec11-9450 and Ec12-0465) isolated after the outbreak clustered with two 2009 isolates from
the Republic of Georgia (2009EL-2050 and 2009EL-2071) forming another distinct clade (non-
outbreak clade B, shortly clade B; highlighted in blue in Figure 1). All three clades are closely related,
suggesting that they shared a common ancestor. Notably, clade A and the outbreak clade were more
closely related to each other than to clade B (Figure 1). The three clades share relatively distant
relationship with three isolates (HUSEC041, 04-8351, 09-7901) and the hypothetical progenitor EAEC
strain 55989, and the clades formed by the four strains were collectively named as “historical clade”
(shown in black in Figure 1). Taken together, the phylogeny of STEC O104:H4 indicated that this
bacterium has diversified into multiple lineages, at least three of them sharing a close relationship
which may represent the dominant population of STEC O104:H4. We note that clade A and B isolates
were obtained from different geographic regions (Table 1), indicating the local expansion of certain
STEC O104:H4 clones.
Clade-specific SNPs
We identified eight canonical SNPs in the core genome that are unique to the outbreak clade (Table
2), suggesting they were acquired by the outbreak clone recently. Mapping these SNPs to the
available sequences of 40 additional outbreak isolates (including German and French isolates)
reported previously (6,7,17) (http://www.hpa-bioinformatics.org.uk/lgp/genomes) supported their
canonical nature. All SNPs located within coding regions and seven of them were non-synonymous.
Comparison of the accessory genome
Plasmids
Plasmids of outbreak strain TY-2482 (pTY1, pTY2 and pTY3), non-outbreak strain 2009EL-2050
(p09EL50) and historical strain HUSEC041 (pHUSEC41-1, pHUSEC41-3, and pHUSEC41-4) were used as
reference to investigate the plasmid content of isolates analyzed here. Substantial variations in the
content of plasmids were observed among strains analyzed (Figure 2). Plasmid pTY1 carrying the
ESBL gene blaCTX-M-15 and a beta-lactamase gene blaTEM-1 is present in all isolates of the outbreak
clade, but not in any isolates of other clades, indicating that pTY1 may be recently acquired by the
outbreak strains resulting in potential adaptive advantages (e.g. antibiotic resistance). The plasmid
pTY2 carries an agg operon encoding AAF/I fimbriae resulting in the enteroaggregative phenotype of
the outbreak strains. The pTY2-like plasmid was not found in any isolates of the historical clade, but
in all isolates of outbreak and non-outbreak clades A and B except Ec11-9450 (due to plasmid loss
during culturing; (18)). Notably, we observed a spontaneous deletion in the pTY2 plasmid of isolate
The plastic genome structure of STEC O104:H4
105
9Z resulting in the loss of the aggR gene, which encodes a transcriptional activator for the fimbriae
expression (Figure S1). This gene was detected in the original isolate (3), and may therefore have
been lost during propagation in vitro. In contrast, another enteroaggregative plasmid p55989 (also
known as pAA from EAEC) encoding AAF/III fimbriae instead of AAF/I fimbriae was exclusively found
in strains of historical clade, suggesting a recent replacement of pAA by pTY2. Plasmid pTY3 is a small
cryptic plasmid only carrying a repA gene, which was found in all isolates of outbreak and non-
outbreak clades except in Ec11-9990. It was not present in the isolates of historical clade. We blasted
the sequence of pTY3 in GenBank to explore the origin of the small cryptic plasmid. Besides plasmids
found in E. coli O104:H4, highly similar plasmids (identity > 90%) were found in other E. coli strains
and also in some other bacterial species (Table S1). Therefore, the origin of the small cryptic plasmid
could not yet been resolved.
The plasmid pHUSEC41-1 from the historical isolate HUSEC041 carries a Tn3-like transposase flanked
by the multiple drug-resistance (MDR) genes blaTEM-1, strA, strB and sul2. Besides HUSEC041,
pHUSEC41-1-like plasmid was found in historical isolate 04-8351, clade-A isolates 381-1, E112/10,
Ec11-9941, Ec11-9990, Ec12-0466, clade-B isolates Ec11-9960 as well as the non-O104 / stx-negative
isolate 381-3 (Figure 2). However, none of the outbreak isolates harbored this plasmid, which may be
caused by the fact that both plasmid pTY1 and pHUSEC41 share the same incompatibility group
(Incl1). Notably, the region containing MDR genes was missing on the pHUSEC41-1-like plasmid in 04-
8351, Ec11-9450, Ec11-9990 and E112/10 (Figure 2). However, such region was replaced by another
carrying the ESBL gene blaCTX-M-15 on the pHUSEC41-1-like plasmid of 381-1. To our knowledge, this is
the first ESBL-producing non-outbreak isolate reported to date. Noteworthy, an almost identical
pHUSEC-41-1-like plasmid as that observed in 381-1 was found in the non-O104 / stx-negative isolate
381-3, both of which were recovered from the same patient (Figure S2). This may result from a
possible transconjugation event between 381-1 and 381-3 or between a common donor and both
isolates, since the plasmid harbored an intact transconjugation operon (trb, tra and pil). This finding
may explain why only 381-1 but not 338 and 381-4 were ESBL positive although the three isolates
were clonal. No significant hit of pHUSEC41-3, pHUSEC41-4 and p09EL50 were found in any of the
isolates studied here except their origins.
Chapter 5
106
Table 1. Isolates analyzed in this study
Isolate IDa Date of
isolate Patient information
Clinical manifestations
Epidemic information
Country of isolation
ESBL Virulence group
b
Reference
7N 2011 Unknown Unknown German outbreak
Germany + Group I Ferdous et al., unpublished
8G 2011 Unknown Unknown German outbreak
Germany + Group I Ferdous et al., unpublished
9Z 2011 Unknown HUS German outbreak
Germany + Group Ic Ferdous et al.,
unpublished TY-2482 2011 16-year-old
female HUS German
outbreak Germany + Group I (2)
2011c-3493
2011 51-year-old male
HUS Germany, Travel, German outbreak period
U.S. + Group I (15)
Ec11-4404 06. 2011 Male HUS French outbreak
France + Group I (7)
Ec11-4632.1
06. 2011 Female HUS French outbreak
France + Group I (7)
Ec12-0466 12. 2011 Child HUS North Africa, Travel
France - Group I (18)
381-4 07. 2013 23-year-old female
Diarrhea Turkey, Travel Netherlands + Group I Ferdous et al., unpublished
381-1 07. 2013 23-year-old female
Diarrhea Turkey, Travel Netherlands - Group I Ferdous et al., unpublished
338 07. 2013 22-year-old female
HUS Turkey, Travel Netherlands - Group I Ferdous et al., unpublished
Ec11-9941 9.2011 Child HUS Unknown France - Group I (18) E112/10 2010 Unknown Unknown Tunisia, Travel Sweden - Group I (18) Ec11-9990 8.2011 Child HUS Unknown France - Group I (18) Ec11-9450 10. 2011 Unknown HUS Turkey, Travel,
Local outbreak France - Group I
c (30)
2009EL-2071
2009 Unknown Bloody diarrhea
Unknown Republic of Georgia
- Group I (31)
Ec12-0465 11. 2011 Child HUS Unknown France - Group I (18) 2009EL-2050
2009 Unknown Bloody diarrhea
Unknown Republic of Georgia
- Group I (31)
04-8351 2004 6-year-old male
Hemorrhagic colitis
Unknown France - Group II [32]
09-7901 2009 Adult male HUS Unknown France - Group II (32) HUSEC041 (01-09591)
2001 Child HUS Unknown Germany - Group II (33)
55989 Late 1990s
HIV patient Diarrhea Unknown Central African Republic
- Group III (13)
381-3d 07. 2013 23-year-old
female Diarrhea Turkey, Travel Netherlands + Group IV Ferdous et al.,
unpublished aThe isolates listed here were grouped in different colours according to the phylogenetic results shown in Fig. 1. The
sequence type and serotype of all isolates is ST678 and O104:H4, with the exception of isolate 381-3 which is ST-10 and O126:H2. bThe virulence groups are defined as Group I (positive for stx2/aggA/aggR/aatA/sigA/pic/iha), Group II (positive for
stx2/agg3A/aggR/aatA/sigA/pic/iha), Group III (positive for agg3A/aggR/aatA/sigA/pic/iha) and group IV (positive for aatA/iha). cStrain 9Z lost a fragment containing aggR (please refer to the text for more details), and strain Ec11-9450 lost pTY2 in vitro
as described previously (18). dThis strain was not included in the phylogenetic analysis but only in the plasmid analysis.
The plastic genome structure of STEC O104:H4
107
Figure 1. Maximum-likelihood phylogeny of Escherichia coli O104:H4. The phylogeny was derived by core-genome analysis using an approximately 4.5-Mbp genome sequence of each sample. The three major clades were respectively referred as to outbreak clade (red), non-outbreak clade A (green), and non-outbreak clade B (blue). The other clades were collectively named ‘historical clade’ (black). The inset shows the close-up phylogenetic tree of the three major clades. The numbers on the nodes represent the percentage of bootstrap support (>90).
Figure 2. Comparison of the plasmid content in Escherichia coli O104:H4 strains. Each ring corresponds to the BLASTn result of one genome relative to the artificial plasmid reference. The reference was composed of numerous plasmids shown by the first outer ring with labels in alternate colors. From outer to inner, the rings were ordered as the sequence shown in the legends (left). Strains were grouped in different colors according to the phylogenetic results shown in Figure 1. The gradients (dark, pale and white) of each color represent the sequence similarity (from 100% to 0%) between samples and reference. The multiple drug-resistance region in pHUSEC41-1 is marked by a purple frame.
Chapter 5
108
Table 2. Eight SNPs distinguishing outbreak isolates from non-outbreak isolates Reference Position
a
SNP (Ob->Nob)
b
Location Annotation Amino acid change
347122 C->T CDS Putative oxidoreductase Arg130Gln
1323393 A->G CDS PTS system, galactitol-specific IIC component GatC
synonymous
1449640 T->C CDS ferredoxin-type protein NapG (periplasmic nitrate reductase)
His47Arg
1768361 T->G CDS uracil phosphoribosyltransferase protein Glu184Asp
2602394 A->G CDS putative calcium/sodium:proton antiporter YrbG Ile108Met
3033847 T->G CDS selenocysteine-specific translation elongation factor
Asn169His
3429136 T->C CDS rhamnulokinase Glu424Gly
4527390 C->T CDS DNA-binding ATP-dependent protease La Type I Thr319Ile
a TY-2482 was used as reference here, of which the start coordinate was reset as described in the text.
b Ob and Nob represent outbreak and non-outbreak, respectively.
Prophages
Frequent gain or loss of prophages occurred across the investigated population. To further analyse
the diversity of prophages among STEC O104:H4 isolates, we used the seven prophages identified
from TY-2482 (named as Phage-I to Phage-VII according to their positions on the chromosome) (18)
as reference to group the others according to sequence identity.
Our analysis found several lineage-specific prophages. Phage-IV was the most diverse prophage
found in this study, which was identified in all isolates except for two isolates of the historical clade
Ec09-7901 and 55989 (Figure 3). All Phage-IV shared the same integration site within the yecE gene
(Figure S3). Phylogenetic analysis using ML trees revealed a striking topological homology with the
core-genome ML tree indicative of co-evolution. Thus the phage-IV of outbreak isolates clustered
tightly in a single clade distant from the clade formed by other isolates, with the exception of Ec12-
0466 that appeared to be more closely related to the one of the outbreak isolates (Figure 4A),
consistent with its outlier position in the core-genome ML tree. This indicates that a replacement of
Phage-IV occurred in the outbreak clone recently, although it remains unknown whether this
prophage is functional or not.
Phage-VII carries the stx2 gene, and so is known as the Stx2-encoding prophage. Except the
progenitor strain 55989, all other strains harbored this prophage which chromosomally located
within wrbA. Remarkably, phylogenetic analysis revealed that Stx2-encoding prophages detected
from clade-B isolates clustered in a single clade separated from the one formed by all other isolates
(Figure 4B). This suggests that a single replacement of the Stx2-encoding prophage occurred in the
ancestor of clade B. Further sequence analysis showed that one of the significant differences
The plastic genome structure of STEC O104:H4
109
between the two clusters of Stx2-encoding prophages was found within the lysis region, where a rha
gene (encoding the Rha family phage regulatory protein) and an unnamed gene (encoding the lytic
protein) were replaced in clade-B isolates by a bor gene (encoding a virulence factor) and another
unnamed gene (encoding lytic protein), respectively (Figure S4). Additionally, Phage-V seems to be
another lineage-specific prophage, which was lost by clade-B isolates except 2009EL-2071 (Figure 3).
Phage-I and Phage-VI were relatively conserved among all isolates, suggesting that these two
prophages were likely to be present in a common ancestor.
Genomic Islands
Multiple genomic islands (GEIs) were detected in each of the isolates. Here we focus on several
highly diverse GEIs only (Figure 3). One such region, referred to as GEI-1 contains the mch operon
(microcin H47 biosynthesis), iha (adhesin), ter operon (tellurium resistance), ag43 (antigen 43) and
yeeV-yeeU pair (toxin-antitoxin system). This GEI was found in all isolates except in the progenitor
strain 55989. However, a significant deletion with the loss of multiple genes of the mch operon (i.e.
mchB and mchC involved in microcin biosynthesis) was found in clade-A isolates with exception of
Ec12-0466 (Figure 3 and Figure S5), again consistent with its outlier position in the core-genome ML
tree.
GEI-2 contains MDR genes (dfrA7, sul2, ebr, strA, strB, mer operon, tetA) as well as the virulence
genes ag43 and yeeV-yeeU pair. This region was detected in all outbreak as well as clade A and B
isolates, but not in any isolates of the historical clade. Syntenic analysis revealed that the structure of
GEI-2 was largely conserved on the intra-clade level, but highly diverse on the inter-clade level
(Figure 3 and Figure S6) supporting the core-genome phylogeny. A large deletion including almost all
of the resistance genes (sul2, strA, strB, mer operon, tetA) occurred in clade-A isolates with the
exception of Ec12-0466, again consistent with its outlier position. In contrast, all MDR genes were
maintained in clade-B isolates except 2009EL-2071, in which the mer operon and tetA were deleted
(Figure S6). This finding suggests a differential antibiotic selection between clade B and the outbreak
clade compared to clade A (except Ec12-0466). The third diverse GEI named GEI-3 mainly contains
the type VI secretion system (T6SS) and an incomplete prophage. A consistent deletion occurred in
the region of the incomplete prophage in clade B isolates as well as in the historical strain 04-8351,
whereas a different deletion within the same region occurred in 55989 (Figure 3).
Chapter 5
110
Figure 3. Genomic comparison of Escherichia coli O104:H4. The core represents the chromosome of TY-2482
(taken as the reference genome and depicted as a black circle) and its GC content (indicated in black) and GC
skew (indicated in purple/green) shown in three circles (in-outside), and the chromosomal position is
numbered in a clockwise direction. Strains were grouped in different colors according to the phylogenetic
results shown in Figure 1. The order of strains followed the direction of the legend from ‘GC Content’ to
‘55989’. The gradients (dark, pale and white) of each color represent the sequence identity (from 100% to 0%)
between samples and reference defined by BLASTn. The prophages (purple) and genomic islands (orange)
identified from reference TY-2482 were labeled by an arc.
Figure 4. Phylogeny of prophages uncovered from Escherichia coli O104:H4 STEC analyzed in this study.
Prophages were marked in different colors according to the phylogenetic results of their hosts shown in Figure
1. Not all analyzed strains are shown. (a) Phylogeny of phage-IV; (b) phylogeny of the Stx2-encoding prophage
(phage-VII). The phage VT2phi_272 (Accession number HQ424691) was used as the outgroup.
The plastic genome structure of STEC O104:H4
111
DISCUSSION
STEC O104:H4 has attained significant public health importance, however, little is known about the
population history of the clone that caused the large outbreak in Germany in 2011. In this study, we
comprehensively investigated the genomes of 23 STEC O104:H4 isolates, including previously
reported outbreak- and non-outbreak-related isolates, in more detail to elucidate their evolutionary
past. In accordance with our findings we propose a model as illustrated in Figure 5. This allows a
more detailed understanding about the steps that have led to the emergence of the STEC O104:H4
outbreak clone (15,18,19). Our model reveals that the STEC O104:H4 population diversified into
multiple lineages, of which two (clade A and B) derived from a recent common ancestor shared by
the outbreak clone. This is the first time that two additional clones that have so far not been
associated with any outbreak have been shown to share close evolutionary relationship with the
2011 outbreak clone. We presume that the three clades may represent the most successful
descendants of STEC O104:H4 to date because of their present abundance among ascertained clinical
cases. Noteworthy, we identified eight canonical SNPs within coding regions which are able to
unambiguously distinguish all of the 2011 outbreak isolates from the remaining population. This
finding may help to setup clinical diagnostics tools (i.e. real-time PCR) to support early identification
and appropriate infection control and public health measures. Additionally, seven of the eight SNPs
are non-synonymous, mostly located within genes whose products involved in metabolisms (i.e.
ferredoxin-type protein NapG, uracil phosphoribosyltransferase protein and rhamnulokinase).
Further work would be worth investigating the role of these SNPs with respect to positive selection
of the outbreak clone.
Our model also describes a set of lineage-specific epidemiological markers of STEC O104:H4, some of
them show the hallmarks of genomic adaptation. These findings may be helpful to identify the
driving forces that lead to the diversification of STEC O104:H4. One of the obvious diversities is
antibiotics, and this is supported by two observations. First, an ESBL-producing (blaCTX-M-15) plasmid
pTY1 was exclusively detected in the 2011 outbreak isolates and appeared to be relatively stable, i.e.
there are no reports of pTY1 loss yet to our best knowledge. This is consistent with previous studies
(15, 18). Second, GEI-2 was only found in the three major clades (outbreak, A and B), and the region
containing MDR genes within GEI-2 was lost in the clade A isolates (except isolate Ec12-0466). Both
findings suggest that diverse antibiotic selective pressures may have shaped the evolution of STEC
O104:H4. However, one may argue the influence of antibiotic-driven evolution of STEC as
conventional guidelines discourages the use of antibiotics in the management of clinical cases of
STEC infections (20), but it cannot be ignored that many patients with diarrhea receive empirical
antibiotic therapy by their physicians (4). Another driving force can be related to the niche
Chapter 5
112
competition. Isolates of clade A lost multiple microcin-biosynthesis genes within GEI-1. Microcin is a
bactericidal antibiotic involved in competitive exclusion of other bacteria to form nutritionally
restricted niches. Therefore, any habitat switch would have impact on the divergence between clade
A and the other clades.
Figure 5. The evolutionary model for STEC O104:H4. Populations are grouped in different colors according to
the phylogenetic results shown in Fig. 1. The gray boxes with dashed outline represent hypothetical
populations not identified yet. The symbol “+” and “-” represents gain and loss of MGEs, respectively. Not all
events of MGE changes observed in this study are shown here.
Although some other lineage-specific MGEs indicated in our model cannot directly be related to any
ecological constraints, valuable information can still be extracted from our findings. For instance, it is
unclear whether the replacement of pAA (AAF/III) by pTY2 (AAF/I) occurring in the three major clades
confers any fitness, however, our observations indicate that pTY2 may not be crucial in adhesion /
colonization of the outbreak clone i.e. the outbreak isolate 9Z lost the globe regulator AggR. In fact,
frequent loss of pTY2 during infection progression was detected previously from other outbreak
isolates (21,22). This differs markedly from the pAA of prototypical EAEC, which is so stable that an 1-
kb fragment of this plasmid has been widely used as a sensitive and specific diagnostic marker
(23).Notably, a more recent study suggested that the pTY2 plasmid may be dispensable for the
The plastic genome structure of STEC O104:H4
113
adhesion / colonization ability of STEC O104:H4 in vivo. This is based on observations that the overall
abundance and intestinal distribution of the plasmid-devoid strains were indistinguishable from the
wild-type strain in a rabbit infection model (24). However, we cannot exclude that in humans the
plasmid may be lost in the course of the infection but may still be crucial for during the early phase of
an infection. The cryptic plasmid pTY3 is one of the smallest plasmids found in E. coli, containing only
a gene repA encoding the plasmid replication protein. Our model suggests that the hypothetical
progenitor of the three major clades acquired the small cryptic plasmid after splitting from the
historical clade (Figure 5). The origin of the small cryptic plasmid is difficult to be tracked due to its
broad host range (Table S1; (25)). A recent study suggests that a pTY3-like plasmid pSERB2 (GenBank
accession number: NG_036178) frequently co-transforms with an Incl1 pHUSEC-1-like plasmid
(GenBank accession number: NG_035985) carrying a type IV pilus system (26). Both plasmids have
been associated with an atypical EAEC strain and are necessary for adherence to abiotic surfaces
required for fully mature biofilm formation in those strains (26). We thus speculate that pTY3 might
co-transform with pTY2 acquired by STEC O104:H4, which may contribute to the pathogenicity of
STEC O104:H4.
It is unclear what caused the replacement of Stx2-encoding prophages in clade B, which has also
previously been reported for the two Georgia isolates 2009EL-2050 and 2009EL-2071 (27). However,
a recent study demonstrated experimentally that Stx2-encoding prophages from the 2011 German
outbreak strains are completely identical to that of HUSEC041, but distinct to those from the two
Georgia isolates with respect to host range and superinfection susceptibility (28). Beutin et al. (2012)
found that the replaced Stx2-encoding phage can only infect the Georgia isolates but not others.
Together with the core-genome (Figure 1) and Stx2-encoding phage (Figure 4B) phylogeny shown in
this study, we suspect that a replacement event of the Stx2-encoding prophages would have
occurred within clade B after the lineage split. Whether the other lineage-specific events, like the
replacement of Phage-IV and acquirement of pTY3 in outbreak clone as well as the loss of Phage-V by
clade B, were caused by additional ecological forces remains to be resolved.
When accepting our model one should be aware of the fact that the genome of STEC O104:H4 is
rather dynamic. Moreover, certain isolates may be able to evolve much more rapidly than other
isolates within the same clade. For example, the accessory genome (i.e. GEI-1, GEI-2 and Phage-IV) of
the clade-A isolate Ec12-0466 was closer related to the outbreak isolates whereas its core genome is
more closely related to other clade-A isolates. Rapid gain or loss of plasmids occurred in the three
2013 Dutch isolates even though they are clonal. Additionally, epidemiological data suggest that
isolates of clade A and B are circulating in different regions, which may result from overlooked
transmissions events between these regions, i.e. by travelling and trading.
Chapter 5
114
Our investigation revealed that the genome of STEC O104:H4 is rather mosaic in nature mainly due
to the frequent loss or gain of MGEs on very short evolutionary time scales. We also suggest multiple
ecological constraints that may have shaped the phylogeny of STEC O104:H4. Our findings further
support the hypothesis that STEC O104:H4 might have evolved to public health importance from
EAEC by exploiting a rather specific cocktail of MGEs (29). This highlights the possibility that further
outbreaks could be triggered if strains attain novel combinations of MGEs. Therefore, molecular
surveillance on STEC O104:H4 is necessary for early identifying of the putative outbreak strains,
especially in regions where they are frequently recovered from patients.
ACKNOWLEDGEMENTS
Barbara Kesztyüs and Wim Niessen are thanked for their helps to obtain the Groningen isolates and
Erwin Raangs for help in obtaining the NGS-data.
REFERENCES
1. Frank C, Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, Bernard H, Fruth A, Prager
R, Spode A, Wadl M, Zoufaly A, Jordan S, Kemper MJ, Follin P, Müller L, King LA, Rosner B,
Buchholz U, Stark K, Krause G; HUS Investigation Team. 2011. Epidemic profile of Shiga-toxin-
producing Escherichia coli O104:H4 outbreak in Germany. N Engl J Med 365:1771-80
2. Rohde H, Qin J, Cui Y, Li D, Loman NJ, Hentschke M, Chen W, Pu F, Peng Y, Li J, Xi F, Li S, Li Y,
Zhang Z, Yang X, Zhao M, Wang P, Guan Y, Cen Z, Zhao X, Christner M, Kobbe R, Loos S, Oh J,
Yang L, Danchin A, Gao GF, Song Y, Li Y, Yang H, Wang J, Xu J, Pallen MJ, Wang J, Aepfelbacher
M, Yang R, E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium. 2011. Open-source
genomic analysis of Shiga-toxin-producing E. coli O104:H4. N Engl J Med 365:718-724.
3. Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth, A, Bauwens A, Peters G, Karch H. 2011.
Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome
in Germany, 2011: A microbiological study. Lancet Infect Dis 11:671–676.
4. Ishii Y, Kimura S, Alba J, Shiroto K, Otsuka M, Hashizume N, Tamura K, Yamaguchi K. 2005.
Extended-spectrum beta-lactamase-producing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a
new concern. J Clin Microbiol 43:1072-5.
5. Rasko DA, Webster DR, Sahl JW, Bashir A, Boisen N, Scheutz F, Paxinos EE, Sebra R, Chin CS,
Iliopoulos D, Klammer A, Peluso P, Lee L, Kislyuk AO, Bullard J, Kasarskis A, Wang S, Eid J, Rank
D, Redman JC, Steyert SR, Frimodt-Møller J, Struve C, Petersen AM, Krogfelt KA, Nataro JP,
Schadt EE, Waldor MK. 2011. Origins of the E. coli strain causing an outbreak of hemolytic-uremic
syndrome in Germany. N Engl J Med 365:709–717.
6. Brzuszkiewicz E, Thürmer A, Schuldes J, Leimbach A, Liesegang H, Meyer FD, Boelter J, Petersen
H, Gottschalk G, Daniel R. 2011. Genome sequence analyses of two isolates from the recent Escherichia
The plastic genome structure of STEC O104:H4
115
coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic
Escherichia coli (EAHEC). Arch Microbiol 193:883–891.
7. Grad YH, Lipsitch M, Feldgarden M, Arachchi HM, Cerqueira GC, FitzGerald M, Godfrey P, Haas
BJ, Murphy CI, Russ C, Sykes S, Walker BJ, Wortman JR, Young S, Zeng Q, Abouelleil A,
Bochicchio J, Chauvin S, Desmet T, Gujja S, McCowan C, Montmayeur A, Steelman S, Frimodt-
Møller J, Petersen AM, Struve C, Krogfelt KA, Bingen E, Weill FX, Lander ES, Nusbaum C, Birren
BW, Hung DT, Hanage WP. 2012. Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in
Europe, 2011. Proc Natl Acad Sci 109:3065–3070.
8. Assefa S, Keane TM, Otto TD, Newbold C, Berriman M. 2009. ABACAS: algorithm-based automatic
contiguation of assembled sequences. Bioinformatics 25:1968-9.
9. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM,
Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D,
Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O.
2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75.
10. Darling AE, Mau B, Perna NT. 2010. progressiveMauve: multiple genome alignment with gene gain, loss
and rearrangement. PLoS One 5:e11147.
11. Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. Nucleic
Acids Res 39:W347-52.
12. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. 2011. BLAST Ring Image Generator (BRIG):
simple prokaryote genome comparisons. BMC Genomics 12:402.
13. Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier
C, Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C,
Karoui ME, Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguénec C, Lescat M,
Mangenot S, Martinez-Jéhanne V, Matic I, Nassif X, Oztas S, Petit MA, Pichon C, Rouy Z, Ruf CS,
Schneider D, Tourret J, Vacherie B, Vallenet D, Médigue C, Rocha EP, Denamur E. 2009. Organised
genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 5.
14. Künne C, Billion A, Mshana SE, Schmiedel J, Domann E, Hossain H, Hain T, Imirzalioglu C,
Chakraborty T. 2012. Complete sequences of plasmids from the hemolytic-uremic syndrome-associated
Escherichia coli strain HUSEC41. J Bacteriol 194:532-3.
15. Ahmed SA, Awosika J, Baldwin C, Bishop-Lilly K. a, Biswas B, Broomall S, Chain PS, Chertkov O,
Chokoshvili O, Coyne S, Davenport K, Detter JC, Dorman W, Erkkila TH, Folster JP, Frey KG,
George M, Gleasner C, Henry M, Hill KK, Hubbard K, Insalaco J, Johnson S, Kitzmiller A, Krepps
M, Lo CC, Luu T, McNew LA, Minogue T, Munk CA, Osborne B, Patel M, Reitenga KG, Rosenzweig
CN, Shea A, Shen X, Strockbine N, Tarr C, Teshima H, van Gieson E, Verratti K, Wolcott M, Xie G,
Sozhamannan S, Gibbons HS; Threat Characterization Consortium. 2012. Genomic Comparison of
Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity,
Including Shiga Toxin Encoding Phage stx2. PLoS One 7.
16. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands
of taxa and mixed models. Bioinformatics 22:2688-90.
17. Guy L, Jernberg C, Ivarsson S, Hedenström I, Engstrand L, Andersson SG. 2012. Genomic diversity
of the 2011 European outbreaks of Escherichia coli O104:H4. Proc Natl Acad Sci U S A 109:E3627-8.
18. Grad YH, Godfrey P, Cerquiera GC, Mariani-Kurkdjian P, Gouali M, Bingen E, Shea TP, Haas BJ,
Griggs A, Young S, Zeng Q, Lipsitch M, Waldor MK, Weill FX, Wortman JR, Hanage WP. 2013.
Comparative genomics of recent Shiga toxin-producing Escherichia coli O104:H4: short-term evolution of
an emerging pathogen. MBio 4:1-10.
Chapter 5
116
19. Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R,
Ji Y, Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM,
Moore RL, Guenther S, Rothberg JM, Karch H. 2011. Prospective genomic characterization of the
German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing
technology. PLoS One 6:e22751.
20. Paton JC, Paton AW. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli
infections. Clin Microbiol Rev 11:450-79.
21. Zangari T, Melton-Celsa AR, Panda A, Boisen N, Smith MA, Tatarov I, De Tolla LJ, Nataro JP,
O'Brien AD. 2013. Virulence of the Shiga toxin type 2-expressing Escherichia coli O104:H4 German
outbreak isolate in two animal models. Infect Immun 81:1562-74.
22. Zhang W, Bielaszewska M, Kunsmann L, Mellmann A, Bauwens A, Köck R, Kossow A, Anders A,
Gatermann S, Karch H. 2013. Lability of the pAA Virulence Plasmid in Escherichia coli O104:H4:
Implications for Virulence in Humans. PLoS One 8:e66717.
23. Baudry B, Savarino SJ, Vial P, Kaper JB, Levine MM. 1990. A sensitive and specific DNA probe to
identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis 161:1249-
51.
24. Munera D, Ritchie JM, Hatzios SK, Bronson R, Fang G, Schadt EE, Davis BM, Waldor MK. 2014.
Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin-producing Escherichia coli
O104:H4. Nat Commun 5:3080.
25. Srivastava P, Nath N, Deb JK. 2006. Characterization of broad host range cryptic plasmid pCR1 from
Corynebacterium renale. Plasmid 56:24-34.
26. Dudley EG, Abe C, Ghigo JM, Latour-Lambert P, Hormazabal JC, Nataro JP. 2006. An IncI1 plasmid
contributes to the adherence of the atypical enteroaggregative Escherichia coli strain C1096 to cultured cells
and abiotic surfaces. Infect Immun 74:2102-14.
27. Guy L, Jernberg C, Arvén Norling J, Ivarsson S., Hedenström I, Melefors Ö, Liljedahl U, Engstrand
L, Andersson SG. 2013. Adaptive Mutations and Replacements of Virulence Traits in the Escherichia coli
O104:H4 Outbreak Population. PLoS One 8:1–13.
28. Beutin L, Hammerl JA, Strauch E, Reetz J, Dieckmann R, Kelner-Burgos Y, Martin A, Miko A,
Strockbine NA, Lindstedt BA, Horn D, Monse H, Huettel B, Müller I, Stüber K, Reinhardt R. 2012.
Spread of a distinct Stx2-encoding phage prototype amongEscherichia coli O104:H4 strains from outbreaks
in Germany, Norway, and Georgia. J Virol 86:10444-55.
29. Baquero F, Tobes R. Bloody coli: a gene cocktail in Escherichia coli O104:H4. 2013. MBio 4:e00066-13.
30. Jourdan-da Silva N, Watrin M, Weill FX, King LA, Gouali M, Mailles A, van Cauteren D, Bataille M,
Guettier S, Castrale C, Henry P, Mariani P, Vaillant V, de Valk H. 2012. Outbreak of haemolytic
uraemic syndrome due to Shiga toxin-producing Escherichia coli O104:H4 among French tourists returning
from Turkey, September 2011. Euro Surveill 17.
31. Scheutz F, Nielsen EM, Frimodt-Møller J, Boisen N, Morabito S, Tozzoli R, Nataro JP, Caprioli A. 2011. Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4
strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. Euro Surveill
16.
32. Monecke S, Mariani-Kurkdjian P, Bingen E, Weill FX, Balière C, Slickers P, Ehricht R. 2011.
Presence of enterohemorrhagic Escherichia coli ST678/O104:H4 in France prior to 2011. Appl Environ
Microbiol 77:8784-6
The plastic genome structure of STEC O104:H4
117
33. Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A, Middendorf B, Harmsen D, Schmidt
MA, Karch H. 2008. Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic
Escherichia coli. Emerg Infect Dis 14:1287-90.
Supplementary Figures
Figure S1. Comparison of the region with agg3 operon on the pTY2 plasmid of TY-2482 and 9Z. Partial components of original pTY2 (TY-2482) are shown. Open-reading frames are shown by blue arrows. The gradients (dark to pale) in the alignment region represent the percentage of sequence identity between samples as defined by BLASTn.
Figure S2. Comparison of the pHUSEC41-1-like plasmid of 381-1and 381-3. The original pHUSEC41-1 from strain HUSEC041 is shown as reference. Open reading frames are indicated by blue arrows. The gradients (dark to pale) of alignment region represent the percentage of sequence identity between samples defined by BLASTn.
Chapter 5
118
Figure S3. Comparison of phage-IV in Escherichia coli O104:H4 strains. The sequence of 55989 shown here represents the conserved flanking region of phage-IV in all analyzed strains. Open reading frames are indicated by blue arrows. The gradients (dark to pale) of alignment region represent the percentage of sequence identity between samples defined by BLASTn. Please note that not all analyzed strains are shown in this figure.
Figure S4. Comparison of Stx2-encoding phage (phage-VII) in Escherichia coli O104:H4 strains. The sequence of 55989 shown here represents the conserved flanking region of phage-IV in all analyzed strains. Open reading frames are indicated by blue arrows. The gradients (dark to pale) of alignment region represent the percentage of sequence identity between samples defined by BLASTn. One of gene replacements between clade A and B is highlighted in red. Please note that not all analyzed strains are shown in this figure.
Figure S5. Comparison of GEI-1 in Escherichia coli O104:H4 strains. The sequence of 55989 shown here represents the conserved flanking region of phage-IV in all analyzed strains. Open reading frames are indicated by blue arrows. The gradients (dark to pale) of alignment region represent the percentage of sequence identity between samples defined by BLASTn. Please note that not all analyzed strains are shown in this figure.
The plastic genome structure of STEC O104:H4
119
Figure S6. Comparison of GEI-2 in Escherichia coli O104:H4 strains. The sequence of 55989 shown here represents the conserved flanking region of phage-IV in all analysed strains. Open reading frames are indicated by blue arrows. The gradients (dark to pale) of alignment region represent the percentage of sequence identity between samples defined by BLASTn. Please note that not all analysed strains are shown in this figure.
Supplementary Table
Table S1. The homologies of pTY3 detected by BLASTn in GenBank
Plasmid ID Accession nr. Cover Identity Source Reference
pCE10D CP003038 100% 99% E. coli O7:K1 1
pO26-S1 EU999782 100% 99% E. coli O26 2
pEC299-1 NG_041600 100% 97% E. coli ST131 3
pSF301-1 JF813186 99% 99% Shigella flexneri strain 2a 301 -
pJJ1886_1 NC_022661 97% 94% E. coli ST131 4
pCR1 X99132 95% 95% Corynebacterium renale 5
Strain ID FHI63 LM996420 100% 97% E. coli O145 -
FHI30 LM995872 100% 96% E. coli O113 -
FHI65 LM996502 99% 99% E. coli O146 -
FHI59 LK999963 99% 95% E. coli O91 -
FHI97 LM997177 97% 97% E. coli O103 -
Note: Only the plasmids and strains with clear information and high similarity to pTY3 (> 90% identity) are listed here.
References for Table S1
1. Lu S, Zhang X, Zhu Y, Kim KS, Yang J, Jin Q. Complete genome sequence of the neonatal-meningitis-associated
Escherichia coli strain CE10. J Bacteriol 2011; 193:7005.
2. Fratamico PM, Yan X, Caprioli A, et al. The complete DNA sequence and analysis of the virulence plasmid and of five
additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30. Int J Med Microbiol 2011;
301:192-203.
3. Brolund A1, Franzén O, Melefors O, Tegmark-Wisell K, Sandegren L. Plasmidome-analysis of ESBL-producing
escherichia coli using conventional typing and high-throughput sequencing. PLoS One 2013; 8: e65793.
Chapter 5
120
4. Andersen PS, Stegger M, Aziz M, et al. Complete Genome Sequence of the Epidemic and Highly Virulent CTX-M-15-
Producing H30-Rx Subclone of Escherichia coli ST131. Genome Announc 2013; 1: pii: e00988-13.
5. Srivastava P, Nath N, Deb JK. Characterization of broad host range cryptic plasmid pCR1 from Corynebacterium renale.
Plasmid 2006; 56: 24-34.
121
CHAPTER 6
Is Shiga Toxin-Negative Escherichia coli O157:H7
Enteropathogenic or Enterohemorrhagic Escherichia
coli? Comprehensive Molecular Analysis Using Whole
Genome Sequencing
Mithila Ferdous 1, Kai Zhou 1, Alexander Mellmann 2, Stefano Morabito 3, Peter D.Croughs4,
Richard F. de Boer 5, Anna M.D. Kooistra-Smid 1, 5, John W.A. Rossen1 # * and Alexander W.
Friedrich 1 #
1Department of Medical Microbiology, University of Groningen, University Medical Center
Groningen, Groningen, the Netherlands. 2Institute of Hygiene, University Hospital of Münster, Münster, Germany. 3Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy. 4Star-MDC, Rotterdam, the Netherlands. 5Certe Laboratory for Infectious Diseases, Groningen, the Netherlands. #These authors contributed equally
Keywords
Shiga Toxin-Producing Escherichia coli (STEC), Enteropathogenic Escherichia coli (EPEC),
Enterohaemorrhagic Escherichia coli (EHEC), Whole genome sequencing, Stx-converting
bacteriophages, Molecular typing, Phylogenetic analysis
J Clin Microbiol (2015) 53:3530 –3538.
Chapter 6
122
ABSTRACT
The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is
related to numerous virulence factors, most notably stx gene encoding Shiga toxin (Stx), carried by a
bacteriophage. Loss of the Stx encoding bacteriophage may occur during infection or culturing of the
strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (non-motile)
isolates from patients with gastrointestinal complaints. Isolates were characterized by whole genome
sequencing (WGS) and their virulence properties and phylogenetic relationship were determined.
Because of the presence of the eae gene but lack of the bfpA gene, the stx-negative isolates were
considered as atypical enteropathogenic E. coli (aEPEC). However, they had similar phenotypic
characteristics as the Shiga toxin producing E. coli (STEC) isolates and belonged to the same sequence
type, ST11. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of
enterocyte effacement region and plasmids. Core-genome phylogenetic analysis using a gene-by-
gene typing approach showed that the sorbitol fermenting (SF) stx-negative isolates clustered
together with an SF STEC isolate and one non-sorbitol fermenting (NSF) stx-negative isolate clustered
together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have
lost the Stx phage or to be a progenitor of STEC O157:H7/NM. As detection of STEC infections is often
based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine
laboratories. Therefore, an improved diagnostic approach is required to manage identification,
treatment strategy, and prevention of transmission of these potentially pathogenic strains.
Comparison of stx-Positive and -Negative E. coli O157:H7
123
INTRODUCTION
Escherichia coli of serotype O157:H7 was first recognized in 1982 as a human pathogen associated
with outbreaks of bloody diarrhea in the US and is now considered as a major cause of foodborne
infections (1, 2). The virulence of E. coli O157:H7 depends on the presence of a number of mobile
genetic elements (MGE) such as Shiga toxin (Stx)-converting bacteriophages carrying different genes
encoding Stx1 and Stx2, the virulence plasmid pO157, the locus of enterocyte effacement (LEE), O
islands, an arginine translocation system and various adhesion factors (3). In Stx producing E. coli
(STEC), Stx is thought to be responsible for the most severe form of the infection causing the life
threatening hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (4). Strains causing these
clinical symptoms are also known as enterohaemorrhagic E. coli (EHEC) (5). The genes encoding Stxs
(stx1 and stx2) are found in lysogenic lambdoid bacteriophages (6), which can integrate into the host
chromosome via specific insertion sites. Integration sites for stx2-converting phages in STEC O157:H7
include wrbA, argW, sbcB, and yecE, whereas stx1-converting phages integrate in the yehV region (7-
11).
STEC O157:H7 strains generally do not ferment sorbitol (NSF) and this feature is widely used to
identify these pathogenic strains. Nevertheless, sorbitol-fermenting (SF) STEC O157:NM (non-motile)
strains as an emerging and important pathogen in Europe have been isolated from patients with HUS
and diarrhea (12). Both SF and NSF O157:H7/NM strains are thought to have evolved from a common
non-pathogenic ancestor of serotype O55:H7 following the modification of the O-antigen genes
cluster and the acquisition of a number of virulence-associated mobile genetic elements (MGE) via
horizontal gene transfers (13). The most current and accepted evolutionary model proposes that E.
coli O157:H7 lost the O55 rfb-gnd gene cluster and acquired the Stx2 bacteriophage and the O157
rfb-gnd gene cluster. Subsequently, SF stx2-producing E. coli O157 separated from this lineage. After
the diversification of the two lineages, E. coli O157:H7 acquired stx1 via acquisition of the
bacteriophage containing the stx1 gene, and lost the ability to ferment sorbitol, while the sorbitol-
fermenting stx2-producing E. coli O157 lost its motility and evolved into non-motile E. coli O157:NM
(14, 15). Most E. coli O157 isolates produce a large outer membrane protein intimin (encoded by the
eae gene) which is the genetic determinant of the formation of attaching and effacing (A/E) lesion, a
central mechanism in the pathogenesis of enteropathogenic E. coli (EPEC) (16). Strains containing eae
but not stx are categorized as EPEC (17).
As described in previous studies, stx-negative E. coli O157:H7/NM isolates were obtained from
patients with HUS and diarrhoea. It was assumed, especially for the HUS cases that these patients
were originally infected with an EHEC strain and that excision of the Stx bacteriophage occurred
during the infection. Those stx-negative O157:H7/NM appeared to be closely related with stx-
Chapter 6
124
positive O157:H7 isolates as determined by conventional molecular typing methods (18-21). In this
study, whole genome sequencing (WGS) was used to get a more detailed molecular characterization
of stx-negative E. coli O157:H7/NM isolates and to reveal their genetic relationship with stx-positive
O157:H7 isolates obtained from patients with gastrointestinal complaints.
MATERIALS AND METHODS
Selection of isolates for the study.
Fecal samples were collected from patients with gastrointestinal complaints in the regions of
Groningen and Rotterdam during the period April 2013-March 2014, as part of a large multicenter
study (STEC-ID-Net, unpublished data). Samples were screened for the presence of stx1, stx2 and
escV (used as an alternative marker for LEE instead of eae gene), and O-serogroup determination
(O26, O103, O104, O111, O121, O145 and O157) by real-time PCR as described previously (22). This
resulted in the collection of 34 E. coli O157 isolates (with or without stx) from 34 different patients.
PCR for the detection of the fliC-H7 gene was performed as described before (23) and only the
isolates positive for fliC-H7 were then subjected to WGS.
The publically available genomes of E. coli O157:H7 strains Sakai, EDL933 and SS52, E. coli O157:H45
strain C639_08, E. coli O127:H6 strain E2348/69 and E. coli O55:H7 CB9615 were also included in the
comparative analysis. Moreover, as no complete genomes of SF STEC O157:NM and stx-negative
O157:H7/NM were available in the NCBI database when the study was performed, we have
sequenced the genome of one SF STEC O157:NM (E09/10) and one stx-negative O157:NM (E09/224)
and included them as control strains in all the analyses. The information on the isolates used in this
study is presented in Table S1 in the supplemental material.
Phenotypic Characterization.
Sorbitol fermentation was determined using CT-SMAC plates (Sorbitol MacConkey agar with Cefixime
and Tellurite) and motility was tested using motility test medium with triphenyltetrazolium chloride
(Mediaproducts BV, Groningen, the Netherlands). The production of beta-glucuronidase and urease
were checked using MacConkey II agar with 4-methylumbelliferryl-β-D-glucuronide (MUG) (BD
Diagnostics, Breda, the Netherlands) and urea- triple sugar iron (TSI) agar (Mediaproducts BV,
Groningen, the Netherlands) respectively. The O and H serotypes of the isolates were determined by
seroagglutination performed in the National Institute for Public Health and the Environment (RIVM,
Bilthoven, the Netherlands).
Comparison of stx-Positive and -Negative E. coli O157:H7
125
DNA Extraction and WGS.
DNA was extracted using the UltraClean® microbial DNA isolation kit (MO Bio Laboratories, Carlsbad,
CA, USA) according to the manufacturer’s protocol. A DNA library was prepared using the Nextera XT
kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions and then run on a
Miseq instrument (Illumina) for generating paired-end 250-bp reads aiming at a coverage of at least
60 fold.
Data analysis.
De novo assembly was performed using CLC Genomics Workbench v7.0.3 (CLC bio A/S, Aarhus,
Denmark) after quality trimming (Qs, ≥ 28) with optimal word sizes based on the maximum N50
value. Annotation was performed by uploading the assembled genome on the RAST server version
2.0 (24). The sequence type (ST) was identified by uploading the assembled genomes to the MLST
(multilocus sequence type) server (version 1.7) (25) and the virulence genes and stx subtypes were
determined by virulence finder 1.2 (26). The serogenotype of the isolates was determined using the
CGE SeroTypeFinder tool (27).
Comparison of the LEE island, plasmids and other genes.
To analyze the sequence homology of the LEE pathogenicity island in the isolates, the contigs of each
sample were subjected to a BLAST search against the LEE region of E. coli O157:H7 strain 71074
(accession no GQ338312) as reference and plotted by BLAST Ring Image Generator (BRIG) (28). To
identify plasmids pO157 (accession no. NC_002128) and pOsak1 (Accession no. AB011548) of the E.
coli strain Sakai and plasmid pSFO157 (accession no. NC_009602) of the E. coli O157:NM strain
3072/96 were used as references for BLAST analyses. For identifying proposed marker genes for
differentiating NSF STEC O157:H7/NM from SF STEC O157:NM, including the complete cdt cluster,
the complete sequence of efa1, the tellurite resistance- and adherence-conferring pathogenicity
island (TAI), and the urease gene cluster (29), contigs were either blasted using blastn
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) or mapped by CLC Genomics Workbench v7.0.3 using default
settings against a reference sequence artificially generated by concatenating sequences of the
marker genes.
Phylogenetic analysis.
To determine the phylogenetic relationship of the isolates, a gene-by-gene typing approach was
performed using SeqSphere+ v1.0 (Ridom GmbH, Münster, Germany). Briefly, an in-house defined
MLST+ scheme was developed using the genome of E. coli O157:H7 strain Sakai as reference genome
to extract open reading frames (ORFs) from the genome of each isolate by SeqSphere+. Only the
Chapter 6
126
ORFs without premature stop codon and ambiguous nucleotides from contigs of assembled genomes
were included. The genes shared by the genomes of all isolates analyzed were defined as the core
genome for phylogenetic analysis (30, 31). A Neighbor Joining (NJ) tree was constructed based on a
distance matrix among the isolates depending on the core genomes of all isolates.
Analyzing phage integration sites.
The most common and well described Stx phage integration sites for STEC O157:H7/NM were
analyzed in our stx-negative isolates to reveal if any phage was occupying these sites or that they
were available for future integration of a phage including one that contains stx genes. Five
integration sites were studied: yehV (for the Stx1 phage), wrbA and argW (for the Stx2a phage), sbcB
(for the Stx2c phage) and yecE (for the Stx2a phage of SF STEC) (11). These five loci were identified in
the contigs and the adjacent regions were extracted and the presence of phage integrases was
detected using the blastn algorithm.
Accession numbers.
This whole-genome shotgun project has been deposited in NCBI under the bioproject PRJNA285020.
The GenBank accession numbers of the isolates analyzed in this study are LDOZ00000000,
LFUA00000000, LFUB00000000, LGAZ00000000, LFUH00000000, LGBA00000000, LGBB00000000,
LGBC00000000, LGBD00000000, LGBE00000000, LGBF00000000, LGBG00000000, LGBH00000000,
LGBQ00000000, LGBI00000000, LGBJ00000000, LGBK00000000, LGBL00000000, LGBM00000000,
LGBN00000000, LGBO00000000, and LGBP00000000.
RESULTS
Selection of Isolates.
Among 34 E. coli O157 isolates initially obtained in this study, 16 were STEC (all were fliC- H7 positive)
and 18 were categorized as EPEC (as they were stx negative but eae positive). Among the 18 EPEC
isolates, four O157:H7, eight O157:H16, two O157:H26 and four O157:H39 were identified. For the
subsequent comparative study, 20 isolates positive for fliC-H7 (16 STEC O157:H7 and 4 EPEC
O157:H7/NM) were used.
Phenotype.
The phenotypic characteristics of the 20 O157:H7 isolates of this study and the two control strains
are shown in Table 1. The 16 STEC O157:H7 isolates did not ferment sorbitol (NSF) and were beta-
Comparison of stx-Positive and -Negative E. coli O157:H7
127
glucuronidase negative whereas among the four EPEC isolates one (EPEC 287) was NSF and beta-
glucuronidase negative. The remaining three (EPEC 393, EPEC 1572, EPEC 1669) and the control
strain (E09/224) were sorbitol fermenting (SF) and beta-glucuronidase positive as the SF STEC control
strain E09/10 (Table S1). Among the NSF STEC isolates, six of the sixteen isolates were non-motile as
were all SF isolates. All isolates of this study were urease negative.
Molecular typing and presence of virulence genes.
stx-subtyping of STEC isolates revealed eight (50%) isolates with stx1a and stx2c subtypes, two
(12.5%) with stx1a and stx2a subtypes and six (37.5%) with stx2c subtype only. All isolates contained
the O serotyping gene wzx-O157 and the flagellar gene fliC-H7 and eae (type gamma) and were
assigned to ST11 (Table 1). The virulence profiles of the isolates are shown in Table 2. All NSF STEC
isolates had identical virulence profiles with exception of isolate STEC 1109 that contained the cdt-v
gene encoding for cytolethal distending toxin. Some of the virulence genes, like serine protease
autotransporter gene espP, toxin encoding gene toxB, catalase peroxidase encoding gene katP,
tellurite resistance and adherence conferring island (TAI) and the urease gene cluster were only
present in NSF STEC and in the only NSF EPEC isolate but not in any SF isolates. On the other hand,
sfpA, the complete efa1 and cdt gene cluster were only present in SF isolates. The SF EPEC isolates
carried almost all the virulence genes carried by the German SF STEC O157:NM isolate with the
exception of the tccP gene (encoding tir cytoskeletal coupling protein) that was present only in SF
EPEC isolates.
Table 1. Phenotypic and molecular characteristics of the isolates.
Pathotype and serotype
a
Number of isolates (n)
Beta glucuronidase activity
Urease production
Motility (no. of isolate)
Stx subtype (no. of isolate)
Intimin type
b
Sequence type by MLST
NSF stx positive O157:H7/NM
16 - - +(10) stx1a+stx2c (8) stx1a+stx2a(2) stx2c(6)
gamma 11
NSF stx negative O157:NM
1 - - - NAc gamma 11
SF stx positive O157:NM
1d + -
- stx2a gamma 11
SF stx negative O157:NM
4e + - - NA gamma 11
a NSF, non-sorbitol fermenting; SF, sorbitol fermenting; NM, non-motile. b The type of the eae gene was determined from WGS data using blastn. c NA, not applicable. d This isolate was obtained from Germany and used as a control strain for SF STEC O157:NM isolates. e One of these four isolates was obtained from Germany and used as a control strain for the stx-negative O157:NM isolates.
Chapter 6
128
LEE pathogenicity island.
All the STEC isolates contained an LEE region highly similar to the LEE of STEC O157:H7 strain 71074,
used as a reference. However, some NSF STEC isolates lacked two genes encoding the mobile
element proteins orfA and orfB located in the insertion sequence IS911. Three STEC isolates (STEC
2257, STEC 2820, and STEC 2821) did not possess the intL gene, which is known to encode an
integrase of the putative prophage 933L carried in the LEEs of other STEC O157:H7 isolates. The
sequences of the LEEs of our NSF and SF EPEC isolates were more similar to those of NSF and SF
STEC, respectively, than to the LEE of EPEC reference genomes E2348/69 and C639_08 (Figure. 1).
Figure 1. Comparison of LEE pathogenicity islands, showing a BLAST comparison of STEC and EPEC isolates,
depicted by each ring, against the reference LEE sequence (core black circle). The color of the rings represents
sequence identity on a sliding scale; the more gray the ring is, the lower the percent identity. Different colors of
the rings represent different groups of isolates. The colors of different groups as well as the order of the rings
for each isolate (from inner to outer) with the color gradient for sequence identity are shown at the right.
Plasmids.
All the NSF isolates analyzed (including EPEC 287) carried a pO157-like plasmid. No sequence
variation was observed in regions carrying putative virulence genes such as, e.g., genes involved in
the type II secretion system, hemolysins, toxins, and catalase peroxidase in pO157 of NSF STEC.
However, with the exception of the two stx2a-positive isolates (STEC 2112 and STEC 2868), all STEC
Comparison of stx-Positive and -Negative E. coli O157:H7
129
isolates lacked the pO157p35 gene, encoding a reverse transcriptase. Only three STEC isolates (STEC
605, STEC 989, and STEC 1109) harbored the plasmid pOSAK1. Among the EPEC isolates, the NSF one
(EPEC 287) had almost the intact pO157 plasmid, lacking only an intact espP gene. The SF EPEC
isolates contained an almost identical copy of plasmid pSFO157 of SF STEC O157:NM (Figure. 2).
Figure 2. Comparison of plasmids, showing a BLAST comparison of STEC and EPEC isolates, depicted by each ring, against
the reference plasmid composed of three plasmids shown in the outermost ring by three different colors (black, blue, and
orange represent plasmids pO157, pOSKA1, and pSFO157, respectively). The color of the rings represents sequence identity
on a sliding scale; the more gray the ring is, the lower the percent identity. Different colors of the rings represent different
groups of isolates. The colors of different groups as well as the order of the rings for each isolate (from inner to outer) with
the color gradient for sequence identity are shown at the right.
Chapter 6
130
Table 2. Distribution of virulence and other genes among stx-positive and stx-negative O157:H7/NM isolates.
Presence of genes (no. of positive strains)
Adhesins genes Fimbrial genes Secretion system genes
Autotran
sporter
gene Toxins genes Other genes
Pathotype and serotype
eae tir efa1a espB lpfA bfpA sfpA prfB espA espF espJ nleA,
B,C
etpD tccP espP astA ehxA cdtb toxB katP TAI
c Urease
clusterd
NSF STEC O157:H7/NM
(n=16)
+ + - + + - - + + + + + + - + + + + (1) + + + +
NSF EPEC O157:NM
(n=1)
+ + - + + - - + + + + + + - +e + + - + + + +
SF STEC O157:NM
f
(n=1)
+ + + + + - + + + - + + + - - + + + - - - -
SF EPEC O157:NM
(n=4)
+ + + + + - + + + + (1) + + + + - + + (3)g - - - -
aComplete efa1 gene bEncoding cytolethal distending toxin A, B and C subunit cTellurite resistance and adherence-conferring island (TAI) encoding adhesin gene iha and putative tellurite resistance genes tlrA, tlrB, tlrC and tlrD dure gene cluster containing ureA, ureB, ureC, ureD, ureE, ureF and ureG eOnly part of the espP gene was present
f
This strain was used as a control strain for SF STEC gcdt was absent in isolate E09/224
Comparison of stx-Positive and -Negative E. coli O157:H7
131
Phylogenetic analysis.
Core genome phylogenetic analysis was performed to evaluate the evolutionary relationship
between the stx-positive (STEC) and stx-negative (EPEC) O157:H7/NM isolates. In total, 3,005 ORFs
were shared by all isolates analyzed in this study, and these were defined as the core genome for
phylogenetic analysis. This analysis separated EPEC C639_08 and EPEC E2348/69 from the
O157:H7/NM isolates in this study (Fig. 3). The latter isolates formed two separated clusters: SF
isolates (cluster 1) and NSF isolates (cluster 2). Remarkably, in cluster 1, four SF EPEC isolates (EPEC
393, EPEC 1572, EPEC 1669, and E09/224) clustered together with the SF STEC isolate. Cluster 2 (NSF
O157:H7 isolates) could be divided into three subclusters: cluster 2a, containing STEC O157:NM
(nonmotile) isolates together with one NSF EPEC isolate (EPEC 287); cluster 2b, containing six of the
motile STEC O157:H7 isolates; and cluster 2c, containing two stx2a-positive isolates (STEC 2112 and
STEC 2868) clustered closely with two previously described STEC outbreak isolates, Sakai and EDL933
(1, 32). The last subcluster also included two other motile isolates (STEC 1109 and STEC 989) and
STEC O157:H7 strain SS52 (stx2a and stx2c positive), isolated from super shedder cattle (33). Taken
together, the data indicate that the EPEC O157:NM isolates clustered with STEC isolates but not with
EPEC isolates (Fig. 3).
Phage insertion sites.
In the SF EPEC strains (EPEC 393, EPEC 1572, EPEC 1669 and EPEC E09/224) the phage insertion sites
analyzed were intact with the exception of argW that was occupied in isolate EPEC 393 and EPEC
1572. In EPEC 287, although yehV and argW were occupied by phages, the wrbA and sbcB loci
(integration sites for Stx2a and Stx2c phage respectively) were unoccupied. Comparison of the
different integration sites is shown in Figure 4.
Chapter 6
132
Figure 3. Neighbor-joining (NJ) phylogenetic tree of STEC and EPEC isolates. Different isolate groups are
indicated in different colors. The NJ tree was constructed based on a distance matrix among the isolates
depending on their core genomes.
Figure 4. Phage integration sites. Genes surrounding phage integration sites of the isolates are shown in boxes.
A red arrow indicates the presence of a phage integrase adjacent to the integration site. (a) SF STEC isolate
E09/10; the yecE region is occupied by phage. (b) SF EPEC isolate EPEC 1572; the yecE region is unoccupied. (c)
NSF EPEC isolate EPEC 287; the wrbA region is unoccupied. (d) NSF EPEC isolate EPEC 287; the sbcB region is
unoccupied. (e) NSF EPEC isolate EPEC 287; the yehV region occupied by phage.
Comparison of stx-Positive and -Negative E. coli O157:H7
133
DISCUSSION
E. coli O157:H7 is one of the major causes of food-borne illness and represents a considerable public
health concern worldwide (34). This study aimed at determining the phylogenetic relationships and
comparing the virulence factors of stx negative E. coli O157:H7/NM with those of stx positive E. coli
O157:H7 with the highest resolution and greatest possible detail using a WGS approach. E. coli
O157:H7 isolates of this study were obtained from patients with diarrhea and other gastrointestinal
complaints from two different regions in the Netherlands (Groningen and Rotterdam). Isolate E09/10
and E09/224 were sequenced and used as control strains for SF STEC O157:NM and stx-negative
O157:NM isolates, respectively. As the stx-negative isolates were found to be positive for eae but
negative for the bfpA gene, they were considered as atypical EPEC. Notably, the EPEC isolates
belonged to ST11 and contained type gamma intimin which are not typical features of EPEC, but
frequently associated with STEC O157:H7 (35).
All STEC isolates shared a similar virulence pattern. Remarkably, all the typical genes (e.g., ehxA, astA,
lpfA,, katP, etpD, espP, the pathogenicity island TAI, and urease gene cluster) of NSF STEC O157:H7
described previously were present in EPEC 287. In contrast, SF EPEC isolates possessed the sfp gene
cluster, the cdt gene cluster and the complete efa1 gene but lacked the genes toxB, katP, espP
carried on plasmid pO157, the urease gene cluster and the pathogenicity island TAI, which are typical
features of SF STEC O157:NM (18, 29). Subsequent analyses showed that EPEC 287 harbored an
almost identical plasmid pO157 as the STEC isolates with sequence variability mostly in mobile
genetic elements. The SF EPEC isolates contained a plasmid similar to the pSFO157 present in the SF
STEC O157:NM isolate strain 3072/96. Additionally, our NSF and SF EPEC isolates shared an almost
identical LEE pathogenicity island with the STEC isolates containing additional ORFs encoding a
putative prophage normally not present in the LEE of EPEC (36). All these results together suggest
that the EPEC O157:H7/NM isolates investigated, shared a virulence profile similar to STEC isolates
rather than to the virulence profile of EPEC.
The genetic relationship of the O157:H7/NM isolates of this study was confirmed by the phylogenetic
analysis using a gene-by-gene comparison analysis by core genome MLST. The NSF EPEC isolate
clustered together with the non-motile NSF STEC isolates and the three SF EPEC clustered together
with the control strain of SF STEC O157:NM. It has already been described that the non-motile
characteristic of SF STEC O157 is due to a 12 base pair deletion in the master regulator of flagellar
biosynthesis, the flhC gene (37). We also observed this deletion in the same gene in our non-motile
SF EPEC isolates, but not in our non-motile NSF STEC isolates, further strengthening the hypothesis of
their derivation from stx-positive SF O157:NM. Taken together, the results of our analyses provide
further evidence that the NSF and SF EPEC isolates were mostly related to the STEC group, but
Chapter 6
134
lacking the Stx-converting phages and might be referred to as EHEC that lost the Shiga-toxin (EHEC-
LST), as has also been proposed previously (19, 20). Studying in more detail the typical Stx phage
insertion sites (yecE for SF EPEC and wrbA and sbcB for NSF EPEC) revealed that they were
unoccupied in the stx-negative isolates. Such unoccupied regions could be an indication of loss of the
Stx bacteriophage, e.g., in course of infection, or during isolation or subculture (38). Conversely, stx-
negative strains could be progenitors of STEC prepared to acquire one or more Stx-converting phages
(18, 39). In our case, the stx genes were not lost in the laboratory during isolation or subculture as
they were not detected in the feces of the patient. Therefore, the patients may have been infected
with an stx-negative variant which could explain the mild symptoms they displayed.
In routine diagnostic testing, these stx-negative isolates may be missed as most of the
microbiological laboratories depend on the molecular screening of STEC by the detection of the stx
genes only. Screening for several additional genes (including eae, saa, sfpA) in routine diagnostics to
identify these pathogens has already been proposed (18). Moreover, our findings bring into question
if classifying pathogenic E. coli into STEC and EPEC based on detection of the stx and eae gene,
respectively, is reliable enough. Due to integrating and interchanging mobile genetic elements, e.g.,
the Stx converting bacteriophages which could integrate into several E. coli pathogroups, it is
sometimes complicated to precisely define the classification of pathogenic E. coli (40, 41). Obviously,
screening a bunch of accessory virulence genes of STEC would be laborious for routine diagnostics. In
our opinion, screening of at least the fliC-H7, together with the O157 encoding gene may help to
identify EHEC-LST of the most virulent clone of EHEC (O157:H7) as all the fliC-H7 positive isolates
were genetically related to STEC O157:H7 in this study. Our results are consistent with the finding
that the proportion of stx-negative variants among SF O157:NM isolates is generally higher than
among NSF O157:H7 (20, 42). Nevertheless, the number of isolates studied was too low to draw firm
conclusions. However, to our best knowledge, this is the first report where the genetic relationship of
stx negative variants of E. coli O157:H7/NM with stx positive O157:H7/NM has been confirmed using
WGS.
In conclusion, stx-negative E. coli O157:H7/NM are a cause of gastrointestinal disease in the
Netherlands, and because of the presence of the complete set of accessory virulence genes, these
isolates should be considered of public health concern similar to their stx-positive variants.
Additional diagnostic approaches should be implemented to identify these EHEC-LST isolates for
surveillance purposes and to allow appropriate treatment measures and for preventing their
transmission.
Comparison of stx-Positive and -Negative E. coli O157:H7
135
ACKNOWLEDGEMENTS
This study was partly supported by the Interreg IVa-funded projects EurSafety Heath-net (III-1-02=73)
and SafeGuard (III-2-03=025) and by a University Medical Center Groningen Healthy Ageing Pilots
grant.
REFERENCES
1. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, Hebert RJ, Olcott ES,
Johnson LM, Hargrett NT, Blake PA, Cohen ML. 1983. Hemorrhagic colitis associated with a rare
Escherichia coli serotype. N Engl J Med 308:681-5.
2. Lim JY, Yoon J, Hovde CJ. 2010. A brief overview of Escherichia coli O157:H7 and its plasmid O157.
J Microbiol Biotechnol 20:5-14.
3. Eppinger M, Mammel MK, Leclerc JE, Ravel J, Cebula TA. 2011. Genomic anatomy of Escherichia
coli O157:H7 outbreaks. Proc Natl Acad Sci U S A 108:20142-7.
4. Law D. 2000. Virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli. J
Appl Microbiol 88:729-45.
5. Levine MM. 1987. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic,
enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis 155:377-89.
6. Schmidt H. 2001. Shiga-toxin-converting bacteriophages. Res Microbiol 152:687-95.
7. De Greve H, Qizhi C, Deboeck F, Hernalsteens JP. 2002. The Shiga-toxin VT2-encoding
bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome. Biochim
Biophys Acta 1579:196-202.
8. Muniesa M, de Simon M, Prats G, Ferrer D, Pañella H, Jofre J. 2003. Shiga toxin 2-converting
bacteriophages associated with clonal variability in Escherichia coli O157:H7 strains of human origin
isolated from a single outbreak. Infect Immun 71:4554-62.
9. Bielaszewska M, Prager R, Zhang W, Friedrich AW, Mellmann A, Tschäpe H, Karch H. 2006.
Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic
Escherichia coli O157:NM. Appl Environ Microbiol. 72:1900-9.
10. Serra-Moreno R, Jofre J, Muniesa M. 2007. Insertion site occupancy by stx2 bacteriophages depends
on the locus availability of the host strain chromosome. J Bacteriol 189:6645-54.
11. Shringi S, Schmidt C, Katherine K, Brayton KA, Hancock DD, Besser TE. 2012. Carriage of stx2a
differentiates clinical and bovine-biased strains of Escherichia coli O157. PLoS One 7:e51572.
12. Rosser T, Dransfield T, Allison L, Hanson M, Holden N, Evans J, Naylor S, La Ragione R, Low
JC, Gally DL. 2008. Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM.
Infect Immun 76:5598-607.
13. Feng PC, Monday SR, Lacher DW, Allison L, Siitonen A, Keys C, Eklund M, Nagano H, Karch H,
Keen J, Whittam TS. 2007. Genetic diversity among clonal lineages within Escherichia coli O157:H7
stepwise evolutionary model. Emerg Infect Dis 13:1701-6.
14. Shaikh N, Tarr PI. 2003. Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,
excisions, truncations, and evolutionary implications. J Bacteriol 185:3596-605.
15. Leopold SR, Magrini V, Holt NJ, Shaikh N, Mardis ER, Cagno J, Ogura Y, Iguchi A, Hayashi T,
Mellmann A, Karch H, Besser TE, Sawyer SA, Whittam TS, Tarr PI. 2009. A precise reconstruction
of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone
concatenomic analysis. Proc Natl Acad Sci U S A 106:8713-8.
16. Blanco M, Blanco JE, Dahbi G, Mora A, Alonso MP, Varela G, Gadea MP, Schelotto F, González
EA, Blanco J. 2006. Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC)
Chapter 6
136
isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin
variants (muB and xiR/beta2B). J Med Microbiol 55:1165-74.
17. Bentancor A, Vilte DA, Rumi MV, Carbonari CC, Chinen I, Larzábal M, Cataldi A, Mercado EC.
2010. Characterization of non-Shiga-toxin-producing Escherichia coli O157 strains isolated from dogs.
Rev Argent Microbiol 42:46-8.
18. Bielaszewska M, Köck R, Friedrich AW, von Eiff C, Zimmerhackl LB, Karch H, Mellmann A.
2007. Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm? PLoS
One 2:e1024.
19. Bielaszewska M, Middendorf B, Köck R, Friedrich AW, Fruth A, Karch H, Schmidt MA,
Mellmann A. 2008. Shiga toxin-negative attaching and effacing Escherichia coli: distinct clinical
associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution. Clin
Infect Dis 47:208-17.
20. Friedrich AW, Zhang W, Bielaszewska M, Mellmann A, Köck R, Fruth A, Tschäpe H, Karch H.
2007. Prevalence, virulence profiles, and clinical significance of Shiga toxin-negative variants of
enterohemorrhagic Escherichia coli O157 infection in humans. Clin Infect Dis 45:39-45.
21. Themphachana M, Nakaguchi Y, Nishibuchi M, Seto K, Rattanachuay P, Singkhamanan K,
Sukhumungoon P. 2014. First report in Thailand of a stx-negative Escherichia Coli 0157 strain from a
patient with diarrhea. Southeast Asian J Trop Med Public Health 45:881-9.
22. de Boer RF, Ferdous M, Ott A, Scheper HR, Wisselink GJ, Heck ME, Rossen JW, Kooistra-Smid
AM. 2015. Assessing the Public Health Risk of Shiga Toxin-Producing Escherichia coli by Use of a
Rapid Diagnostic Screening Algorithm. J Clin Microbiol 53:1588-98.
23. Perelle S, Dilasser F, Grout J, Fach P. 2004. Detection by 5'-nuclease PCR of Shiga-toxin producing
Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's
most frequent clinical cases. Mol Cell Probes 18:185-92.
24. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM,
Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D,
Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko
O. 2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75.
25. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-
Pontén T, Ussery DW, Aarestrup FM, Lund O. 2012. Multilocus sequence typing of total-genome-
sequenced bacteria. J Clin Microbiol 50:1355-61.
26. Joensen KG, Scheutz F, Lund O, Hasman H, Kaas RS, Nielsen EM, Aarestrup FM. 2014. Real-
time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic
Escherichia coli. J Clin Microbiol 52:1501-10.
27. Joensen KG, Tetzschner AM, Iguchi A, Aarestrup FM, Scheutz F. 2015. Rapid and easy in silico
serotyping of Escherichia coli using whole genome sequencing (WGS) data. J Clin Microbiol pii:
JCM.00008-15.
28. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. 2011. BLAST Ring Image Generator (BRIG):
simple prokaryote genome comparisons. BMC Genomics 12:402.
29. Friedrich AW, Köck R, Bielaszewska M, Zhang W, Karch H, Mathys W. 2005. Distribution of the
urease gene cluster among and urease activities of enterohemorrhagic Escherichia coli O157 isolates
from humans. J Clin Microbiol 43:546-50.
30. Maiden MC, Jansen van Rensburg MJ, Bray JE, Earle SG, Ford SA, Jolley KA, McCarthy ND.
2013. MLST revisited: the gene by-gene approach to bacterial genomics. Nat Rev Microbiol 11:728-36.
31. Leopold SR, Goering RV, Witten A, Harmsen D, Mellmann A. 2014. Bacterial whole-genome
sequencing revisited: portable, scalable, and standardized analysis for typing and detection of virulence
and antibiotic resistance genes. J Clin Microbiol 52:2365-70.
32. Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii K, Yokoyama K, Han CG,Ohtsubo E,
Nakayama K, Murata T, Tanaka M, Tobe T, Iida T, Takami H, Honda T, Sasakawa C,
Ogasawara N, Yasunaga T, Kuhara S, Shiba T, Hattori M, Shinagawa H. 2001. Complete genome
Comparison of stx-Positive and -Negative E. coli O157:H7
137
sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory
strain K-12. DNA Res 8:11-22.
33. Katani R, Cote R, Raygoza Garay JA, Li L, Arthur TM, DebRoy C, Mwangi MM, Kapur V. 2015.
Complete genome sequence of SS52, a strain of Escherichia coli O157:H7 recovered from supershedder
cattle. Genome Announc 3:e01569-14. doi:10.1128/genomeA.01569-14.
34. Olaimat AN, Holley RA. 2012. Factors influencing the microbial safety of fresh produce: a review.
Food Microbiol 32:1-19.
35. Tennant SM, Tauschek M, Azzopardi K, Bigham A, Bennett-Wood V, Hartland EL, Qi W,
Whittam TS, Robins-Browne RM. 2009. Characterisation of atypical enteropathogenic E. coli strains
of clinical origin. BMC Microbiol 9:117.
36. Perna NT, Mayhew GF, Pósfai G, Elliott S, Donnenberg MS, Kaper JB, Blattner FR. 1998.
Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect
Immun 66:3810-7.
37. Monday SR, Minnich SA, Feng PC. 2004. A 12-base-pair deletion in the flagellar master control gene
flhC causes nonmotility of the pathogenic German sorbitol-fermenting Escherichia coli O157:H- strains.
J Bacteriol 186:2319-27.
38. Feng P, Dey M, Abe A, Takeda T. 2001. Isogenic strain of Escherichia coli O157:H7 that has lost both
Shiga toxin 1 and 2 genes. Clin Diagn Lab Immunol 8:711-7.
39. Karch H, Bielaszewska M. 2001. Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157:H(-
) strains: epidemiology, phenotypic and molecular characteristics,and microbiological diagnosis. J Clin
Microbiol 39:2043-9.
40. Kaper JB, Nataro JP, Mobley HL. 2004. Pathogenic Escherichia coli. Nat Rev Microbiol 2:123-40.
41. Tozzoli R, Grande L, Michelacci V, Ranieri P, Maugliani A, Caprioli A, Morabito S. 2014. Shiga
toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion. Front
Cell Infect Microbiol 4:80.
42. Mellmann A, Bielaszewska M, Zimmerhackl LB, Prager R, Harmsen D, Tschäpe H, Karch H.
2005. Enterohemorrhagic Escherichia coli in human infection: in vivo evolution of a bacterial pathogen.
Clin Infect Dis 41:785-92.
Chapter 6
138
Supplementary Table
Table S1. Information of the patients and isolates used in this study
Isolate ID Serotype a
stx type
Isolation period
Isolation region
Patient Age(Year)/Sex
Clinical symptom
Genbank accession number
References
NSF STEC
STEC 343 O157:H7 stx2c July 2013 Groningen, NL 27/Female Diarrhoea LDOZ00000000 This study
STEC 605 O157:H7 stx2c August 2013 Groningen, NL 52/Male Unknownb LFUA00000000 This study
STEC 623 O157:NM stx1a+stx2c
August 2013 Groningen, NL 25/Male Bloody Diarrhoea
LFUB00000000 This study
STEC 771 O157:H7 stx1a+stx2c
September 2013
Groningen, NL 30/Female Diarrhoea LGAZ00000000 This study
STEC 915 O157:NM stx1a+stx2c
September 2013
Groningen, NL 8/Male Bloody Diarrhoea
LFUH00000000 This study
STEC 989 O157:H7 stx1a+stx2c
October 2013
Groningen, NL 58/Male Bloody Diarrhoea
LGBA00000000 This study
STEC 994 O157:H7 stx2c October 2013
Groningen, NL 43/Male Bloody Diarrhoea
LGBB00000000 This study
STEC 1109 O157:H7 stx2c October 2013
Groningen, NL 78/Male Abdominal pain
LGBC00000000 This study
STEC 2075 O157:H7 stx2c May 2013 Rotterdam, NL 1/Male Diarrhoea LGBD00000000 This study
STEC 2112 O157:H7 stx1a+stx2a
June 2013 Rotterdam, NL 10/Female Diarrhoea LGBE00000000 This study
STEC 2257 O157:NM stx1a+stx2c
July 2013 Rotterdam, NL 4/Female Unknownb LGBF00000000 This study
STEC 2410 O157:H7 stx2c August 2013 Rotterdam, NL 14/Female Diarrhoea LGBG00000000 This study
STEC 2667 O157:NM stx1a+stx2c
October 2013
Rotterdam, NL 4/Female Unknownb LGBH00000000 This study
STEC 2820 O157:NM stx1a+stx2c
November 2013
Rotterdam, NL 3 months/unknown
Unknownb LGBQ00000000 This study
STEC 2821 O157:NM stx1a+stx2c
November 2013
Rotterdam, NL 72/Female Diarrhoea LGBI00000000 This study
STEC 2868 O157:H7 stx1a+stx2a
November 2013
Rotterdam, NL 14/Female Bloody Diarrhoea
LGBJ00000000 This study
SF STEC
E09/10c O157:NM stx2a 2009 Münster
Germany 4 /unknown
HUS LGBK00000000 This study
NSF EPEC
EPEC 287 O157:NM NA July 2013 Groningen, NL 4/Male Abdominal pain
LGBL00000000 This study
SF EPEC
EPEC 393 O157:NM NA July 2013 Groningen, NL 37/Male diarrhoea LGBM00000000 This study
EPEC 1572 O157:NM NA February 2014
Groningen, NL 13/Female Abdominal pain
LGBN00000000 This study
EPEC 1669 O157:NM NA March 2014 Groningen, NL 9/Female Abdominal pain
LGBO00000000 This study
E09/224c O157:NM NA 2009 Lübeck
Germany 3/unknown
Diarrhoea LGBP00000000 This study
STEC Reference genomes
EDL933d O157:H7 stx1a+
stx2a 1982 Michigan, USA Isolated
from ground beef
CP008957 (1)
Comparison of stx-Positive and -Negative E. coli O157:H7
139
Isolate ID Serotype a
stx type
Isolation period
Isolation region
Patient Age(Year)/Sex
Clinical symptom
Genbank accession number
References
Sakaid O157:H7 stx1a+
stx2a 1996 Japan Unknown HUS NC_002695 (2)
SS52d O157:H7 stx2a+
stx2c unknown USA Super
shedder cattle
CP010304 (3)
EPEC Reference genomes
C639_08d,e
O157:H45 NA unknown Denmark unknown Diarrhoea AIBH00000000 (4)
CB9615d,e
O55:H7 NA 2003 Germany infant Diarrhoea CP001846 (5)
E2348/69d,
f
O127:H6 NA 1969 Taunton, United Kingdom
unknown Diarrhoea FM180568 (6)
NA= Not applicable, NL= the Netherlands a All the isolates used in this study are positive for fliC H7 gene. b Information from the patients was not available. c These isolates were collected from Germany and used as control strains. d The genome was obtained from NCBI database and used for comparison. e This is an atypical EPEC f This is a typical EPEC
References for supplementary Table S1
1. Latif H, Li HJ, Charusanti P, Palsson BØ, Aziz RK. 2014. A Gapless, Unambiguous Genome Sequence of the
Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933. Genome Announc 2: pii: e00821-14. doi:
10.1128/genomeA.00821-14.
2. Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii K, Yokoyama K, Han CG,Ohtsubo E, Nakayama K,
Murata T, Tanaka M, Tobe T, Iida T, Takami H, Honda T, Sasakawa C, Ogasawara N, Yasunaga T, Kuhara S,
Shiba T, Hattori M, Shinagawa H. 2001. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7
and genomic comparison with a laboratory strain K-12. DNA Res 8:11-22.
3. Katani R, Cote R, Raygoza Garay JA, Li L, Arthur TM, DebRoy C, Mwangi MM, Kapur V. 2015. Complete
genome sequence of SS52, a strain of Escherichia coli O157:H7 recovered from supershedder cattle. Genome Announc
3:e01569-14. doi:10.1128/genomeA.01569-14.
4. Hazen TH, Sahl JW, Fraser CM, Donnenberg MS, Scheutz F, Rasko DA. 2013. Draft Genome Sequences of Three
O157 Enteropathogenic Escherichia coli Isolates. Genome Announc 1: pii: e00516-13. doi:10.1128/genomeA.00516-13.
5. Zhou Z, Li X, Liu B, Beutin L, Xu J, Ren Y, Feng L, Lan R, Reeves PR, Wang L. 2010. Derivation of Escherichia
coli O157:H7 from its O55:H7 precursor. PLoS One 5: e8700. doi:10.1371/journal.pone.0008700.
6. Iguchi A, Thomson NR, Ogura Y, Saunders D, Ooka T, Henderson IR, Harris D, Asadulghani M, Kurokawa K,
Dean P, Kenny B, Quail MA, Thurston S, Dougan G,Hayashi T, Parkhill J, Frankel G. 2009. Complete genome
sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J Bacteriol
191: 347-54.
Chapter 6
140
141
CHAPTER 7
Virulence, Antimicrobial Resistance Properties and
Phylogenetic Background of Non-H7 Enteropathogenic
Escherichia coli O157
Mithila Ferdous1, Anna M.D. Kooistra-Smid1,2, Kai Zhou1,3, John W.A. Rossen1# and Alexander W. Friedrich1#
1Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
2Department of Medical Microbiology, Certe Laboratory for Infectious Diseases, Groningen, the Netherlands.
3State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Centre for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
#These authors equally contributed.
Keywords
Enteropathogenic Escherichia coli (EPEC), Antimicrobial resistance, Virulence, Mobile genetic
elements, Whole genome sequencing, Phylogenetic relationship
Front. Microbiol (2016). 7:1540
Chapter 7
142
ABSTRACT
Escherichia coli (E. coli) O157 that do not produce Shiga toxin and do not possess flagellar antigen H7
are of diverse H serotypes. In this study, the antibiotic resistance properties, genotype of a set of
virulence associated genes and the phylogenetic background of E. coli O157:non-H7 groups were
compared. Whole genome sequencing was performed on fourteen O157:non-H7 isolates collected in
the STEC-ID-net study. The genomes were compared with E. coli O157 genomes and a typical
Enteropathogenic E. coli (tEPEC) genome downloaded from NCBI. Twenty-six (86%) of the analyzed
genomes had the intimin encoding gene eae but of different types mostly correlating with their H
types, e.g., H16, H26, H39 and H45 carried intimin type ε, β, κ and α, respectively. They belonged to
several E. coli phylogenetic groups, i.e., to phylogenetic group A, B1, B2 and D. Seven (50%) of our
collected O157:non-H7 isolates were resistant to two or more antibiotics. Several mobile genetic
elements, such as plasmids, insertion elements and pathogenicity islands, carrying a set of virulence
and resistance genes were found in the E. coli O157:non-H7 isolates. Core genome phylogenetic
analysis showed that O157:non-H7 isolates probably evolved from different phylogenetic lineages
and were distantly related to the E. coli O157:H7 lineage. We hypothesize that independent
acquisition of mobile genetic elements by isolates of different lineages have contributed to the
different molecular features of the O157:non-H7 strains. Although distantly related to the STEC
O157, E. coli O157:non-H7 isolates from multiple genetic background could be considered as
pathogen of concern for their diverse virulence and antibiotic resistance properties.
Molecular diversity of EPEC O157
143
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC) was first identified in the United Kingdom in the 1940s as
the cause of outbreaks of infantile diarrhea (1). Although such outbreaks are now rare in developed
countries, EPEC strains continue to be a leading cause of diarrhea among infants from developing
countries (2). The most important feature of EPEC pathogenesis is its ability to produce characteristic
histopathological intestinal lesions known as "attaching and effacing" (A/E) lesions that are also
characteristic for some Shiga toxin-producing E. coli (STEC) (3). The genes responsible for this activity
in EPEC are encoded on a 35-kb pathogenicity island (PAI) called the locus of enterocyte effacement
(LEE), whereas the LEE of STEC contains some additional genes encoded within a putative prophage
designated 933L (4). Both EPEC and STEC LEE encode a type III secretion system, multiple secreted
proteins, and a bacterial adhesin called intimin (5). The 5′ regions of intimin-encoding gene eae are
conserved in both EPEC and STEC, whereas the 3′ regions are heterogeneous even within the same
pathotype (6). Several variants of the eae gene encoding different intimin types and subtypes are
described (7-9) and it has been suggested that different intimins may be responsible for different
host tissue cell tropisms (10).
EPEC, carrying EPEC adherence factor plasmid (pEAF) is named as typical EPEC (tEPEC), while those
without the pEAF as atypical EPEC (aEPEC) (11, 12). aEPEC are thought to be a concern as they can
acquire Shiga toxin (Stx) converting bacteriophages, thereby obtaining the ability to cause more
serious illness (13). Besides the virulence genes encoded on LEE, EPEC strains also carry other
virulence genes including astA (heat stable enterotoxin), the cdt (cytolethal distending toxin) gene
cluster, efa1 (enterohemorrhagic E. coli factor for adherence) and paa (porcine attaching-effacing
associated protein) (14-17).
Antibiotic resistance is a global concern due to the increased use of antibiotics, especially for the
intestinal organisms as the gut is a heavily populated niche and resistance genes can be transmitted
horizontally via these resistant organisms in the gut (18). High prevalence of antimicrobial resistance
among EPEC strains has been reported in different countries but the genetic basis for this resistance
and the evolutionary consequences are rarely studied (19-21).
The serotyping of O antigens (together with the H-flagellar antigen) is used as an effective method to
identify various pathogenic clones (22). The serotype O157 is a predominant serotype of the
documented STEC related outbreaks worldwide (5, 23) and is frequently associated with the H7
antigen (encoded by fliC H7). E. coli O157:H7 strains that do not possess Stx are presumably the Stx-
lost variants or progenitors of STEC as they share the same phylogenetic lineage (24). However, the
large and diverse O157 serogroup also includes many non-H7 serotypes that are commonly found in
animals, food and clinical samples. EPEC strains of the O157:H45 serotype have been noted as agents
Chapter 7
144
of diarrhea in outbreaks and in sporadic cases in Germany, Japan, Korea and Thailand (25, 26).
Strains of serotype O157:H8 and O157:H16 have been isolated from cases of diarrhea in human,
whereas the latter serotype was also isolated from cattle, beef and water (22, 27). As these strains
do not carry the stx gene, they are often not detected or no detailed characterization is carried out
(27). Recently, two studies were performed on comparative genomics analysis and phylogenetic
relationship of E. coli O157:H7 and non-H7 strains (28, 29). However, there is a lack of information
regarding their virulence, antimicrobial resistance properties and some other molecular features. In
our current study, apart from the phylogenetic and evolutionary relationship of E. coli O157:non-H7
groups, a comprehensive characterization was performed focusing on their diversity in virulence and
antimicrobial resistance properties.
MATERIALS AND METHODS
Selection of isolates for the study.
A total of fourteen E. coli O157 strains that were negative for fliC H7 gene were selected for this
study. Isolates were obtained from fecal samples of patients with gastrointestinal complaints in the
regions of Groningen and Rotterdam during the period April 2013-March 2014, as part of a large
multicenter study (STEC-ID-net, unpublished data).
Additionally, publically available genomes of sixteen E. coli including two O157:H7, one O55:H7,
twelve O157:non-H7 and one tEPEC strain (E2348/69; O127:H6) were included in the comparative
analyses. The information of isolates and downloaded genomes used in this study are presented in
Table 1.
Phenotypic characterization and antibiotic resistance profile
Sorbitol fermentation was determined using CT-SMAC plates (sorbitol MacConkey agar with cefixime
and tellurite, Mediaproducts BV, Groningen, the Netherlands). Motility was tested using Motility test
medium with triphenyltetrazolium chloride (Mediaproducts BV, Groningen, the Netherlands). The
production of beta-glucuronidase and urease were checked by using MacConkey II agar with 4-
methylumbelliferryl-β-D-glucuronide (MUG) (BD Diagnostics, Breda, the Netherlands) and urea-triple
sugar iron (TSI) agar (Mediaproducts BV, Groningen, the Netherlands), respectively. The O and H
serotypes of the isolates were determined by seroagglutination performed at the National Institute
for Public Health and the Environment (RIVM, Bilthoven, the Netherlands). Antibiotic resistance
patterns of the isolates were determined using VITEK2 (bioMérieux, Marcy l'Etoile, France) following
EUCAST guidelines.
Molecular diversity of EPEC O157
145
Whole genome sequencing
From all the isolates DNA was extracted using the UltraClean® microbial DNA isolation kit (MO BIO
Laboratories, Carlsbad, CA, US) according to the manufacturer’s protocol. A DNA library was
prepared using the Nextera XT kit (Illumina, San Diego, CA, US) according to the manufacturer’s
instructions and then ran on a Miseq (Illumina) for generating paired-end 250-bp reads aiming at a
coverage of at least 60 fold as described previously (24, 30).
Data analysis and molecular typing
De novo assembly was performed using CLC Genomics Workbench v7.0.3 (CLC bio A/S, Aarhus,
Denmark) after quality trimming (Qs ≥ 28) with optimal word sizes based on the maximum N50 value
(24, 30). All the assembled genomes of this study and the assembled genomes downloaded from
NCBI were subjected to further analyses. Annotation was performed by uploading the assembled
genomes onto the RAST server version 2.0 (31). The sequence type (ST) was identified by uploading
the assembled genomes to the Center for Genomic Epidemiology (CGE) MLST finder website (version
1.7) (32). For four of the isolates the allele numbers were submitted to the E. coli MLST databases
(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) to obtain a ST for them. The virulence genes were
determined by virulence finder 1.2 (33), antibiotic resistance genes were determined by ResFinder
2.1 (34) and the serotyping genes of the isolates were confirmed using the SeroTypeFinder tool (35)
from the CGE server. Intimin types of the isolates were determined using blastn. Isolates were
assigned to one of the major E. coli phylogenetic groups A, B1, B2 or D using the genetic markers
chuA, yjaA and the DNA fragment TspE4.C2 (36).
Synteny analysis of resistance and virulence genes
To determine the location of virulence and antibiotic resistance genes in the isolates, the contigs
containing the targeted genes were subjected to BLAST in NCBI to look for the most related genomic
regions which were later used as reference to confirm their presence in our analyzed isolates.
To compare the sequences of the LEE region, the LEE of the E. coli O157:H7 strain 71074 (accession
no GQ338312) was used as the reference and the contigs of each sample were subjected to BLAST
against the reference and plotted by BLAST ring image generator (BRIG) (37).
Phylogenetic analysis.
To determine the phylogenetic relationship of the isolates, a gene-by-gene approach was performed
using SeqSphere+ v3.0 (Ridom GmbH, Münster, Germany). For this, an ad hoc core genome MLST
(cgMLST) scheme was used as described previously (38). Briefly, the genome of E. coli O157:H7 strain
Chapter 7
146
Sakai was taken as a reference genome and ten additional E. coli genomes were used as query
genomes to extract open reading frames (ORFs) using MLST+ Target Definer 2.1.0 of SeqSphere+.
Only the ORFs without premature stop codon and ambiguous nucleotides from contigs of assembled
genomes were included. The genes shared by the genomes of all isolates analyzed in this study were
defined as the core genome for phylogenetic analysis (38). A Neighbor Joining (NJ) tree was
constructed based on a distance matrix among differences in the core genome of the isolates.
RESULTS
Phenotype
Ten of the fourteen O157 isolates collected in our study were motile and were of serotype O157:H16
(n=8) or O157:H26 (n=2); the other four were non-motile. All the isolates were positive for beta-
glucuronidase and negative for urease. Except two isolates (EPEC 2646 and EPEC 2669) all grew on
CT-SMAC agar and fermented sorbitol.
Molecular typing
The molecular typing results of the E. coli O157 isolates included in this study are summarized in
Table 1. The four non-motile isolates of this study were confirmed to carry the fliC H39 gene and
were assigned to a new ST (ST4554). All of the O157:H16 isolates belonged to E. coli phylogenetic
group A except strain Santai which belonged to phylogenetic group D (phylogenetic group of STEC
O157:H7). None of the strains were positive for the stx gene.
Virulence profiling and intimin typing
The virulence profiles of the isolates and of the genomes are presented in Figure 1. Four of the
genomes obtained from NCBI (N1, T22, 3006 and Santai) did not have the eae gene. The rest carried
the gene but different types of it were found mostly correlating to their genoserotypes, e.g.,
O157:H16 and O157:H39 isolates carried intimin type ε and κ respectively (Table 1). None of the
isolates carried intimin γ. Like tEPEC E2348/69, all O157:H45 strains had intimin α.
Besides the virulence genes located on the LEE PAI, isolates contained other virulence genes carried
on several mobile genetic elements (MGEs), e.g., the plasmid-borne genes etpD, toxB, sepA, the
prophage encoded genes espI, tccP, cif, the PAI related genes iha and espC, and other virulence
determinants carried on insertion elements or transposons (Figure 1). All O157:H39 isolates carried
the etpD gene located on pO55 like (O55:H7 strain CB9615) plasmid encoding the type II secretion
system proteins (etp gene cluster) and the conjugal transfer proteins (tra gene cluster)
(Supplementary figure S1), and adhesion gene iha presumably on a transposon. All of the O157:H45
Molecular diversity of EPEC O157
147
and O157:H39 strains carried the espC PAI (Supplementary figure S2). All the isolates analyzed in this
study were negative for the pillin subunit encoding gene sfpA.
Table 1. E. coli isolates used in this study.
Isolate ID Isolation region
a
Source Symp toms
b
Genosero -type
MLST Phylogenetic group
Intimin type
Accession No References
EPEC 400 Groningen (NL) Human D O157:H16 10 A ε LZDU00000000 This study EPEC 536 Groningen (NL) Human ND O157:H16 10 A ε LZDV00000000 This study
EPEC 631 Groningen (NL) Human N O157:H16 10 A ε LZDW00000000 This study
EPEC 720 Groningen (NL) Human N O157:H16 10 A ε LZDX00000000 This study
EPEC 1316 Groningen (NL) Human D O157:H16 10 A ε LZDY00000000 This study
EPEC 2646 Rotterdam (NL) Human N O157:H16 10 A ε LZDZ00000000 This study
EPEC 2669 Rotterdam (NL) Human N O157:H16 10 A ε LZEA00000000 This study
EPEC 3029 Rotterdam (NL) Human N O157:H16 10 A ε LZEB00000000 This study
EPEC 1150 Groningen (NL) Human D O157:H39 4554 B2 κ LZEC00000000 This study
EPEC 1554 Groningen (NL) Human D O157:H39 4554 B2 κ LZED00000000 This study
EPEC 2252 Rotterdam (NL) Human D O157:H39 4554 B2 κ LZEE00000000 This study
EPEC 2272 Rotterdam (NL) Human D O157:H39 4554 B2 κ LZEF00000000 This study
EPEC 2081 Rotterdam (NL) Human N O157:H26 189 A β LZEG00000000 This study
EPEC 2827 Rotterdam (NL) Human D O157:H26 189 A β LZEH00000000 This study
Sakai Japan Human HUS O157:H7 11 D γ NC_002695 (39) SS52 USA Cattle NA O157:H7 11 D γ CP010304 (40)
CB9615 Germany Human D O55:H7 335 D γ CP001846 (41)
Santai China Duck NA O157:H16 1011 D NA CP007592 Cheng et al., unpublished
3006 USA Human N O157:H16 5502 A NA AMUN01000000
(28)
TW15901 France Food NA O157:H16 10 A ε AMUK01000000 (28)
TW00353 USA Human N O157:H16 10 A ε AMUM01000000
(28)
C639_08 Denmark Human N O157:H45 725 B2 α AIBH00000000 (42)
C844_97 Japan Human N O157:H45 725 B2 α AIBZ01000000 (42) RN587/1 Brazil Human N O157:H45 725 B2 α ADUS01000000 (42)
ARS4.2123 USA Water NA O157:H45 725 B2 α AMUL01000000 (28) TW07793 Unknown Water NA O157:H39 1041 B2 κ AFAG0200000 Sanka et al.,
unpublished 7798 Argentina Human N O157:H39 5611 B2 κ AMUP00000000 (28) N1 Unknown Food NA O157:H29 515 B1 NA AMUQ0100000
0 (28)
T22 Hungary Human N O157:H43 155 B2 NA AHZD02000000 (28)
E2348/69 Taunton, United Kingdom
Human D O127:H6 15 B2 α FM180568 (43)
aNL, the Netherlands.
bD, Diarrhea; ND, Abdominal pain and other gastrointestinal problems without diarrhea; N, Not available; HUS, Hemolytic
uremic syndrome; NA, Not applicable.
Chapter 7
148
Comparison of LEE
Figure 2 shows the variations in the sequence of the LEE PAI in EPEC O157 isolates with different H
types. The first 8 kb region, encoding the phage associated integrase and IS elements in STEC LEE, is
not intact in EPEC isolates and some of them only contain part of it. For example, EPEC O157:H16
isolates and EPEC 2827 contain the insertion element IS66 and O157:H26 isolates contain a similar
but not identical phage integrase as present in the STEC LEE. Among the core LEE genes, EPEC
secreted proteins encoding genes (esp genes) and the genes encoding the proteins for adhesion (e.g.,
eae, tir-encoding translocated intimin receptor) of the EPEC isolates are different from those of STEC.
Other genes encoding type III secretion system (e.g., esc and sep genes) and the regulator gene ler
are similar in O157:H39 and O157:H45 EPEC isolates (represented by dark blue and dark pink color in
figure 2, respectively) to those of STEC but are different in O157:H16 and O157:H26 (represented by
the relatively light green and orange colors in figure 2, respectively). The sequence of the tir gene is
conserved between O157:H16 and O157:H26 serotypes (97-98% similar), and between O157:H39
and O157:H45 serotypes (98-99% similar). Notably, the tir gene of H39 and H45 is similar (97-98%) to
that of tEPEC E2348/69, whereas in H16 and H26 it is different (<78% similarity) from all other
serotypes analyzed in this study (data not shown). Clearly, the sequence of the LEE region is variable
in different O157 EPEC strains.
Antibiotic resistance profile and presence of resistance genes
Among the fourteen O157 isolates collected in this study, all O157:H39 and O157:H26 isolates and
one of the O157:H16 isolates (EPEC 1316) were resistant to two or more antibiotics and EPEC 2827
was resistant to several antibiotics. The presence of antibiotic resistance genes was determined for
all the genomes analyzed in this study (including those downloaded from NCBI without available
phenotypic data) and the results are presented in Table 2. More than ten resistance genes were
found in two isolates, EPEC 2827 and Santai. EPEC 2827 carried the resistance gene strA on a plasmid
similar to pSTU288 (accession no. CP004059) of Salmonella enterica (Figure 3(A)), and dfrA, aphA,
aadA, blaOXA, and catB on an integron (accession no. HQ386835) of Proteus sp. VIITMP5 (Figure
3(B)). EPEC 1554 carried the tetA gene on a transposon similar to Tn1721 (accession no. X61367)
(Figure 3(C)). The O157:H39 strains had a plasmid similar to pCERC1 (accession no. JN012467) of E.
coli strain S1.2.T2R carrying a cluster of antibiotic resistance genes including strA-B, sul2 and dfrA14
(Figure 3(D)). EPEC 1316 carried the strA and blaTEM genes on a plasmid similar to pVR50A
(accession no. CP011135) (data not shown) of E. coli VR50 caused an asymptomatic bacteriuria.
Molecular diversity of EPEC O157
149
Figure 1. Virulence profiles of the isolates analyzed in this study. Predicted locations of the virulence genes
are indicated. The red color indicates the presence of a gene in the corresponding isolate.
Figure 2. Comparison of LEE in E. coli isolates analyzed in this study. The figure shows BLAST comparison of E. coli isolates against the reference LEE sequence (core black circle). Each ring represents one isolate, different colors of the rings represent different genoserotypes. The gradients (dark, pale and white) of each color represent the sequence similarity (from 100% to 0%) between samples and reference. The colors of different isolates as well as the order of the rings (from inner to outer) with the color gradient for sequence identity are shown in the legend (right). Please note that, for each genoserotype only representative isolates showing variations in the LEE sequences are presented in the figure.
Chapter 7
150
Table 2. Antibiotic resistance profiles of the analyzed isolates
Isolate IDa Genoserotype Phenotypical
resistanceb
Presence of Resistance genesc
EPEC 1316 O157:H16 AMP, SXT, TMP blaTEM-1B, dfrA8, strA/B, sul2
EPEC 1150 O157:H39 AMP, SXT, TMP blaTEM-1B, dfrA14, strA/B, sul2 EPEC 2252 O157:H39 AMC, AMP, SXT, TMP blaTEM-1B, dfrA14, strA/B, sul2
EPEC 2272 O157:H39 AMC, AMP, SXT, TMP blaTEM-1B, dfrA14, strA/B, sul2 EPEC 1554 O157:H39 AMP, TET blaTEM-1B, tetA EPEC 2081 O157:H26 AMP, NOR, TET, TMP blaTEM-1B, dfrA8, tetA, EPEC 2827 O157:H26 AMP, CIP, FOX, GEN,
NIT, NOR, SXT, TMP, TET, TOB
aadB, aph(3’) IIA/XV, blaOXA-4, blaTEM-1B, catB, dfrA1, mphA, strA/B, sul2, tetA
C639_08 O157:H45 NT sul2
RN587/1 O157:H45 NT blaTEM-116
Santai O157:H16 NT aac(3)-IId, aac(6')Ib-cr, aadA, armA, ARR-3, strA/B, blaOXA-1, blaTEM-1B, catA/B, dfrA12, floR, fosA, mphA/E, msrE, sul1/2, tetA
TW00353 O157:H16 NT blaTEM-1C ARS4.2123 O157:H45 NT strA/B, sul2
E2348/69 O127:H6 NT strA/B, sul2
aThe first seven rows shaded grey are isolates collected in our study and their resistance profile was determined using
VITEK2. bAMP, ampicillin; AMC, amoxicillin-clavulanic acid; FOX, cefoxitin; CIP, ciprofloxacin; GEN, gentamicin; NIT, nitrofurantoin;
NOR, norfloxacin; TET, tetracycline; TOB, tobramycin; TMP, trimethoprim; SXT, trimethoprim-sulfamethoxazole. NT, Not
tested as only the genomes of these strains were available. cResistance genes mentioned in the table confer resistance to antibiotics of the following categories:
aac(3)-IId, aac(6')Ib-cr, aadA, aph(3’), arma, strA/B against aminoglycosides; ARR-3 against
rifampicin; blaOXA, blaTEM against beta lactum antibiotics; catA/B, floR against phenicols; fosA against fosfomycin;
mphA/E, msrE against macrolides; dfrA against trimethoprim; sul1/2 against sulfonamides; tet against tetracycline.
Phylogenetic analysis
Figure 4 shows a NJ tree based on 2683 genes (core genome). The tree shows that strain Santai
(phylogenetic group D) shared a common ancestor with the E. coli O157:H7 lineage and its
progenitor O55:H7. The tEPEC O157:H45 (RN587/1 and ARS4.2123) and the aEPEC O157:H45
(C639_08 and C844_97) genomes clustered together and O157:H45 was the serogroup most closely
related to tEPEC reference strain E2348/69. O157:H39 isolates were more closely related with tEPEC
isolates than the O157:H16 and O157:H26 isolates. Three of the O157:H39 isolates (EPEC 1150, EPEC
2252 and EPEC 2272) appeared to be exactly similar in their core genome. Remarkably, except strain
3006 and Santai, the other ten O157:H16 isolates belonging to ST10 were closely related to each
other with allele differences ranging from 0 to 41, whereas Santai and 3006 had a minimum of 2176
allele differences (data not shown) with the other O157:H16 isolates of this study indicating that only
one fifth of their core genome contains identical alleles.
Molecular diversity of EPEC O157
151
Figure 3. Comparison of mobile genetic elements containing antibiotic resistance genes. Resistance genes of EPEC 2827 are carried on a plasmid similar to pSTU288-2 of Salmonella enterica (A) and on an integron similar to VIITMP5 1 of Proteus sp. (B). (C) Presence of a transposon like Tn1721 in EPEC 1554. (D) Presence of plasmid like pCERC1 in O157:H39 strains is shown by a representative isolate (EPEC 2252). For all the figures, the color represents sequence identity on a sliding scale, the lighter the color, the lower the percentage identity.
Figure 4. Phylogenetic relationship of the isolates analyzed in this study. This Neighbor Joining tree was
constructed based on a distance matrix among differences in the core genome of the isolates. Each isolate Id is
followed by its genoserotype, sequence type and major phylogenetic group.
Chapter 7
152
DISCUSSION
E. coli O157 strains are mostly known as enterohemorrhagic E. coli (EHEC) that have been associated
with food born outbreaks worldwide. But they are also comprised of other E. coli pathogroups,
including ones containing eae and lacking stx, which are considered as EPEC. E. coli O157:H7 isolates
lacking stx gene are assumed to be members of STEC that lost the Stx encoding bacteriophages (24).
The current study was performed to compare the molecular features and virulence properties of
EPEC O157 having different H antigens (other than H7) with those of STEC O157:H7 isolates and to
determine their phylogenetic relationship. STEC O157 isolates generally belong to ST11 and carry the
fliC H7 gene encoding flagellar antigen H7 but the EPEC O157 isolates appeared to be from different
sequence and H serotypes. The stx negative O157 isolates in this study were usually sorbitol
fermenting (SF) and some of them were non-motile. Therefore, they could be misidentified as STEC
SF O157:NM if proper molecular tests are not performed (27). For some draft genomes obtained
from NCBI, prediction of the O157 serotyping gene wzx was difficult probably because of the
breaking of contigs in the middle of the gene sequence, therefore a lower threshold of the
percentage of minimum overlapping gene length was applied to confirm the genoserotype.
Intimin types of the isolates correlated to their genoserotypes; four of the genomes did not possess
intimin and therefore were not considered to be EPEC. Our phylogenetic analysis based on cgMLST
confirmed that E. coli O157:H7 belong to a different lineage than the non-H7 group, the latter
belonging to diverse phylogenetic lineages (28). Our results support previous findings that horizontal
transfer of the O157 antigen gene cluster has occurred independently among different E. coli
lineages (22). Interestingly, isolates of O157:H45 clustered together independent of being a tEPEC or
aEPEC. However, strain C844-97 was previously described to have lost its EAF plasmid (42), and this
may also be the case for strain C639-08 explaining their aEPEC features in spite of clustering together
with tEPEC strains. Although the whole bfp gene cluster was present in tEPEC strains RN587/1 and
ARS4.2123, they did not contain the complete EAF plasmid.
Almost all of the O157:H39 isolates included in this study belonged to recently evolved STs (e.g.,
ST4554 and ST5611). Alternatively, they were not reported previously, because no further
characterization was performed as they did not carry the stx gene. We observed the presence of
several plasmids, insertion elements and PAIs in O157:H39 isolates contributing to their virulence
and resistance features. Three of the O157:H39 isolates of the new sequence type ST4554, shared an
identical core genome but no epidemiological link could be identified among the patients from whom
they were isolated.
Molecular diversity of EPEC O157
153
The distribution of virulence genes almost perfectly correlated to the STs although slight intra-ST
variations in virulence patterns were observed. The virulence genes were often carried on plasmids,
PAIs, prophages and insertion elements suggesting that these virulence factors were acquired from
numerous sources via MGE, found specifically in the genome of pathogenic strains (44). The lineage
specific acquisition of specific combinations of virulence factors may confer selective advantages
contributing to the fitness of the pathogens favoring their establishment and transmission as new
virulent clones (45). It was observed that ten of the O157:H16 isolates of ST10 (including one isolate
from the US, one non-human isolate from France, and the rest from the Netherlands) shared almost
identical virulence properties and were closely related in cgMLST with a maximum of a 41-allele
difference (data not shown). A similar finding was observed in a previous study where O157:H16
strains were described as representatives of a widespread clone (27). However, the other two
O157:H16 isolates, 3006 and Santai, belonged to two different STs, ST5502 and ST1011, respectively,
and possessed almost no virulence genes. They were not classified as EPEC supporting the idea that
the O and H serotyping might not represent the virulence status of the isolates and that the
distribution of O and H antigen encoding genes is irrespective of the E. coli pathotype (25).
Moreover, aEPEC isolates analyzed in this study were distributed in two completely different
phylogenetic branches; one containing O157:H39 and O157:H45 (closer related to tEPEC) and
another containing O157:H16 and O157:H26. This may be explained by horizontal transfer of the LEE
PAI to different E. coli groups contributing to their EPEC feature (27).
Strain Santai belonging to the phylogenetic group D as does STEC O157:H7, appears to be more a
multidrug resistant strain than a virulent one. The resistance genes were integrated into the
chromosome of this organism through insertion elements and transposons from different species.
EPEC 2827 was also resistant to several antibiotics and carried the drug resistance genes on a plasmid
very similar to that of Salmonella enterica (46) and on an insertion element similar to a class1
integron of Proteus sp. (47). Isolates of ST4554 carried a plasmid similar to pCER1, a small, globally
disseminated plasmid harboring sulphonamide and streptomycin resistance genes (48). EPEC 1316
carried the resistance genes on a plasmid similar to pVR50A which also carries a tra gene cluster
encoding conjugal transfer proteins (49). Clearly, the transmission of MGEs plays a role in the
evolution of new MDR lineages (50, 51). EPEC infections can normally be recovered without
antimicrobial therapy, therefore, the persistence of resistant EPEC strains is more likely to be related
to selective pressure from antimicrobials applied at the population level or present in the
environment (18). Indeed the presence of resistance genes in E coli that originated from the
environment have been described before (52).
Chapter 7
154
In conclusion, E. coli O157:non-H7 isolates have evolved from multiple genetic backgrounds. They are
distantly related to the most virulent EHEC clonal lineage EHEC O157:H7. Diversity in virulence and
antibiotic resistance patterns have been observed among these O157:non-H7 isolates and mobile
elements carried by these organisms could play a role in the evolution and dissemination of these
virulent and resistant clones.
ACKNOWLEDGEMENTS
We would like to thank the project members of the STEC-ID-net study, technicians for performing the
whole genome sequencing and Sigrid Rosema for helping in data analysis.
FUNDING
This study was partly supported by the Interreg IVa-funded projects EurSafety Heath-net (III-1-02 =
73) and SafeGuard (III-2-03 = 025).
REFERENCES
1. Bray, J. 1945. Isolation of antigenically homogenous strains of Bact. coli Neapolitanum from summer
diarrhoea of infants. J. Pathol. Bacteriol. 57:239-247.
2. Chen, HD, Frankel, G. 2005. Enteropathogenic Escherichia coli: unravelling pathogenesis. FEMS Microbiol.
Rev. 29:83-98. doi: S0168-6445(04)00052-X [pii].
3. Jerse, AE, Yu, J, Tall, BD, Kaper, JB. 1990. A genetic locus of enteropathogenic Escherichia coli necessary
for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. U. S. A.
87:7839-7843.
4. Perna, NT, Mayhew, GF, Posfai, G, Elliott, S, Donnenberg, MS, Kaper, JB, Blattner, FR. 1998.
Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect. Immun.
66:3810-3817.
5. Nataro, JP, Kaper, JB. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.
6. Blanco, M, Blanco, JE, Blanco, J, de Carvalho, VM, Onuma, DL, Pestana de Castro, AF. 2004. Typing
of intimin (eae) genes in attaching and effacing Escherichia coli strains from monkeys. J. Clin. Microbiol.
42:1382-1383.
7. Oswald, E, Schmidt, H, Morabito, S, Karch, H, Marches, O, Caprioli, A. 2000. Typing of intimin genes in
human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin
variant. Infect. Immun. 68:64-71.
Molecular diversity of EPEC O157
155
8. Zhang, WL, Kohler, B, Oswald, E, Beutin, L, Karch, H, Morabito, S, Caprioli, A, Suerbaum, S,
Schmidt, H. 2002. Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains. J. Clin.
Microbiol. 40:4486-4492.
9. Blanco, M, Blanco, JE, Dahbi, G, Mora, A, Alonso, MP, Varela, G, Gadea, MP, Schelotto, F, Gonzalez,
EA, Blanco, J. 2006. Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated
from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (muB and
xiR/beta2B). J. Med. Microbiol. 55:1165-1174. doi: 55/9/1165 [pii].
10. Torres, AG, Zhou, X, Kaper, JB. 2005. Adherence of diarrheagenic Escherichia coli strains to epithelial
cells. Infect. Immun. 73:18-29. doi: 73/1/18 [pii].
11. Trabulsi, LR, Keller, R, Tardelli Gomes, TA. 2002. Typical and atypical enteropathogenic Escherichia
coli. Emerg. Infect. Dis. 8:508-513. doi: 10.3201/eid0805.010385 [doi].
12. Hernandes, RT, Elias, WP, Vieira, MA, Gomes, TA. 2009. An overview of atypical enteropathogenic
Escherichia coli. FEMS Microbiol. Lett. 297:137-149. doi: 10.1111/j.1574-6968.2009.01664.x [doi].
13. Bolton, DJ, Ennis, C, McDowell, D. 2014. Occurrence, virulence genes and antibiotic resistance of
enteropathogenic Escherichia coli (EPEC) from twelve bovine farms in the north-east of Ireland. Zoonoses
Public. Health. 61:149-156. doi: 10.1111/zph.12058 [doi].
14. Dulguer, MV, Fabbricotti, SH, Bando, SY, Moreira-Filho, CA, Fagundes-Neto, U, Scaletsky, IC. 2003.
Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association
between enteroaggregative E. coli heat-stable enterotoxin and diarrhea. J. Infect. Dis. 188:1685-1694. doi:
JID30876 [pii].
15. Bouzari, S, Varghese, A. 1990. Cytolethal distending toxin (CLDT) production by enteropathogenic
Escherichia coli (EPEC). FEMS Microbiol. Lett. 59:193-198.
16. Badea, L, Doughty, S, Nicholls, L, Sloan, J, Robins-Browne, RM, Hartland, EL. 2003. Contribution of
Efa1/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells. Microb. Pathog. 34:205-215.
doi: S0882401003000263 [pii].
17. Batisson, I, Guimond, MP, Girard, F, An, H, Zhu, C, Oswald, E, Fairbrother, JM, Jacques, M, Harel,
J. 2003. Characterization of the novel factor paa involved in the early steps of the adhesion mechanism of
attaching and effacing Escherichia coli. Infect. Immun. 71:4516-4525.
18. Scaletsky, IC, Souza, TB, Aranda, KR, Okeke, IN. 2010. Genetic elements associated with antimicrobial
resistance in enteropathogenic Escherichia coli (EPEC) from Brazil. BMC Microbiol. 10:25-2180-10-25. doi:
10.1186/1471-2180-10-25 [doi].
19. Senerwa, D, Mutanda, LN, Gathuma, JM, Olsvik, O. 1991. Antimicrobial resistance of enteropathogenic
Escherichia coli strains from a nosocomial outbreak in Kenya. Apmis. 99:728-734.
20. Medina, A, Horcajo, P, Jurado, S, De la Fuente, R, Ruiz-Santa-Quiteria, JA, Dominguez-Bernal, G,
Orden, JA. 2011. Phenotypic and genotypic characterization of antimicrobial resistance in enterohemorrhagic
Escherichia coli and atypical enteropathogenic E. coli strains from ruminants. J. Vet. Diagn. Invest. 23:91-95.
doi: 23/1/91 [pii].
21. Abe, CM, Trabulsi, LR, Blanco, J, Blanco, M, Dahbi, G, Blanco, JE, Mora, A, Franzolin, MR, Taddei,
CR, Martinez, MB, Piazza, RM, Elias, WP. 2009. Virulence features of atypical enteropathogenic Escherichia
coli identified by the eae(+) EAF-negative stx(-) genetic profile. Diagn. Microbiol. Infect. Dis. 64:357-365. doi:
10.1016/j.diagmicrobio.2009.03.025 [doi].
Chapter 7
156
22. Iguchi, A, Shirai, H, Seto, K, Ooka, T, Ogura, Y, Hayashi, T, Osawa, K, Osawa, R. 2011. Wide
distribution of O157-antigen biosynthesis gene clusters in Escherichia coli. PLoS One. 6:e23250. doi:
10.1371/journal.pone.0023250 [doi].
23. Caprioli, A, Morabito, S, Brugere, H, Oswald, E. 2005. Enterohaemorrhagic Escherichia coli: emerging
issues on virulence and modes of transmission. Vet. Res. 36:289-311. doi: 10.1051/vetres:2005002 [doi].
24. Ferdous, M, Zhou, K, Mellmann, A, Morabito, S, Croughs, PD, de Boer, RF, Kooistra-Smid, AM,
Rossen, JW, Friedrich, AW. 2015. Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or
Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing. J.
Clin. Microbiol. 53:3530-3538. doi: 10.1128/JCM.01899-15 [doi].
25. Blank, TE, Lacher, DW, Scaletsky, IC, Zhong, H, Whittam, TS, Donnenberg, MS. 2003.
Enteropathogenic Escherichia coli O157 strains from Brazil. Emerg. Infect. Dis. 9:113-115. doi:
10.3201/eid0901.020072 [doi].
26. Park, JH, Oh, SS, Oh, KH, Shin, J, Jang, EJ, Jun, BY, Youn, SK, Cho, SH. 2014. Diarrheal outbreak
caused by atypical enteropathogenic Escherichia coli O157:H45 in South Korea. Foodborne Pathog. Dis.
11:775-781. doi: 10.1089/fpd.2014.1754 [doi].
27. Feng, PC, Keys, C, Lacher, D, Monday, SR, Shelton, D, Rozand, C, Rivas, M, Whittam, T. 2010.
Prevalence, characterization and clonal analysis of Escherichia coli O157: non-H7 serotypes that carry eae
alleles. FEMS Microbiol. Lett. 308:62-67. doi: 10.1111/j.1574-6968.2010.01990.x [doi].
28. Sanjar, F, Rusconi, B, Hazen, TH, Koenig, SS, Mammel, MK, Feng, PC, Rasko, DA, Eppinger, M.
2015. Characterization of the pathogenome and phylogenomic classification of enteropathogenic Escherichia
coli of the O157:non-H7 serotypes. Pathog. Dis. 73:10.1093/femspd/ftv033. Epub 2015 May 10. doi:
10.1093/femspd/ftv033 [doi].
29. Kossow, A, Zhang, W, Bielaszewska, M, Rhode, S, Hansen, K, Fruth, A, Ruter, C, Karch, H,
Mellmann, A. 2016. Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic
Escherichia coli O157 Reveals High Diversity. J. Clin. Microbiol. 54:1357-1363. doi: 10.1128/JCM.02897-15
[doi].
30. Zhou, K, Ferdous, M, de Boer, RF, Kooistra-Smid, AM, Grundmann, H, Friedrich, AW, Rossen, JW.
2015. The mosaic genome structure and phylogeny of Shiga toxin-producing Escherichia coli O104:H4 is driven
by short-term adaptation. Clin. Microbiol. Infect. 21:468.e7-468.18. doi: 10.1016/j.cmi.2014.12.009 [doi].
31. Aziz, RK, Bartels, D, Best, AA, DeJongh, M, Disz, T, Edwards, RA, Formsma, K, Gerdes, S, Glass,
EM, Kubal, M, Meyer, F, Olsen, GJ, Olson, R, Osterman, AL, Overbeek, RA, McNeil, LK, Paarmann, D,
Paczian, T, Parrello, B, Pusch, GD, Reich, C, Stevens, R, Vassieva, O, Vonstein, V, Wilke, A, Zagnitko, O.
2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 9:75-2164-9-75. doi:
10.1186/1471-2164-9-75 [doi].
32. Larsen, MV, Cosentino, S, Rasmussen, S, Friis, C, Hasman, H, Marvig, RL, Jelsbak, L, Sicheritz-
Ponten, T, Ussery, DW, Aarestrup, FM, Lund, O. 2012. Multilocus sequence typing of total-genome-
sequenced bacteria. J. Clin. Microbiol. 50:1355-1361. doi: 10.1128/JCM.06094-11 [doi].
33. Joensen, KG, Scheutz, F, Lund, O, Hasman, H, Kaas, RS, Nielsen, EM, Aarestrup, FM. 2014. Real-
time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic
Escherichia coli. J. Clin. Microbiol. 52:1501-1510. doi: 10.1128/JCM.03617-13 [doi].
34. Zankari, E, Hasman, H, Cosentino, S, Vestergaard, M, Rasmussen, S, Lund, O, Aarestrup, FM,
Larsen, MV. 2012. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother.
67:2640-2644. doi: 10.1093/jac/dks261 [doi].
Molecular diversity of EPEC O157
157
35. Joensen, KG, Tetzschner, AM, Iguchi, A, Aarestrup, FM, Scheutz, F. 2015. Rapid and Easy In Silico
Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data. J. Clin. Microbiol. 53:2410-
2426. doi: 10.1128/JCM.00008-15 [doi].
36. Clermont, O, Bonacorsi, S, Bingen, E. 2000. Rapid and simple determination of the Escherichia coli
phylogenetic group. Appl. Environ. Microbiol. 66:4555-4558.
37. Alikhan, NF, Petty, NK, Ben Zakour, NL, Beatson, SA. 2011. BLAST Ring Image Generator (BRIG):
simple prokaryote genome comparisons. BMC Genomics. 12:402-2164-12-402. doi: 10.1186/1471-2164-12-402
[doi].
38. Ferdous, M, Friedrich, AW, Grundmann, H, de Boer, RF, Croughs, PD, Islam, MA, Kluytmans-van
den Bergh, MF, Kooistra-Smid, AM, Rossen, JW. 2016. Molecular characterization and phylogeny of Shiga
toxin-producing Escherichia coli isolates obtained from two Dutch regions using whole genome sequencing.
Clin. Microbiol. Infect. 22:642.e1-642.e9. doi: 10.1016/j.cmi.2016.03.028 [doi].
39. Hayashi, T, Makino, K, Ohnishi, M, Kurokawa, K, Ishii, K, Yokoyama, K, Han, CG, Ohtsubo, E,
Nakayama, K, Murata, T, Tanaka, M, Tobe, T, Iida, T, Takami, H, Honda, T, Sasakawa, C, Ogasawara,
N, Yasunaga, T, Kuhara, S, Shiba, T, Hattori, M, Shinagawa, H. 2001. Complete genome sequence of
enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res.
8:11-22.
40. Katani, R, Cote, R, Raygoza Garay, JA, Li, L, Arthur, TM, DebRoy, C, Mwangi, MM, Kapur, V.
2015. Complete Genome Sequence of SS52, a Strain of Escherichia coli O157:H7 Recovered from Supershedder
Cattle. Genome Announc. 3:10.1128/genomeA.01569-14. doi: 10.1128/genomeA.01569-14 [doi].
41. Zhou, Z, Li, X, Liu, B, Beutin, L, Xu, J, Ren, Y, Feng, L, Lan, R, Reeves, PR, Wang, L. 2010.
Derivation of Escherichia coli O157:H7 from its O55:H7 precursor. PLoS One. 5:e8700. doi:
10.1371/journal.pone.0008700 [doi].
42. Hazen, TH, Sahl, JW, Fraser, CM, Donnenberg, MS, Scheutz, F, Rasko, DA. 2013. Draft Genome
Sequences of Three O157 Enteropathogenic Escherichia coli Isolates. Genome Announc.
1:10.1128/genomeA.00516-13. doi: 10.1128/genomeA.00516-13 [doi].
43. Iguchi, A, Thomson, NR, Ogura, Y, Saunders, D, Ooka, T, Henderson, IR, Harris, D, Asadulghani, M,
Kurokawa, K, Dean, P, Kenny, B, Quail, MA, Thurston, S, Dougan, G, Hayashi, T, Parkhill, J, Frankel,
G. 2009. Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli
O127:H6 strain E2348/69. J. Bacteriol. 191:347-354. doi: 10.1128/JB.01238-08 [doi].
44. Donnenberg, MS, Whittam, TS. 2001. Pathogenesis and evolution of virulence in enteropathogenic and
enterohemorrhagic Escherichia coli. J. Clin. Invest. 107:539-548. doi: 10.1172/JCI12404 [doi].
45. Reid, SD, Herbelin, CJ, Bumbaugh, AC, Selander, RK, Whittam, TS. 2000. Parallel evolution of
virulence in pathogenic Escherichia coli. Nature. 406:64-67. doi: 10.1038/35017546 [doi].
46. Hooton, SP, Timms, AR, Cummings, NJ, Moreton, J, Wilson, R, Connerton, IF. 2014. The complete
plasmid sequences of Salmonella enterica serovar Typhimurium U288. Plasmid. 76C:32-39. doi: S0147-
619X(14)00061-4 [pii].
47. Guo, X, Xia, R, Han, N, Xu, H. 2011. Genetic diversity analyses of class 1 integrons and their associated
antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China. Lett. Appl.
Microbiol. 52:667-675. doi: 10.1111/j.1472-765X.2011.03059.x [doi].
48. Anantham, S, Hall, RM. 2012. pCERC1, a small, globally disseminated plasmid carrying the dfrA14
cassette in the strA gene of the sul2-strA-strB gene cluster. Microb. Drug Resist. 18:364-371. doi:
10.1089/mdr.2012.0008 [doi].
Chapter 7
158
49. Beatson, SA, Ben Zakour, NL, Totsika, M, Forde, BM, Watts, RE, Mabbett, AN, Szubert, JM, Sarkar,
S, Phan, MD, Peters, KM, Petty, NK, Alikhan, NF, Sullivan, MJ, Gawthorne, JA, Stanton-Cook, M, Nhu,
NT, Chong, TM, Yin, WF, Chan, KG, Hancock, V, Ussery, DW, Ulett, GC, Schembri, MA. 2015.
Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary
tract by gene acquisition. Infect. Immun. 83:1749-1764. doi: 10.1128/IAI.02810-14 [doi].
50. Beceiro, A, Tomas, M, Bou, G. 2013. Antimicrobial resistance and virulence: a successful or deleterious
association in the bacterial world? Clin. Microbiol. Rev. 26:185-230. doi: 10.1128/CMR.00059-12 [doi].
51. Punina, NV, Makridakis, NM, Remnev, MA, Topunov, AF. 2015. Whole-genome sequencing targets
drug-resistant bacterial infections. Hum. Genomics. 9:19-015-0037-z. doi: 10.1186/s40246-015-0037-z [doi].
52. Hamelin, K, Bruant, G, El-Shaarawi, A, Hill, S, Edge, TA, Fairbrother, J, Harel, J, Maynard, C,
Masson, L, Brousseau, R. 2007. Occurrence of virulence and antimicrobial resistance genes in Escherichia coli
isolates from different aquatic ecosystems within the St. Clair River and Detroit River areas. Appl. Environ.
Microbiol. 73:477-484. doi: AEM.01445-06 [pii].
Molecular diversity of EPEC O157
159
Supplementary Figures
Supplementary Figure S1. pO55 like plasmid in O157:H39 Isolates. The figure shows BLAST comparison of the isolates against the reference plasmid pO55 of strain CB9615 (core black circle). The color of the rings, each representing one isolate indicates the sequence identity on a sliding scale, the more grey it gets, the lower the percentage identity. The order of the rings (from inner to outer) with the color gradient for sequence identity are shown in the legend (right).
Supplementary Figure S2. espC pathogenicity island in O157:H45 and O157:H39 isolates. The figure shows BLAST comparison of the isolates against the reference espC pathogenicity island of strain E2348/69 (core black circle). The color of the rings, each representing one isolate indicates the sequence identity on a sliding scale, the more grey it gets, the lower the percentage identity. The order of the rings (from inner to outer) with the color gradient for sequence identity are shown in the legend (right).
Chapter 7
160
161
CHAPTER 8
General Discussion, Summary and Future Perspectives
Chapter 8
162
General Discussion and Summary
The research performed in this thesis describes rapid molecular diagnosis of Shiga toxin-producing E.
coli (STEC) together with risk assessment and detailed characterization of the bacterium using
modern, high resolution whole genome sequencing (WGS). This research focused on two serotypes
O157:H7 and O104:H4, which are clinically important and relevant to public health. It also describes
the plasticity in virulence and antibiotic resistance properties and the molecular typing of E. coli
O157:non-H7 strains lacking the Shiga toxin (Stx) encoding gene stx to reveal their relation with STEC
O157:H7.
STEC infections are usually diagnosed through laboratory testing of stool specimens. Most
laboratories can identify STEC O157:H7 by cultural techniques. To detect non-O157 STEC infections,
molecular methods, in particular detection of the stx gene, is the most reliable way. Improved
diagnosis and classification of STEC is necessary for the diagnostic laboratories to implement proper
infection control measures in hospitals and to alert public health services, which on their turn need
to prevent (further) dissemination of this pathogen in the community. In chapter 2, a rapid screening
algorithm including both molecular and conventional methods of STEC detection was applied on
direct fecal materials. Risk assessment of STEC infection was performed using a presumptive
categorization of STEC based on serotyping and presence of virulence genes together with stx as
described by the European Food Safety Authority (EFSA) in 2013 with slide modifications. Risk groups
for disease severity were defined and ranged from high risk pathotype (PT) group I to low risk PT
group III, whereas PT group IV consists of not culture confirmed stx qPCR-positive samples. It was
observed that PT I, defined to have the escV or agg and/or aat gene and belonging to one of the
major O serotypes (O26, O103, O104, O111, O121, O145, O157), was significantly associated with
bloody diarrhea. stx subtypes 2a and 2c were more associated with PT I confirming the association of
these subtypes with severe clinical outcomes, which is in concordance with previous findings (1, 2).
Therefore, subtyping of the stx gene directly on DNA extracts from enriched stool samples could help
to increase the speed to obtain data for risk assessment, compared to subtyping performed on
isolates. Moreover, it can also determine the subtype in case the stx gene is present in a free Stx
converting bacteriophage rather than in E. coli. In this chapter, it was also observed that an
enrichment step of fecal samples increased the chance to obtain a pure isolate for further
characterization. Moreover, our study showed the effective use of selective agar medium (CHROM
Agar STEC) for easier identification and isolation of eae positive STEC as well as enteroaggregative E.
coli (EAEC) and enteropathogenic E. coli (EPEC) pathotypes (3, 4).
General Discussion
163
In chapter 3, we performed an overall molecular characterization using WGS of the STEC
isolates. We confirmed that WGS is a reliable and robust one-step process for characterization of
STEC. The most common serotypes found in this study were O91:H14 (14%), O157:H7 (13%),
O26:H11 (11%), O103:H2 (8%), O128:H2 (5%) and the most predominant (MLST) sequence types
(STs) were ST33 (14%), ST11 (13%), ST21 (13%) , ST17 (7%), and ST25 (4%). Previous studies in the
Netherlands also observed the predominance of non-O157 over O157 STEC (5-7). Over the period
2007–2012 the most reported non-O157 serotypes were O26 (12%), O63 (10%), O91 (9%), O113
(6%), O103 (5%) and O146 (4%) (7).
The presence of the pathogenicity island LEE (locus of enterocyte effacement) is thought to
be an important virulence determinant in STEC. This is also observed in our study as the presence of
virulence genes located on LEE were significantly more frequently found in isolates obtained from
patients with bloody diarrhea. In addition, several virulence genes were significantly more often
present in eae negative strains confirming their role in the virulence of these strains. In contrast, no
correlation between the severity of the disease and any specific phylogenetic background (e.g.
particular ST) of the STEC isolates was observed. Clearly, there is no single factor that could predict
the disease outcome. Several factors related to the bacterial isolate, such as its heterogeneity in
phage content and competition with intestinal microflora as well as host and environmental factors
may play a role in the disease development (8, 9). Therefore, it is difficult to predict if a specific STEC
strain can cause an outbreak or not, only based on sequence data. An example of this is E. coli
O104:H4, supposed not to be associated with outbreaks previously. Indeed, only few sporadic cases
with Hemolytic Uremic Syndrome (HUS) in Germany, Korea and Georgia were reported in 2001, 2005
and 2009, respectively, before the outbreak in Germany in 2011 (10). The 2011 outbreak strain
showed an unusual combination of virulence factors typically associated with both STEC and EAEC.
By comparing our collection of STEC isolates with a collection of diarrheagenic E. coli (DEC)
reference isolates in chapter 3, it was revealed that many of the STEC isolates shared a common
ancestor with other E. coli pathogroups. This finding suggests that STEC cannot be considered as a
single E. coli pathogroup in evolutionary history, rather originated from multiple pathogroups that
have acquired the Stx phage (11, 12). The genomic diversity pattern of STEC isolates of this study was
similar to that of DEC isolates but was less diverse than the extended spectrum beta-lactamase
(ESBL) producing E. coli suggesting that Stx phages integrate preferentially in specific E. coli types.
Such limitation in host range of the Stx phage can be explained by a preference of this phage for
specific integration sites not present in all E. coli strains. Alternatively, specific bacteriophages
Chapter 8
164
provide selective advantages for certain E. coli strains to let them survive in the environment (8, 13,
14).
As already shortly mentioned, in 2011 one of the largest and most severe outbreaks of STEC
infections occurred in Germany (10, 15) which forced scientists and clinical microbiologists to change
the STEC detection scheme into a new direction. Because the outbreak strain was not a classical STEC
strain, but actually an EAEC strain with an O104:H4 serotype possessing the Stx encoding
bacteriophage, the name Enteroaggregative Hemorrhagic E. coli (EAHEC) was proposed for the strain
(15). In chapter 4, we have described three similar EAHEC strains obtained in the Netherlands from
two friends who had been travelling to Turkey just before one of them was diagnosed with HUS. One
of the EAHEC O104:H4 strains was ESBL positive as the German 2011 outbreak strain. Interestingly,
an stx negative ESBL positive strain was also isolated from the friend of the HUS patient indicating a
possible transfer of resistance genes between bacteria inside the gut. In fact, by comparing the core
genome of the isolates it was observed that the stx negative strain was genetically at a large distance
from the stx positive ones. Although we were not able to prove that the STEC strains originated from
Turkey, there are several studies describing EAHEC O104:H4 strains associated with traveling to
Turkey, Tunisia, Egypt, and North Africa (16). In the same chapter we used WGS to differentiate
between the very closely related isolates that was not possible using conventional methods. Thus,
WGS is a powerful and helpful tool for hospitals and public health organizations and facilitates taking
the appropriate strategies for infection control especially during outbreak situations.
There were several studies performed during the 2011 outbreak describing the role of WGS
to identify and fully characterize outbreak strains within short time periods and to reveal the
phylogenetic background of them. Several hypotheses have been made on the evolution of the
EAHEC O104:H4 including the derivation of this strain from an EAEC that acquired an stx2-phage by
horizontal gene transfer (15, 17) and the idea that it originated from an ancestor STEC O104:H4 by
stepwise gain and loss of chromosomal and plasmid-encoded virulence factors (18). In chapter 5, we
have proposed an evolutionary model based on the phylogenetic analysis results of 23 O104:H4
genomes including the outbreak and non-outbreak clones. According to our model, the evolution of
three successful clades, including the one containing the 2011 outbreak strain and two non-outbreak
clades of EAHEC O104:H4, occurred from a recent common ancestor. Furthermore, we could identify
some of the driving forces that lead to evolution of successful clones, the most important ones being
use of antibiotics and niche competition. Frequent gain and loss of mobile genetic elements (MGEs)
could give rise to a new combination of virulence factors in a pathogen, which could trigger a future
outbreak. The data obtained in chapter 4 and 5 revealed that E. coli O104:H4 strains, similar to the
General Discussion
165
2011 outbreak clone are still circulating in Europe pointing out the necessity for proper molecular
surveillance of STEC.
Another and the best studied STEC serotype is O157:H7 associated with large food-borne
outbreaks worldwide. Thirteen percent of our collected STEC isolates were O157:H7, whereas we
had four stx negative O157:H7 isolates. In chapter 6, we have described a detailed genomic
comparison of stx positive and negative E. coli O157:H7, the latter not attracting the attention of
most clinical laboratories. These stx negative isolates can be considered as EPEC if they contain the
eae gene. Using WGS and subsequent phylogenetic analysis, we proved that stx negative variants of
E. coli O157:H7 carrying all other accessory virulence genes except stx, are in fact closely related to
STEC O157:H7. Either they may have lost the Stx encoding bacteriophage or they may be a
progenitor for STEC O157:H7 being prepared to acquire the Stx phage. Indeed, stx negative isolates
were also reported in previous studies even from feces of HUS patients and they may have lost this
gene during infection (19). As the stx gene is encoded on a bacteriophage that could be gained or lost
by other E. coli pathogroups, it is not always reliable enough to predict the virulence potential of a
strain only based on traditional classification of E. coli. Therefore, we suggest to screen for the
presence of other additional virulence genes to get an idea about the pathogenic potential of an E.
coli isolate.
Little is known about stx negative E. coli O157 that carry a flagellar antigen other than H7. As
stx negative O157 isolates are usually sorbitol fermenting (SF) and could be non-motile, they could be
misidentified as STEC SF O157:HNM if no proper molecular characterization is performed (20). E. coli
O157 lacking the stx gene are very diverse and belong to several STs and H types as described in
chapter 7. They can be classified as typical or atypical EPEC but also as non-EPEC. However, all of
them are distantly related to STEC O157:H7. They appeared to have different virulence properties
and some possess genes conferring resistance to multiple antibiotics. We observed the presence of
several plasmids, pathogenicity islands, and insertion elements which originated from different E. coli
types or even from other species. Some of the isolates may not be considered as pathogenic, but the
presence of resistance genes in the isolates could play a role in dissemination of these genes to other
pathogenic strains in the gut or in the environment.
In this thesis, we have tried to combine advanced diagnostic approaches and comprehensive
research in the field of diarrheagenic E. coli focusing on STEC and to a lesser extent on EPEC. Instead
of using conventional cultural techniques we implemented molecular schemes for rapid diagnosis
together with a presumptive risk categorization of STEC that facilitates the health care authorities
and scientific communities to get prepared to protect the community from a large epidemic.
Chapter 8
166
Although some serotypes of STEC are known to cause outbreaks and mortality, none of the other
serotypes should be neglected as MGEs carrying virulence markers could be integrated in E. coli
chromosomes and a combination of them could give rise to new pathogenic clones even of a rare
serotype (21). The dissemination of MGEs is rather dynamic and gain and loss of these elements
make it hard to define if the organism is a threat for public health or not. Furthermore, we showed
that 25% of our STEC isolates carried one or more antibiotic resistance genes, which they may
transfer to other clinically significant pathogens. Most of the characterization was performed by
WGS, a tool enabling us to study a broad range of characteristics and applicable to a wide range of
pathogens (22, 23). Without applying WGS it would not have been possible to compare the detailed
virulence, resistance and other molecular features together with the phylogenetic background of so
many isolates within a relatively short time span. As WGS is now-a-days becoming less expensive and
turnaround times decrease, it is perfectly applicable in routine diagnostics and clinical laboratories.
Indeed, primers and probes specific for (outbreak) specific signature sequences to setup a rapid
molecular screening test can be developed based on the WGS data. This is of great help for the rapid
identification of an outbreak strain (24) enabling taking infection control measures to prevent further
spread.
The knowledge obtained from this thesis will be helpful for the rapid identification, risk
assessment and understanding of the genomics of STEC leading towards a broad and diverse
research field on STEC.
Future Perspective
As most of the large STEC outbreaks and several sporadic cases of STEC infections are linked to food,
screening of food, water and food producing animals for the presence of STEC and characterization
of the isolates from these sources will contribute to reveal the source of STEC within the food chain
and will help to prevent its transmission. Also screening of the healthy human population for the
presence of STEC will give insight into possible transmission routes of STEC via asymptomatic carriers.
Stx phage heterogeneity may be responsible for converting the pathogenic profiles of their
bacterial hosts (25). The expression of Stx phage genes can be regulated by the presence of other
phages in the host genome (26, 27). Therefore, it is worth to analyze the complete phage properties
of the isolates of different serotypes that may be of help to define relatively more virulent STEC.
To establish infection, pathogens have mechanisms to interact and compete with the
resident microbial community at the site of infection as well as with numerous host factors. Studies
have described changes in Stx expression in the presence of other organisms, indicating that the
microbial balance has an impact on growth and establishment of STEC infection (28-30). However,
General Discussion
167
there is a significant gap in our knowledge regarding the way the intestinal microbiota affects
STEC/EPEC persistence, Stx activation and disease outcome. For this, a metagenomics approach
based on RNA sequencing can be used. It will help us to understand how the intestinal microbiota
affects the likelihood of disease development with regard to a STEC/EPEC infection and will also
allow us to observe host mRNA expression. In addition, proteomics and transcriptomics of the
selective E. coli strains will enable us to observe the gene expression to get an idea about their
relevance in disease development and their contribution to survival mechanisms.
References
1. Friedrich, AW, Bielaszewska, M, Zhang, WL, Pulz, M, Kuczius, T, Ammon, A, Karch, H. 2002.
Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J.
Infect. Dis. 185:74-84. doi: JID010725 [pii].
2. Feng, PC, Jinneman, K, Scheutz, F, Monday, SR. 2011. Specificity of PCR and serological assays in the
detection of Escherichia coli Shiga toxin subtypes. Appl. Environ. Microbiol. 77:6699-6702. doi:
10.1128/AEM.00370-11 [doi].
3. Hirvonen, JJ, Siitonen, A, Kaukoranta, SS. 2012. Usability and performance of CHROMagar STEC
medium in detection of Shiga toxin-producing Escherichia coli strains. J. Clin. Microbiol. 50:3586-3590. doi:
10.1128/JCM.01754-12 [doi].
4. Wylie, JL, Van Caeseele, P, Gilmour, MW, Sitter, D, Guttek, C, Giercke, S. 2013. Evaluation of a new
chromogenic agar medium for detection of Shiga toxin-producing Escherichia coli (STEC) and relative
prevalences of O157 and non-O157 STEC in Manitoba, Canada. J. Clin. Microbiol. 51:466-471. doi:
10.1128/JCM.02329-12 [doi].
5. van Duynhoven, YT, Friesema, IH, Schuurman, T, Roovers, A, van Zwet, AA, Sabbe, LJ, van der
Zwaluw, WK, Notermans, DW, Mulder, B, van Hannen, EJ, Heilmann, FG, Buiting, A, Jansen, R,
Kooistra-Smid, AM. 2008. Prevalence, characterisation and clinical profiles of Shiga toxin-producing
Escherichia coli in The Netherlands. Clin. Microbiol. Infect. 14:437-445. doi: 10.1111/j.1469-
0691.2008.01963.x [doi].
6. Friesema, I, van der Zwaluw, K, Schuurman, T, Kooistra-Smid, M, Franz, E, van Duynhoven, Y, van
Pelt, W. 2014. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E. coli
(STEC) infections in the Netherlands, January 2008 to December 2011. Euro Surveill. 19:26-32. doi: 20787
[pii].
7. Franz, E, van Hoek, AH, Wuite, M, van der Wal, FJ, de Boer, AG, Bouw, EI, Aarts, HJ. 2015. Molecular
hazard identification of non-O157 Shiga toxin-producing Escherichia coli (STEC). PLoS One. 10:e0120353.
doi: 10.1371/journal.pone.0120353 [doi].
Chapter 8
168
8. Gamage, SD, Patton, AK, Hanson, JF, Weiss, AA. 2004. Diversity and host range of Shiga toxin-encoding
phage. Infect. Immun. 72:7131-7139. doi: 72/12/7131 [pii].
9. Launders, N, Byrne, L, Jenkins, C, Harker, K, Charlett, A, Adak, GK. 2016. Disease severity of Shiga
toxin-producing E. coli O157 and factors influencing the development of typical haemolytic uraemic syndrome:
a retrospective cohort study, 2009-2012. BMJ Open. 6:e009933-2015-009933. doi: 10.1136/bmjopen-2015-
009933 [doi].
10. Scheutz, F, Nielsen, EM, Frimodt-Moller, J, Boisen, N, Morabito, S, Tozzoli, R, Nataro, JP, Caprioli,
A. 2011. Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4
strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. Euro Surveill.
16:19889. doi: 19889 [pii].
11. Tozzoli, R, Grande, L, Michelacci, V, Ranieri, P, Maugliani, A, Caprioli, A, Morabito, S. 2014. Shiga
toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion. Front. Cell.
Infect. Microbiol. 4:80. doi: 10.3389/fcimb.2014.00080 [doi].
12. Nyholm, O, Halkilahti, J, Wiklund, G, Okeke, U, Paulin, L, Auvinen, P, Haukka, K, Siitonen, A. 2015.
Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli
(STEC/ETEC) Strains. PLoS One. 10:e0135936. doi: 10.1371/journal.pone.0135936 [doi].
13. Laing, CR, Zhang, Y, Gilmour, MW, Allen, V, Johnson, R, Thomas, JE, Gannon, VP. 2012. A
comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the
German E. coli O104:H4 outbreak strain. PLoS One. 7:e37362. doi: 10.1371/journal.pone.0037362 [doi].
14. Schmidt, CE, Shringi, S, Besser, TE. 2016. Protozoan Predation of Escherichia coli O157:H7 Is Unaffected
by the Carriage of Shiga Toxin-Encoding Bacteriophages. PLoS One. 11:e0147270. doi:
10.1371/journal.pone.0147270 [doi].
15. Brzuszkiewicz, E, Thurmer, A, Schuldes, J, Leimbach, A, Liesegang, H, Meyer, FD, Boelter, J,
Petersen, H, Gottschalk, G, Daniel, R. 2011. Genome sequence analyses of two isolates from the recent
Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-
Haemorrhagic Escherichia coli (EAHEC). Arch. Microbiol. 193:883-891. doi: 10.1007/s00203-011-0725-6
[doi].
16. European Centre for Disease Prevention and Control and European Food Safety Authority. 2011.
Shiga toxin/verotoxin-producing Escherichia coli in humans, food and animals in the EU/EEA, with special
reference to the German outbreak strain STEC O104. . doi: doi:10.2900/55055.
17. Rasko, DA, Webster, DR, Sahl, JW, Bashir, A, Boisen, N, Scheutz, F, Paxinos, EE, Sebra, R, Chin, CS,
Iliopoulos, D, Klammer, A, Peluso, P, Lee, L, Kislyuk, AO, Bullard, J, Kasarskis, A, Wang, S, Eid, J,
Rank, D, Redman, JC, Steyert, SR, Frimodt-Moller, J, Struve, C, Petersen, AM, Krogfelt, KA, Nataro,
JP, Schadt, EE, Waldor, MK. 2011. Origins of the E. coli strain causing an outbreak of hemolytic-uremic
syndrome in Germany. N. Engl. J. Med. 365:709-717. doi: 10.1056/NEJMoa1106920 [doi].
18. Mellmann, A, Harmsen, D, Cummings, CA, Zentz, EB, Leopold, SR, Rico, A, Prior, K, Szczepanowski,
R, Ji, Y, Zhang, W, McLaughlin, SF, Henkhaus, JK, Leopold, B, Bielaszewska, M, Prager, R, Brzoska,
PM, Moore, RL, Guenther, S, Rothberg, JM, Karch, H. 2011. Prospective genomic characterization of the
German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.
PLoS One. 6:e22751. doi: 10.1371/journal.pone.0022751 [doi].
19. Bielaszewska, M, Kock, R, Friedrich, AW, von Eiff, C, Zimmerhackl, LB, Karch, H, Mellmann, A.
2007. Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm? PLoS One.
2:e1024. doi: 10.1371/journal.pone.0001024 [doi].
General Discussion
169
20. Feng, PC, Keys, C, Lacher, D, Monday, SR, Shelton, D, Rozand, C, Rivas, M, Whittam, T. 2010.
Prevalence, characterization and clonal analysis of Escherichia coli O157: non-H7 serotypes that carry eae
alleles. FEMS Microbiol. Lett. 308:62-67. doi: 10.1111/j.1574-6968.2010.01990.x [doi].
21. Muniesa, M, Hammerl, JA, Hertwig, S, Appel, B, Brussow, H. 2012. Shiga toxin-producing Escherichia
coli O104:H4: a new challenge for microbiology. Appl. Environ. Microbiol. 78:4065-4073. doi:
10.1128/AEM.00217-12 [doi].
22. Hasman, H, Saputra, D, Sicheritz-Ponten, T, Lund, O, Svendsen, CA, Frimodt-Moller, N, Aarestrup,
FM. 2014. Rapid whole-genome sequencing for detection and characterization of microorganisms directly from
clinical samples. J. Clin. Microbiol. 52:139-146. doi: 10.1128/JCM.02452-13 [doi].
23. Fournier, PE, Dubourg, G, Raoult, D. 2014. Clinical detection and characterization of bacterial pathogens
in the genomics era. Genome Med. 6:114-014-0114-2. eCollection 2014. doi: 10.1186/s13073-014-0114-2 [doi].
24. Zhou, K, Lokate, M, Deurenberg, RH, Tepper, M, Arends, JP, Raangs, EG, Lo-Ten-Foe, J,
Grundmann, H, Rossen, JW, Friedrich, AW. 2016. Use of whole-genome sequencing to trace, control and
characterize the regional expansion of extended-spectrum beta-lactamase producing ST15 Klebsiella
pneumoniae. Sci. Rep. 6:20840. doi: 10.1038/srep20840 [doi].
25. Smith, DL, Rooks, DJ, Fogg, PC, Darby, AC, Thomson, NR, McCarthy, AJ, Allison, HE. 2012.
Comparative genomics of Shiga toxin encoding bacteriophages. BMC Genomics. 13:311-2164-13-311. doi:
10.1186/1471-2164-13-311 [doi].
26. Serra-Moreno, R, Jofre, J, Muniesa, M. 2008. The CI repressors of Shiga toxin-converting prophages are
involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga
toxin 2. J. Bacteriol. 190:4722-4735. doi: 10.1128/JB.00069-08 [doi].
27. Fogg, PC, Saunders, JR, McCarthy, AJ, Allison, HE. 2012. Cumulative effect of prophage burden on
Shiga toxin production in Escherichia coli. Microbiology. 158:488-497. doi: 10.1099/mic.0.054981-0 [doi].
28. Carey, CM, Kostrzynska, M, Ojha, S, Thompson, S. 2008. The effect of probiotics and organic acids on
Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157:H7. J. Microbiol. Methods. 73:125-
132. doi: 10.1016/j.mimet.2008.01.014 [doi].
29. de Sablet, T, Chassard, C, Bernalier-Donadille, A, Vareille, M, Gobert, AP, Martin, C. 2009. Human
microbiota-secreted factors inhibit shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7. Infect.
Immun. 77:783-790. doi: 10.1128/IAI.01048-08 [doi].
30. Iversen, H, Lindback, T, L'Abee-Lund, TM, Roos, N, Aspholm, M, Stenfors Arnesen, L. 2015. The gut
bacterium Bacteroides thetaiotaomicron influences the virulence potential of the enterohemorrhagic Escherichia
coli O103:H25. PLoS One. 10:e0118140. doi: 10.1371/journal.pone.0118140 [doi].
Chapter 8
170
171
APPENDICES
Nederlandse Samenvatting
Acknowledgements
Biography
List of publication
172
173
Nederlandse Samenvatting
Appendices
174
Nederlandse Samenvatting
Dit proefschrift beschrijft het onderzoek naar snelle moleculaire diagnostiek van Shiga toxine
producerende E. coli (STEC) in combinatie met risico-inschatting en een gedetailleerde
karakterisering van de bacterie. Hierbij is gebruik gemaakt van een moderne techniek met een hoog
discriminerend vermogen: whole genome sequencing (WGS). Het onderzoek richt zich met name op
de STEC serotypes O157:H7 en O104:H4. Daarnaast beschrijft dit proefschrift de plasticiteit van
virulentie- en antibioticaresistentie eigenschappen, de moleculaire typering van E. coli O157:non-H7
isolaten zonder het Shiga toxine (Stx) coderende gen stx en hun genetische verwantschap met STEC
O157:H7.
Hoofdstuk 2 beschrijft de screening van fecale monsters op STEC met behulp van een snel
screeningsalgoritme gebaseerd op zowel moleculaire als conventionele diagnostiek. Op basis van de
pathogeniciteit van STEC werden risicogroepen gedefinieerd die varieerden van de hoog risico
pathotype (PT) groep I tot de laag risico PT groep III. Stx-PCR positieve monsters die niet met een
bacteriële kweek bevestigd konden worden, werden ingedeeld in een vierde PT-groep. Het bleek
dat de PT I groep, gedefinieerd door de aanwezigheid van escV of agg en/of aat genen en eveneens
behorend bij de belangrijkste O-serotypes (O26, O103, O104, O111, O121, O145 en O157),
significant geassocieerd is met bloederige diarree. Ook de Stx subtypes 2a en 2c bleken
geassocieerd te zijn met PT I, waarmee de relatie van deze subtypes met ernstige klinische
uitkomsten zoals bloederige diarree werd bevestigd.
Hoofdstuk 3 beschrijft een algemene moleculaire karakterisering van de STEC isolaten, uitgevoerd
met behulp van WGS. Voor de karakterisering van STEC blijkt WGS een betrouwbare en robuuste
methode te zijn. Een specifiek fylogenetische achtergrond (bijvoorbeeld bepaalde sequence type
(ST)) van het isolaat bleek niet te correleren met de ernst van de ziekte. Aangetoond werd dat veel
STEC isolaten een gemeenschappelijke voorouder delen met andere E. coli pathogroepen. Dit
suggereert dat in de evolutionaire geschiedenis STECs niet als één enkele E. coli pathogroep
beschouwd kunnen worden, maar dat ze zijn ontstaan uit meerdere pathogroepen welke de Stx
faag hebben verkregen. In deze studie kwam de genomische diversiteit van de STEC isolaten
overeen met die van een referentie collectie van diarree veroorzakende E. coli (DEC) isolaten, maar
bleek minder divers dan die van beta-lactamase (ESBL) producerende E. coli’s. Dit suggereert dat de
Stx faag bij voorkeur integreert in bepaalde E. coli types.
In hoofdstuk 4 worden EAHEC (Enteroaggregatieve hemorragische E. coli) isolaten beschreven.
Deze isolaten zijn verkregen uit fecale monsters van twee Nederlandse vriendinnen die op vakantie
waren geweest in Turkije, vlak voordat één van hen gediagnostiseerd werd met het hemolytisch-
uremisch syndroom (HUS). Eén van de EAHEC O104:H4 isolaten was ESBL positief, net zoals de
Nederlandse Samenvatting
175
Duitse uitbraak stam uit 2011. Uit de feces van de vriendin van de HUS patiënt werd ook een stx
negatieve ESBL-positieve bacterie geïsoleerd, hetgeen een aanwijzing kan zijn voor mogelijke
overdracht van resistentie genen tussen bacteriën in de darm.
In Hoofdstuk 5 wordt een evolutionair model gepresenteerd gebaseerd op de fylogenetische
analyse van 23 O104:H4 isolaten, inclusief uitbraak en niet-uitbraak isolaten. Volgens dit model
hebben drie succesvolle E. coli clusters, te weten de uitbraakstam uit 2011 en twee niet-uitbraak
EAHEC O104:O4 clusters, een recente gemeenschappelijke voorouder. Het frequent opnemen en
verliezen van mobiel genetische elementen (MGE) kan resulteren in een nieuwe combinatie van
virulentie factoren in een pathogeen. Dit kan een mogelijke trigger zijn voor een toekomstige
uitbraak. De data verkregen in hoofdstuk 4 en 5 laat zien dat E. coli O104:H4, vergelijkbaar met die
van de uitbraakstam in 2011, nog steeds in Europa circuleert en benadrukt het belang van een
goede moleculaire surveillance van STEC.
Hoofdstuk 6 beschrijft een gedetailleerde genetische vergelijking van stx positieve met negatieve E.
coli O157:H7. Deze stx negatieve isolaten kunnen als EPEC worden beschouwd indien ze het eae gen
bevatten. Met behulp van WGS en fylogenetische analyse werd aangetoond dat de stx negatieve
varianten van E. coli O157:H7 wel alle overige aan stx geassocieerde virulentie genen bevatten en
daarmee nauw verwant zijn aan STEC O157:H7. Of zij hebben de Stx coderende bacteriofaag
verloren of zij zijn mogelijk een voorganger van STEC O157:H7, in staat om de Stx faag op te nemen.
Aangezien stx negatieve O157:non-H7 isolaten gewoonlijk sorbitol fermenterend (SF) zijn en
onbeweeglijk kunnen zijn, kunnen ze ten onrechte als STEC SF O157:HNM geïdentificeerd worden
indien geen gedegen moleculaire karakterisering wordt uitgevoerd. E. coli O157:non-H7 isolaten
zonder het stx gen blijken erg divers te zijn en behoren tot verschillende ST’s en H-types, zoals
beschreven in hoofdstuk 7. Deze isolaten kunnen geclassificeerd worden als typische en atypische
EPEC maar ook als non-EPEC verre verwant aan STEC O157:H7. Ze blijken verschillende virulentie
eigenschappen te hebben en enkelen bevatten resistentiegenen tegen meerdere antibiotica. De
aanwezigheid van verschillende plasmiden, pathogeniciteitseilanden en insertie elementen, die een
rol kunnen spelen bij de verspreiding van virulentie en resistentiegenen naar andere
pathogenetische bacteriën in de darmen of in het milieu, zijn waargenomen.
Ten behoeve van de gezondheidszorgautoriteiten en de wetenschappelijke gemeenschap zijn in
plaats van conventionele kweektechnieken, moleculaire schema’s voor een snelle diagnostiek van
STEC geïmplementeerd, tezamen met een voorlopige risicoclassificatie. Door goed voorbereid te
zijn op een mogelijke epidemie wordt de volksgezondheid beschermt. De verspreiding van MGE’s is
dynamisch en het opnemen of verliezen van deze genetische elementen maakt het moeilijk om het
organisme te classificeren als bedreiging voor de volksgezondheid of juist niet. De karakterisering is
Appendices
176
voornamelijk uitgevoerd met behulp van WGS, een methode geschikt voor een groot aantal
pathogenen en die ons in staat stelt om een uitgebreid aantal kenmerken van isolaten te
bestuderen. Zonder het gebruik van WGS was het niet mogelijke geweest om van een groot aantal
isolaten in een relatief korte tijd de virulentie, resistentie, andere moleculaire eigenschappen en de
fylogenetische verwantschap tot in detail te bestuderen. Aangezien WGS steeds goedkoper wordt
en de doorlooptijd korter, is het goed toepasbaar in de routine diagnostiek en klinische laboratoria.
Aangezien de meeste grote STEC uitbraken en verschillende sporadische STEC infecties gelinkt zijn
aan voedsel, zal het screenen van voedsel, water en voor voedsel bestemde dieren op de
aanwezigheid van STEC, en de karakterisering van de isolaten afkomstig uit deze bronnen, bijdragen
aan het opsporen van de bron in de voedselketen en een bijdrage leveren aan het voorkomen van
transmissie. Ook het screenen van ”gezonde” mensen op de aanwezigheid van STEC geeft inzicht in
mogelijke transmissie routes via asymptomatische dragers. Daarom is het van belang om de
complete faag-eigenschappen van verschillende isolaten met verschillende serotypes in kaart te
brengen om relatief hoog-virulente STEC varianten te kunnen definiëren. Daarnaast geven
proteomics en transcriptomics van geselecteerde STEC isolaten ons inzage in de genexpressie die
mogelijk relevant kan zijn voor de ontwikkeling van ziekte en betrokken kunnen zijn bij
overlevingsmechanismes van de bacterie.
177
Acknowledgements
Appendices
178
Acknowledgements
First and foremost thank to Almighty Allah, my creator, all praises for him.
The successful completion of my thesis would not have been possible without the help and support
of many people whom I wish to acknowledge below.
My sincere thanks to my promoter Prof. Alexander Friedrich and my co-promoter Dr. John
Rossen. This research would not be possible without their esteemed support and guidance.
Dear Alex, first of all, thank you for offering me a Ph.D. position that ended up with this nice book
with our successful publications. You always encouraged my research with brilliant and nice ideas. I
have learned from you how to think very positively and divert everything to a successful ending.
You always encouraged me to be very independent, and from this I learned much. It was really a
great opportunity for me to work in this multispectral department with your precise guidance. I
appreciate your support, your resourcefulness, your enthusiasm and your optimistic attitude, all of
which have a positive influence on me.
Dear John, for me its next to impossible to express my humble gratitude to you. From the
beginning of my Ph.D. project, your assistance, ideas and suggestions helped me to perform these
works. It’s a pleasure to work with you. You are more a friend than a supervisor for me. I dared to
knock your office and disturb you whenever I had a question and I was so lucky, you were never
disturbed by my questions. Being such a friendly supervisor you gave me the opportunities to share
my problems any time I want, as you always told me that I can contact you 24/7 . And you know,
immediately after you appear my problems are somehow solved sometimes even before sharing
with you.
I can’t but thank to Prof. Jan Maarten van Dijl; in another way, it’s you, for whom I am here
today. Dear Jan Maarten, although I did not have an opportunity to directly work with you but you
were the person who invited me to visit you when I applied for a job in UMCG . Till now whenever I
see you I remember my first day in UMCG, I will be always grateful to you. You gave me the
opportunity to meet Dr. Hermie Harmsen. Dear Hermie, I started initially working with you in our
department. It was a great pleasure for me to work with you that helped me a lot in gathering
research experiences and developing my skills. You were always so friendly and co-operative.
I would like to acknowledge all the members of ‘’STEC-ID-net’’ project especially Dr. Mirjam
Kooistra-Smid for coordinating such a nice project. Thank you Dr. Richard de Boer for helping me in
collecting strains and providing me the information regarding the study. My thanks to my dear
colleagues Harmen, Pascal and Hamideh who did a great job to start up the project.
Acknowledgements
179
I would like to thank the members of my thesis reading committee, Prof. Jan Maarten van
Dijl, Prof. dr. H.J. (Henkjan) Verkade and Prof. dr. E.J. (Ed) Kuijper for sparing their precious time to
read and evaluate the thesis.
Thank you Sigrid and Monika for being my paranymph and for all the discussion regarding
my thesis, for the translation of dutch summary, for helping me preparing this book, helping me in
designing the cover page, and many more….. My special thanks to the ex and current members of
my office (2.034); Sigrid, Kai, Silvia, Monika, Linda, Jan-Willem, Ruud, Mehdi, Carien, Rudi, Jelte
(although the people are reshuffled but I remained in the same office). I always got support from
you whenever I had problems. I would like to thank the residents of ‘’de Brug’’ (Bhanu, Erik,
Adriana, Ieneke, Greetje, Coretta, Marjolein, Corina, Ana, Maria, Erley, Randy, Mart and who are
not mentioned by name). I would like to thank Ank, Marchine, Anja, Caroline, Judith, Johan and
Henk for their cordial help. Special thanks to Ank for helping me in arranging everything for the
completion of the thesis including printing, invitations, arranging the symposium and so on. Thanks
to Erwin, Paula, Fenna, Willy, Brigitte, Yvette, Mohammed, Karuna, Gini for helping me in the lab
works. I would like to thank Prof. Hajo Grundmann for our nice and fruitful discussion. Thank you
Ieneke for your nice suggestion on the dutch summary. Thanks to all the members of the Medical
Microbiology department for their help and support during my PhD period. Surely, there are also
many people to acknowledge who are not mentioned here by name.
Most importantly, my family : my parents, this achievement would not be possible without
their inspiration and sacrifices. My father, who waited so eagerly for this day and finally his dreams
came to true. Unfortunately, you two are not here with me in this big day, I could not make it but I
know your affection and prayers for my happiness and prosperity is always with me. I would like to
thank my parents-in-law, my brother (Ritom), brothers-in-law (Rasel vaia, Rajib and Shojib) for their
encouragements. My husband Hasan Mahmud, who was always beside me, made my difficult times
easier and was always very positive that inspired me a lot. The way would have never been easier
without him. In every step of my PhD, in every situation, I got his intense support and care. My
thanks to all of my relatives and friends in Bangladesh from whom I always got support and
inspiration. In such a big achievement I must remember my grandfather (Late) Ramzan Ali Shaikh,
from whom I got a lot of inspiration and guidance during the early journey of my education.
I would like to thank all of my Bangladeshi friends here specially Asad vai, Mila vabi and
their children; they were like my family here in all the situations. Thank you Atiq vai for always
being with us and inspiring with suggestions. I would also like to thanks my friends whom I meet in
Groningen (Khokon vai, Nasrin vabi, Zilee, Khan, Maruf, Sabil, Rushmi, Faruq vai, Tushar, Jasmine,
Soumen, Soutri, Anjala, Ingrid and Simmona) for their encouragements and discussion.
Appendices
180
The last one, the most important one to acknowledge, my son Rayn who sacrificed his days
for my work. My dear Rayn, I remember clearly your first day in the day care when you were exactly
three months and I had to come for my work leaving you with some unknown persons, it was so
painful for me (I am grateful to all of them who took care of Rayn in ‘Picasso’ since his early
months). You always wanted to play and spend your time with me and every morning when we
dropped you in the day care you cried and I had to start my day looking at your crying face; believe
me, it was not easy for me at all, I had to cry. So all of my achievements are for you my boy. You are
the inspiration of my life.
181
Biography
Appendices
182
Biography
Mithila Ferdous was born on 18th of November, 1984 in Kushtia, Bangladesh. She completed her
Higher Secondary School Certificate in 2002 from Kushtia Government College. She studied
Bachelor in the department of Microbiology at the University of Dhaka from 2003-2007. In 2009 she
completed her Masters in the same department. During her masters, she performed her thesis on
‘Prevalence of Extended Spectrum Beta Lactamase producing E. coli and Klebsiella spp. isolated
from hospitalized patients’ in the International Centre for Diarrhoeal Disease Research, Bangladesh
(ICDDRB). Subsequently, she continued to work as a senior research assistant (from August 2009-
July 2010) and as a research officer (August 2010- July 2011) in the Clinical Microbiology Laboratory
in ICDDRB.
From December 2011 Mithila started to work as a visiting scientist in the department of Medical
Microbiology at the University Medical Center Groningen, the Netherlands under the supervision of
Dr. Hermie Harmsen. During that period she worked in the project of quantitative analysis of
intestinal microbiota in chemotherapy-induced gastrointestinal mucositis in a rat model using
Fluorescent In situ Hybridization (FISH). In April 2012 she started to work as a Ph.D. student in the
genomic for infection prevention group of the Medical microbiology department under the
supervision of Prof. Alex Friedrich and Dr. John Rossen. In her Ph.D. project, she worked on Shiga
toxin producing E. coli (STEC) to establish rapid molecular diagnosis, and to perform risk
assessment and detailed characterization of STEC by using high-resolution whole genome
sequencing (WGS). During her research period, she learned multiple experimental techniques and
gathered a lot of experiences in the field of WGS data analysis and interpretation using different
bioinformatics software. Her PhD thesis will be defended on 13 February 2017 in Groningen.
Currently, she is working in the same department as a postdoctoral researcher on genomics of
gram-negative bacteria focusing on pathogenic E. coli.
183
List of Publications
Appendices
184
List of Publication
1. Ferdous M, Kooistra-Smid AM, Zhou K, Rossen JW, Friedrich AW. Virulence, Antimicrobial
Resistance Properties and Phylogenetic Background of Non-H7
Enteropathogenic Escherichia coli O157. Front Microbiol. 2016 Sep 28;7:1540.
2. Ferdous M, Friedrich AW, Grundmann H, de Boer RF, Croughs PD, Islam MA, Kluytmans-van
den Bergh MF, Kooistra-Smid AM, Rossen JW. Molecular characterization and phylogeny of
Shiga toxin-producing Escherichia coli isolates obtained from two Dutch regions using whole
genome sequencing. Clin. Microbiol. 2016 Infect. 22:642.e1-642.e9.
3. Ferdous M, Zhou K, de Boer RF, Friedrich AW, Kooistra-Smid AM, Rossen JW.
Comprehensive Characterization of Escherichia coli O104:H4 Isolated from Patients in the
Netherlands. Front Microbiol. 2015;6:1348.
4. Ferdous M, Zhou K, Mellmann A, Morabito S, Croughs PD, de Boer RF, Kooistra-Smid AM,
Rossen JW, Friedrich AW. Is Shiga Toxin-Negative Escherichia coli O157:H7
Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular
Analysis Using Whole-Genome Sequencing. J Clin Microbiol. 2015;53(11):3530-8.
5. de Boer RF, Ferdous M, Ott A, Scheper HR, Wisselink GJ, Heck ME, Rossen JW, Kooistra-
Smid AM. Assessing the public health risk of Shiga toxin-producing Escherichia coli by use of
a rapid diagnostic screening algorithm. J Clin Microbiol. 2015;53(5):1588-98.
6. Zhou K, Ferdous M, de Boer RF, Kooistra-Smid AM, Grundmann H, Friedrich AW, Rossen JW.
The mosaic genome structure and phylogeny of Shiga toxin-producing Escherichia coli
O104:H4 is driven by short-term adaptation. Clin Microbiol Infect. 2015;21(5):468.e7-18.
7. Fijlstra M, Ferdous M, Koning AM, Rings EH, Harmsen HJ, Tissing WJ. Substantial decreases
in the number and diversity of microbiota during chemotherapy-induced gastrointestinal
mucositis in a rat model. Support Care Cancer. 2015 Jun;23(6):1513-22.
8. Deurenberg RH, Bathoorn E, Chlebowicz MA, Couto N, Ferdous M, García-Cobos S, Kooistra-
Smid AMD, Raangs EC, Rosema S, Veloo ACM, Zhou K, Friedrich AW, Rossen JWA.
Application of next generation sequencing in clinical microbiology and infection prevention.
Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.12.022.
9. Moran-Gilad J, Rokney A,. Danino D, Ferdous M, Elsana F,. Baum M, Dukhan L, Agmon V,
Grotto I, Rossen JWA AND Gdalevich M. Real-Time Genomic Investigation Underlying the
Public Health Response to a Shiga Toxin-Producing Escherichia Coli O26:H11 Outbreak in a
Nursery. Submitted.
