“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
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