Identifying the enemy: The use of

molecular sub-typing to solve the

foodsafety puzzle

Atin Datta, Ph.D.

CFSAN, USFDA

September 17, 2012

2

Estimated annual number of episodes of

domestically acquired foodborne illnesses,

United States* Pathogen # cases % Foodborne** B.cereus 64K 100

Campylobacter 850K 80

C.botulinum 55 100

C.prefirgens 960K 100

STEC-O157 64K 68

STEC-nonO157 112K 82

L.monocytogenes 1591 99

Salmonella 1M 94

Shigella 132K 31

S.aureus 241K 100

V.cholerae 84 100

V.vulnificus 96 47

V.parahaemolyticus 35K 86

Y.enterocolitica 97K 90

*Scallan et al. Emerging Infectious Diseases. Jan.2011

3

Estimated annual number of hospitalization

and deaths due to domestically acquired

foodborne illnesses, United States* Pathogen Death % Hospitalization B.cereus 0 0.4

Campylobacter 76 17.1

C.botulinum 9 82.6

C.prefirgens 26 0.6

STEC-O157 20 46.2

STEC-nonO157 0 12.8

L.monocytogenes 255 94

Salmonella 378 27.2

Shigella 10 20.2

S.aureus 6 6.4

V.cholerae 0 43.1

V.vulnificus 36 91.3

V.parahaemolyticus 4 22.5

Y.enterocolitica 29 34.4

*Scallan et al. Emerging Infectious Diseases. Jan.2011

Investigation

• Circumstantial evidence: Epidemiology

• Forensic evidence: Phenotypic and

genotypic footprints related to disease

causing organisms

4

Challenges in outbreak

investigation • Wide spread distribution of foods

• Complex nature of food production

• Involvement of multiple strains

• Wide spread distribution of illnesses

• Newer foods often pose newer challenges

for contaminant detection

5

6

Why Subtyping?

• Outbreak investigation

• Food association/Attribution

• Persistent clones

• Disease manifestation (virulence potential) and Risk assessment

• Regulatory action

L.monocytogenes Subtyping:

Hierarchical Approach • Serotyping

• Ribotyping/PFGE/Optical mapping

• MLVA

• MLST

• Microarray based typing

• Whole genome sequencing based typing

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Discriminatory Power(D)

8

Higher types not necessarily means high

Discrimination Index(DI)

Discrimination indices for some C. albicans - typing methods

Methods No.of types Size(%)largest type DI

DrugR 16 25 0.899

DNA typing 10 35 0.868

Killer system 25 52 0.724

Immunoblotting 16 41 0.679

Enzyme biovars 4 64 0.549

Serotyping 2 68 0.438

Numerical Index of the Discriminatory Ability of Typing Systems :an Application of Simpson's Index of Diversity

PAUL R. HUNTER* AND MICHAEL A. GASTON. JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1988, p. 2465-

2466

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Human Listeriosis Outbreaks: Changes

over the years Year Cases Implicated Food Serotype

1985 142(66% pregnancy related) Jalisco cheese 4b

1992 279 (33% pregnancy related) Pork tongue in Jelly 4b

1998-99 108 (15% pregnancy related) Hot dog 4b

2008 57 (>80% non-pregnancy related) Deli meat 1/2a

2011 146 (> 95% non-pregnancy related) Cantaloupe 1/2a, 1/2b

10

Genome size increases (roughly) with

evolutionary complexity of organism

Organism Genome (kb) Form

Virus MS2 4 RNA

Virus l 50 Linear DNA

Other viruses 5-300 Circular DNA

E.coli 4500 Circular DNA

Yeast 13,000 Linear DNA

Arabidopsis (plant) 100,000 arranged

Fruit fly 165,000 as

Mouse 3,000,000 several

Human 3,000,000 chromosomes

Listeria monocytogenes: Genomes

Strain Serotype Size GC% Gene Protein

EGDe 1/2a 2.94Mb 38 2940 2846

10403S 1/2a 2.9 38 2910 2814

F2365 4b 2.91 38 2934 2821

Finland 3a 2.87 38.1 2,847 2,762

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Sequence Based Serotypes* Serotype lmo1118 lmo0737 ORF2110 ORF2819 hlyA

1/2a,3a - + - - +

1/2b,3b,7 - - - + +

1/2c,3c + + - - +

4b,4d,4e - - + + +

Others - - - - +

*Burall LS, Simpson AC, Datta AR. Journal of Food Protection, March 2011.

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L. monocytogenes PCR Serotyping (Burall LS, Simpson AC, Datta AR. Journal of Food Protection, March 2011)

DN

A

La

dd

er

Lw C

Lm

1/2a

Lm

1/2a

Lm

1/2b

Lm

3c

Lm

4b

Lm

4c Lin

100bp

200bp

300bp

400bp

500bp

650bp

850bp

1650bp

1000bp

hlyA

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PFGE profile Riboprint

PFGE-XbaI

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212587

212722

211907

220032

222356

254582

257993

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E1 (O 3,10)

E1 (O 3,10)

E1 (O 3,10)

E1 (O 3,10)

E1 (O 3,10)

E1 (O 3,10)

E2 (O 3,15)

A

B

C

D

E

F

G

PFGE Profile

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Facility Producing Ready-to-Eat Seafood

Products

PFGE-AscI PFGE-ApaI

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Finished Product 2005

Finished Product 2005

Finished Product 2005

Swab 2005

Swab 2005

Swab 2005

Swab 2005

Swab 2004

Swab 2004

Swab 2004

Swab 2004

Swab 2006

Swab 2006

17

Optical Mapping

• High-resolution, ordered, whole genome restriction maps

• Discover outbreak strain markers

• Understand changes in food borne pathogens by looking at entire chromosome

• Measure diversity in food borne pathogens sample across entire chromosome .

18

Optical Mapping Overview

Bacterial Cells

gently lysed to

release genomic

DNA

DNA captured in

parallel arrays of

long single DNA

molecules using a

microfluidic device

Restriction

enzymes cleave

the DNA at

specific sites

Gaps in the DNA indicate location

of cut sites

19

Overlapping single molecule maps are aligned to produce a

map assembly covering an entire chromosome

Optical Mapping: Fragment Analysis

Image analysis software measures size and order of restriction fragments

Converts “optical” data into digital data - barcodes

20

Analysis of Outbreak Strains

287

285

281

Food

Patient

21

Multiple Locus Variable Tandem

Repeat Analysis( MLVA)

• Presence of short tandem repeats: dispersed on

chromosome

• Identification of suitable loci: whole genome

sequence provides useful resource

• 43 VNTR loci in EGDe and F2365 sequence

• Repeat size vary from 6-15bp and the numbers

vary from 0-42

• Pilot study to determine identity and number of

loci: all loci are not suitable for MLVA assay

22

MLVA Data*

Serotype MLVA (8 loci) PFGE(2 enzyme)

1/2a,1/2c (32) 7 12

1/2b,3b (31) 7 12

4b (60) 9 26

* Sperry et al. Journal of Clinical Microbiology, 2008

23

Multi-Locus Genotyping

Assay:SNP-Based Subtyping Strains(#) Primer sets Probes(SNPs)

Lineage I(241) 9 60

Lineage II(125) 7 64

Lineage III, IV(48) 5 51

Ducey et al. Applied and Environmental Microbiology, 2007, Ward et al. Applied and Environmental

Microbiology,2008

24

Multi Locus Sequence Typing (MLST)

• Seven house keeping genes

• 62 strains: 4b (40), 1/2a(12), 1/2b(10)

• MLST : 29

• PFGE (ApaI): 47

Salcedo et al. Journal of Clinical Microbiology, 2003

25

MLST Vs MVLST

Assay Strains Loci ST PFGE(ApaI)

1MLST 175 4 122 57

2MVLST 28 6 28 23

Revazishvilli et al. Journal of Clinical Microbiology,2004

Zhang et al. Applied and Environmental Microbiology, 2004

26

Comparison of Subtyping Assays

Lineage Serotype PFGE* MLGT MLST

I 1/2a (53) 33 16 6

4b (9) 6 4 6

II 1/2a(14), 1/2c(5), 3c(1) 17 12 11

Diversity Index 0.97 0.91 0.80

* With ApaI and AscI

Use of sequence-based technologies

for outbreak investigation

• Listeriosis outbreak associated with

cantaloupe in US:DNA Microarray

• Discovery of L.monocytogenes outbreak/s

in Canada

• E.coli O104:H4 outbreak in Germany

27

L. monocytogenes

Overnight Culture

DNA extraction

3-5 hr

DNA fragmentation

30 min Labeling

4 hr

Hybridization

16 hr

Genomic analysis using microarray: Work flow

Washing and staining

3 hr

Scanning

9min/chip

CEL files and data analyses

Rapid genomic-scale analysis of E. coli O104:H4 by using high resolution alternative methods to Next-Generation

sequencing – Jackson et al, AEM. 78: 1601-1605, 2012

Listeria GeneChip Design

Represent 64,539 annotated genes sequences from 24 sequenced strains (May2009)

Expression array: 253,361 25-mer nucleotides, representing 18,630 probe-sets

Each probe-set: up to 28 nucleotide-probes (up to 14PM and 14MM)

18,630 probe-

sets:

Gene and

intergenic regions

28 oligonucleotide probes

(14PM and 14MM)

Gene A GeneC

PM MM

TAGTGAAGATTAGGATTATATTAGC TGATAGTGAAGATTAGGATTATATTAGCAGGTA

TAGTGAAGATTACGATTATATTAGC

Gene B

PM

MM

What Listeria Gene chip can do

• Identify present/absent of DNA sequences

including genes, intergenic regions (non-

coding RNA genes), phage genome

• Identify single nucleotide

polymorphism(SNP)

• Identify expression profiles of genes in

different strains, under different growth

conditions 30

Scatter plots of the summarized Robust Multi-array Averaging

(RMA)

Comparison of the summarized Robust Multi-array Averaging (RMA)

intensities by scatter plots

Same Outbreak: Food and Clinical Same Outbreak: Food and Clinical

Different Outbreaks: Same serotype Different Outbreaks: Same serotype

Hierarchical clustering dendrogram and heat map analysis based on the

summarized Robust Multi-array Averaging (RMA) intensities

Relatedness analysis of the compatible parsimony informative genes from the

38 strains of L. monocytogenes

Relatedness analysis constructed

from the gene contents from 31

strains belonging to the two

serotypes 1/2b and 4b

Multistate Outbreak of Listeriosis Linked to

Whole Cantaloupes, 2011

Summary of the Outbreak

• 146 people infected

• 30 deaths, 1 miscarriage

• Median age 77 years; mostly >60 years

• 7 illnesses were pregnancy associated

• 99% were hospitalized

• Serotypes 1/2a and 1/2b were involved

38

PFGE profiles of Listeria monocytogenes cantaloupe outbreak strains

AscI patterns ApaI patterns

Lane 1 in both panels is

Salmonella braenderup

H9812 standard (digested

by XbaI). The Roman

numerals represent the

PFGE pattern combination

Distribution of Listeria monocytogenes isolates producing PFGE pattern combinations

Relatedness analysis of the compatible parsimony informative genes (Pongpan et al.

PlosOne, 2012. The numbers in red are the cantaloupe outbreak strains)

Red numbers are cantaloupe

related isolates

Relatedness analysis of the compatible parsimony informative genes

Serotype 1/2a (Pongpan et al. PlosOne, 2012. The numbers in red are the cantaloupe outbreak strains)

Diagrammatic representation of the distribution of unique probe-sets of the

serotype 1/2a 2011 cantaloupe outbreak strains from different PFGE pattern

combination groups based on their functions

Probe sets uniquely present in PCI Probe sets uniquely present in PC III & IV

Hierarchical clustering based on the Robust Multi Array (RMA) analysis

Serotype 1/2a (Pongpan et al. PlosOne, 2012. The numbers in red are the

cantaloupe outbreak strains)

Comparison of the RMA summarized probe-set intensities

Diagrammatic representation of the distribution of unique probe-sets of the

serotype 1/2a, PFGE pattern combination IV and absent in the PFGE pattern

combination I and III strains based on their functions

Pongpan et al. PlosOne, 2012.

Relatedness analysis of the compatible parsimony informative genes

Serotype 1/2b

Pongpan et al. PlosOne, 2012. The numbers in red

are the cantaloupe outbreak strains

Hierarchical clustering based on the Robust Multi Array (RMA) analysis

Serotype 1/2b

Pongpan et al. PlosOne, 2012. The numbers in red are the cantaloupe outbreak strains

49

Summary

• L.monocytogenes are routinely sub-typed by a variety of phenotypic and genotypic methods

• DNA based method e.g. PFGE is the current method of choice

• Molecular sub-typing provides important tools for outbreak/traceback investigation and regulatory actions

• Newer methods such as Optical mapping, MLVA, MLST and MLGT(SNP-based assay) may offer important advancement in sub-typing

• DNA microarray based format may provide a platform for identification and subtyping

• A common set of strains needs to be evaluated by different methods for definitive evaluation

Sequence typing confirms that a predominant Listeria

monocytogenes clone caused human listeriosis cases

and outbreaks in Canada from 1988 to 2010. Knabel SJ, Reimer A, Verghese B, Lok M, Ziegler J, Farber J, Pagotto

F, Graham M, Nadon CA; Canadian Public Health Laboratory Network

(CPHLN), Gilmour MW.

J.Clin. Microbiol. 50: 1748-1751, 2012

50

BMC GENOMICS 2010, 11:120

High-throughput genome sequencing of two Listeria

monocytogenes clinical isolates during a large foodborne

outbreak

Matthew W Gilmour1,2*, Morag Graham1,2, Gary Van

Domselaar1, Shaun Tyler1, Heather Kent1, Keri M Trout-Yakel1,

Oscar Larios2, Vanessa Allen3, Barbara Lee4 and Celine

Nadon1,2

51

Minimum spanning tree analysis of Canadian human clinical L. monocytogenes isolates

obtained between 1988 and 2010 based on the MLST method of Ragon et al.

Knabel S J et al. J. Clin. Microbiol. 2012;50:1748-1751

E.Coli O104:H4 Outbreak

• Germany. May, 2011

• 4100 cases; 50% hospitalized

• 49 deaths; mostly old age

• Incubation period: 8 days

• HUS cases: mostly female

XbaI profile of non-human O104:H4 isolates

Previous O104:H4 cases

• 2001: 2 HUS cases in Germany. Stx2+ but

different from the current strain

• 2004: 1 case. Korea stx1, stx2

• 2004: France, 2 cases, stx2+

• 2009: Georgia 2 cases stx2a; similar

fingerprint to the German outbreak strain

• 2010: Finland 1 case; similar fingerprint to

the German outbreak strain

Non-O157 STEC in US

• 2/3rd US STEC infections are from non-O157

• 93% sporadic and 7% outbreak: non-O157:H7

• 2009:non-O157 surpassed O157

• 35% foodborne; 1318 isolates in last 10 years

• Multiple etiology?

• Big six: O26, O45, O103, O111, O121, O145

Source: Human as reservoir of

EAC • O104:H4 only found associated with humans

and with food contaminated by human source

• EAEC may include strains that are not

pathogenic to humans: asymptomatic carrier?

• Sprout farm investigation revealed 3 with clinical

infection with 1 positive for O104:H4 and

developed HUS

• Two asymptomatic excretors of O104:H4

Fenugreek seed

Fenugreek sprouts

The Strain

• Stx2a positive

• eae and hly negative

• Complete genome sequencing: 5.2 Mb

• 93% sequence similarity with the EAEC 55989

E.coli strain isolated from a HIV patient in

Central Africa in 1995/1996

• Most frequently isolated eae- STEC serotypes

from food are stx2a

Phylogentic placement of German EHEC O104:H4

outbreak strain.

Mellmann A, Harmsen D, Cummings CA, Zentz EB, et al. (2011) Prospective Genomic Characterization of the German

Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology. PLoS ONE 6(7):

e22751. doi:10.1371/journal.pone.0022751

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022751

A new E.coli Pathotype:Entero-Aggregative-

Haemorrhagic Escherichia coli (EAHEC)

Natural Transformation Facilitates Transfer of Transposons, Integrons

and Gene Cassettes between Bacterial Species*

Sara Domingues1,2, Klaus Harms2, W. Florian Fricke3, Pa°l J. Johnsen2, Gabriela J. da Silva1, Kaare

Magne Nielsen2,4

Natural transformation of integron harboring populations of competent

bacteria revealed that interspecies exchange of gene cassettes can be

highly efficient, and independent on genetic relatedness between donor

and recipient. In conclusion, natural transformation provides a much

broader capacity for horizontal acquisitions of genetic elements and

hence, resistance traits from divergent species than previously

assumed

*PLoS Pathogens: August 2012

63

Summary • PFGE still provides a very useful tool for cluster identification and

strain characterization at a very basic level

• Sequencing entire genome is quite feasible; text base data are easy

to share, analyze and store

• Bottle neck is in is data storage, analysis and interpretation

• Newer analytical bioinformatics tools and cloud based storage and

analysis will be useful

• Whole genome sequencing:Outbreak investigation and a whole lot

more about the outbreak strains

• Consensus needs to be developed on standardized protocols for

strain comparison

• One protocol may not fit all organisms

64

Acknowledgement

• PFGE data: Christine Keys, CFSAN, FDA

• Optical Mapping daa: Michael Kotewicz, CFSAN, FDA

• Serotyping PCR: Alex Simpson, Laurel Burall, CFSAN,

FDA

• DNA Microarray:Pongpan Laksanalamai, Mark Mammel,

Scott Jackson, CFSAN, FDA

65

Disclamour

• The views expressed in this presentation

is my personal and may not reflect official

FDA position

• Mention of any product and/or services

may not be construed as an endorsement

by FDA

66

OBRIGADO

Thank You

67