“Cronobacter spp. isolates in Argentina:

characterization and subtyping by

Pulsed Field Gel Electrophoresis (PFGE)

and proposal for the standardization of a

PFGE protocol”

1st International Conference on Cronobacter

(Enterobacter sakazakii)

University College Dublin

January 22 – 23, 2009

Day 2 – Session 5

Norma Binsztein

INEI – ANLIS “Carlos G. Malbrán”

Argentina

ObjectivesObjectives

� To evaluate the use of PFGE for Cronobacter

spp. subtyping, to establish the genetic

relationships between the isolates recovered in

Argentina and characterize them by biochemical

and antimicrobial tests

� To present the proposal of a PFGE protocol

Materials and MethodsMaterials and Methods

�� Cronobacter Cronobacter spp.spp. was isolated from powdered infant formulae (PIF) was isolated from powdered infant formulae (PIF) from eight batches of three different imported brands, 2005 to 2from eight batches of three different imported brands, 2005 to 2008. 008.

Method applied: USDA/FDA 2002 Method applied: USDA/FDA 2002

Brand A: 20 isolates, 2005Brand A: 20 isolates, 2005

Brand B: 1 isolate, 2006Brand B: 1 isolate, 2006

Brand C: 2 isolates, 2007 and 2008Brand C: 2 isolates, 2007 and 2008

�� 23 isolates23 isolates were characterized by biochemical tests, antimicrobial were characterized by biochemical tests, antimicrobial susceptibility following CLSI recommendations for susceptibility following CLSI recommendations for EnterobacteriaceaeEnterobacteriaceae

�� PFGEPFGE was performed applying the PulseNet protocol for was performed applying the PulseNet protocol for Shigella sonnei Shigella sonnei

with enzymes with enzymes XbaXbaI and I and SpeSpeII. .

Materials and MethodsMaterials and Methods

PFGE protocolPFGE protocolPulseNet PulseNet E. coli/S. sonneiE. coli/S. sonnei protocolprotocol

OD=1, electrophoresis conditions= 2.2 OD=1, electrophoresis conditions= 2.2 –– 54.2 sec.54.2 sec.

Enzymes (30 units, overnight)Enzymes (30 units, overnight)

First: First: XbaXbaI I

Second: Second: SpeSpeI: selected based on previous work of Dr. Arduino, CDC I: selected based on previous work of Dr. Arduino, CDC USA. Used only for isolates with identical USA. Used only for isolates with identical XbaXbaII--patternpattern

Other enzymes tested: Other enzymes tested: SfiSfiI, I, NotNotI I

Control strain:Control strain:

PulseNet Universal Standard PulseNet Universal Standard SalmonellaSalmonella Braenderup H9812Braenderup H9812

Analysis:Analysis:

BioNumerics version 4.6 (Applied Maths). Dice coefficient, UPGMABioNumerics version 4.6 (Applied Maths). Dice coefficient, UPGMA. . PulseNet script for PulseNet script for S. sonneiS. sonnei

Cronobacter spp.

Evaluation of enzymes for PFGE - SpeI and SfiI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

PFGE-SpeI, (30 U, O.N. at 37ºC) PFGE-SfiI, (30 U, O.N. at 50ºC)

Lanes 1, 5, 10 and 15: PulseNet standard strain S. Braenderup H9812

Results Results –– Biochemical testsBiochemical tests

�� The twentyThe twenty--three isolates studied showed two different three isolates studied showed two different

biochemical patterns compatible with biochemical patterns compatible with C. C. ssakazakiiakazakii and and

C. malonaticusC. malonaticus

Cronobacter sakazakii:Cronobacter sakazakii: indol/dulcitol/malonate Negativeindol/dulcitol/malonate Negative

CronobacterCronobacter malonaticus:malonaticus: indol/dulcitol indol/dulcitol NegativeNegative

malonate malonate PositiivePositiive

BrandBrand NNºº of colonies of colonies

studiedstudiedNNºº of colonies of colonies

C. sakazakiiC. sakazakii

NNºº of colonies of colonies

C. malonaticusC. malonaticus

AA 2020 1313 77

BB 11 11 00

CC 22 22 00

Results Results –– Antimicrobial patternAntimicrobial pattern

�� All the isolates were susceptible to the All the isolates were susceptible to the

antimicrobial agents tested: ampicillin, antimicrobial agents tested: ampicillin,

trimethoprimtrimethoprim--sulfamethoxazole, nalidixic acid, sulfamethoxazole, nalidixic acid,

tetracycline, ciprofloxacin, gentamicin, tetracycline, ciprofloxacin, gentamicin,

chloramphenicol, cefuroxime, amoxicilinchloramphenicol, cefuroxime, amoxicilin--

clavulanic acid, cefotaxime and cefoxitinclavulanic acid, cefotaxime and cefoxitin

D ic e (O p t :1 .50 % ) ( T o l 1 .5 % -1 .5 % ) (H > 0.0 % S > 0 .0 % ) [ 0 .0% - 9 8.3 % ]

P F G E - X b a I

10

0

90

80

70

60

50

P F G E - X b a I

Brand C, (2007)

Brand C, (2008)

Brand A, batch 1

Brand A, batch 2

Brand A, batch 1

Brand A, batch 3

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand B

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 4

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 5

Pattern 1

Pattern 2

Pattern 4

Pattern 3

Pattern 6

Pattern 5

Pattern 7

Pattern 8

Results Results –– PFGE PFGE XbaXbaII

Pattern 3

Brand B

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 4

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 1

Brand A, batch 2

Brand A, batch 1

Brand A, batch 3

Brand A, batch 1

Brand A, batch 1

Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-98.3%]

PFGE-SpeI

100

90

80

70

60

PFGE-SpeI

Esak1114/06 A1

Esak1114/06 A3

Esak1114/06 A4

Esak1325/06

Esak1326/06

Esak1328/06

Esak1329/06

Esak1331/06

Esak1333/06

Esak1923/05

Esak1904/05

Esak1905/05

Esak1674/05 B

Esak1746/05 A1

Esak1747/05 B1

Esak1834/05

Esak1914/05

Esak1926/05

Pattern 1

Pattern 2

Results Results –– PFGE PFGE SpeSpeII

Conclusions IConclusions I

�� The The Cronobacter Cronobacter spp.spp. isolates studied were genetically diverseisolates studied were genetically diverse

�� Different PFGE patterns among isolates from a single Different PFGE patterns among isolates from a single

batch (Brand A, batch 1)batch (Brand A, batch 1)

-- Different sources of contaminationDifferent sources of contamination

-- Important !Important ! to analyze more than one colony from each to analyze more than one colony from each

batch of PIF batch of PIF

�� A same subtype was found in different batches (pattern 3 in A same subtype was found in different batches (pattern 3 in

batches 1, 2, 3 and pattern 6 in batches 1,4) of Brand Abatches 1, 2, 3 and pattern 6 in batches 1,4) of Brand A

Possible common source/s of contamination of different Possible common source/s of contamination of different

batches during the manufacturing process.batches during the manufacturing process.

�� The same subtype was found in two dThe same subtype was found in two different brands ifferent brands

(A and B)(A and B)

Possible common source/s of contamination between Possible common source/s of contamination between the two companies. For example, could be the same the two companies. For example, could be the same supplier of an additivesupplier of an additive

�� Need for a standardized international PFGE Need for a standardized international PFGE protocolprotocol

VALIDATIONVALIDATION

Conclusions II Conclusions II

Argentine Research TeamArgentine Research Team

�� INEI INEI –– ANLIS ANLIS ““Dr. Carlos G. MalbrDr. Carlos G. Malbráánn””::

�� Raquel TerragnoRaquel Terragno

�� Mariana PichelMariana Pichel

�� Angela SalveAngela Salve

�� Silvina BrengiSilvina Brengi

�� Norma BinszteinNorma Binsztein

�� DIRECCION GENERAL DE HIGIENE Y DIRECCION GENERAL DE HIGIENE Y SEGURIDAD ALIMENTARIA, GOBIERNO DE SEGURIDAD ALIMENTARIA, GOBIERNO DE LA CIUDAD DE BUENOS AIRES:LA CIUDAD DE BUENOS AIRES:

�� Sergio EpszteynSergio Epszteyn

�� Celia MelamedCelia Melamed

In the frame of PulseNet International, the objectives of In the frame of PulseNet International, the objectives of standarization and validation of a PFGE protocol for standarization and validation of a PFGE protocol for subtyping subtyping Cronobacter Cronobacter spp. are:spp. are:

-- to have internationally comparable and reproducible results, to have internationally comparable and reproducible results,

-- to create National and International DataBases, to create National and International DataBases,

in order to compare the circulating subtypes between the in order to compare the circulating subtypes between the isolates of human and food in different countries and isolates of human and food in different countries and provide a tool for tracing sources of contamination and provide a tool for tracing sources of contamination and

for outbreak investigationfor outbreak investigation

Standardization of a PFGE protocol for subtyping

Cronobacter spp.

� The following electrophoresis conditions were compared, on eleven Cronobacter spp. isolates from Argentina (recovered from PIF ), using XbaI enzyme ::

Standardization of a PFGE protocol for subtyping

Cronobacter spp.

PulseNet standardized

S. sonnei

Developed by CDC, USA

PulseNet standardized

Yersinia pestis modified

Developed by CDC, USA

Running conditions:

Switch time: 2.2 - 54.2 s.

Running time: 18 h.

Running conditions:

Switch time: 1.8 - 25 s.

Running time: 18 hs.

First Steps

PulseNet standardized protocol for

S. sonneiPulseNet standardized protocol for

Yersinia pestis (modified)

SB 2 3 4 SB 6 7 8 9 SB 11 12 13 14 SB

S. sonnei protocol vs. Yersinia modified protocol

PFGE-XbaI

SB 2 3 4 5 SB 7 8 9 10 SB 12 13 14 SB

SB: PulseNet Standard S. Braenderup H9812

PulseNet standardized protocol for

S. sonneiPulseNet standardized protocol for

Yersinia pestis (modified)

S. sonnei protocol vs. Yersinia modified Protocol

PFGE-SpeI

SB 2 3 4 SB 6 7 8 9 SB 11 12 13 14 SB SB 2 3 4 SB 6 7 8 9 SB 11 12 13 14 SB

SB: PulseNet Standard S. Braenderup H9812

� For both enzymes, the PFGE conditions evaluated (S. sonnei

and Y. pestis modified) generated good quality gels, with low

background and clearly defined bands.

� Yersinia pestis modified protocol generated better resolution

and distribution of the bands in the gels for the isolates tested and

both enzymes.

Standardization of a PFGE protocol for subtyping

Cronobacter spp.

However

PFGE protocol forPFGE protocol for CronobacterCronobacter spp.spp.

International ValidationInternational Validation

� It was agreed to start an International Validation Process

� Based on the previous results, the conditions of

Yersinia pestis modified protocol were selected, using XbaI

as primary enzyme, and SpeI as secondary enzyme.

� A set of 44 strains from the Centres for Food – Safety & Foodborne Zoonoses, UCD Veterinary Sciences Centre, Univesity College Dublin was selected and sent to the participating laboratories

CanadaCanada Health Canada Health Canada

Franco PagottoFranco Pagotto

IrelandIreland Centres for Food Centres for Food –– Safety & FoodSafety & Food--borne borne

Zoonoses,UCD Veterinary Sciences Centre, Zoonoses,UCD Veterinary Sciences Centre,

Univesity College DublinUnivesity College Dublin

SSééamus Fanning, Carol Iversenamus Fanning, Carol Iversen

Switzerland Switzerland Institute of Food Safety & Hygiene, Vetsuisse Institute of Food Safety & Hygiene, Vetsuisse

Faculty, University of ZurichFaculty, University of Zurich

Roger StephanRoger Stephan

USAUSA CDC CDC

Matthew Arduino, Anna Bowen, Bette JensenMatthew Arduino, Anna Bowen, Bette Jensen

ArgentinaArgentina INEI INEI –– ANLIS ANLIS ““Carlos G. MalbrCarlos G. Malbráánn””

Mariana Pichel, Silvina Brengi, Norma BinszteinMariana Pichel, Silvina Brengi, Norma Binsztein

PFGE protocol forPFGE protocol for CronobacterCronobacter spp.spp.

International Validation International Validation

ParticipantsParticipants

With the collaboration of PulseNet USA: Peter Gerner Smidt, Efrain Ribot, Kara Cooper