CRL - Coagulase Positive Staphylococci (CRL-CPS ... › ... ›...

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update French Food

Safety Agency

CRL - Coagulase Positive Staphylococci(CRL-CPS) : Activity Update

Benjamin FELIXAFSSA Maisons Alfort

[email protected]

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Safety Agency

Summary

I) general introduction

II) CRL-CPS skills, tasks and perspectives (typing Unit)

Development of SE gene detection

PFGE Subtyping

Spa-typing, MLST, SCCmec and MLVA (VNTR) typing

III) MRSA in French fattening pigs at slaughterhous e

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Safety Agency

I) IntroductionI.1) CRL-CPS mandate

• Nominated in May 2006 for 5 years– Regulation EC 776/2006

• Mandate of CRLs– To co-ordinate the network of the NRLs (31 NRLs from 26

member states (EU+EFTA) see map– To check the good use of the Official Methods require d for the

criteria– To train the NRL if needed– To improve the official methods (validation, analytica l

development)– To dispatch methods if no standard methods – To ensure a scientific watch– To ensure technical and scientific support to EC

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Safety Agency

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Safety Agency

I.1.2) Criteria

Regulation EC 2073/2005 microbiological criteria for fo odstuffs :

improve hygiene1000 cfu/g100 cfu/gcooked crutaceans and

molluscan shellfish

improve hygiene -

if >105 SET detection

during process when CPS high

105 cfu/g104 cfu/g

25

Cheese from raw milk

improve hygieneend of processSET not detected in

25g05

milk & milk products

If CPS > 105

100 cfu/g10 cfu/gmilk and whey powder end of process

100 cfu/g10 cfu/gfresh unpasteurisedcheese

1000 cfu/g100 cfu/grippened

unpasteurisedcheese

ActionwhenMmcnMatrix

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Safety Agency

• Laboratory for Study & Research on Quality of Foods and on Food Processes

• Food Hygiene sector : physico-chemical and microbiological contaminants

• Accredited by COFRAC– EN ISO 17025– for most of its activities– in particular analytical sectors involved in CRL

activities

I.1.3) CRL-CPS location : AFSSA-LERQAP

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Safety Agency

I.2) CRL organizational chartActing Director

Laurent LALOUX

Co-ordinatorBertrand LOMBARD

Deputy Co-ordinatorAdrien ASSERE

Bacteriology

Scientific LeaderMarie-Laure DE BUYSER

CPS detection/enumerationHMPA/MB

TypingMolecular

typing team CEB

Investigation food-poisoning

CEB

Risk Assessment

MQER

Enterotoxins

Scientific LeaderJacques-Antoine HENNEKINNE

TOP-BAC

Administrativeaffairs

Akpéné PLACCA

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Safety Agency

I.3) Bacterial characterization and epidemiology Unit (CEB) staff involved in the CRL-CPS activities

TypingMolecular typing team

CEB

TechnicianTrinh Tam DAO

Team manager Sophie ROUSSEL

TechnicianJoël GROUT

Team managerSophie GRANIER

Antibioresistance

Scientific leaderMarie-Laure DE BUYSER

Unit leaderAnne

BRISABOIS

Project leaderBenjamin

FELIX

Investigation food-poisoning

CEB

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Safety Agency

I.4) CRL-CPS activities of the Typing Unit

2010 - 20112009

- In house method diffusion - Training PFGE Harmony

- PFGE Optimisation following the Harmony protocolPFGE Harmony

- In house method diffusionMLST

- spa / PFGE comparison- In house method diffusionspa typing

-Training- Inter-laboratory trial

- In house method diffusionSE gene detection

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Safety Agency

II) CRL CPS skills, tasks and perspectives

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Safety Agency

II.1.1) se gene detection for food poisonning investigations

Background: 21 21 Staphylococal EnterotoxinStaphylococal Enterotoxin (SE) types have (SE) types have beenbeen identified identified

SE SE-like

A JB KC : C1,C2,C3 LD ME N

F � TSST (Toxique Shock Syndrom Toxin) OG PH QI U U andand U2U2R VVST

Emetic effect described Non emetic or not tested

Recently described (Ono et al. 2008)

Classical SEs

SEA is the main SE involved in food poisoning

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Safety Agency

II.1.2 Development of se genes detection by PCR

• PCR (simplex): protocol sent to NRLs in 2008

• Characterisation of S.aureus 23S rRNA gene simplex PCR

• 2 multiplex PCR covering 11 se genes

sea seb sec sed see ser seg seh sei sej sep

Multiplex 1Primers from

Mehrotra et al., 2000 (sea-e)

Chiang et al., 2008 (ser)

Multiplex 2

Primers and procedure from Bania et al., 2006

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Safety Agency

05-1

02 S

TA

(sea

)

05-8

0 S

TA

(seb

)

06-8

3 S

TA

(sec

)

06-7

1 S

TA

(sed

)

FR

I 326

(see

)

FR

I 137

(sec

-sed

-ser

)

08 C

EB

325

ST

A

08 C

EB

326

ST

A

08 C

EB

327

ST

A

FR

I S6

(sea

-seb

)

FR

I 137

(sec

-sed

-ser

)

T-

ladd

er10

0 pb

ser (123 pb)

seb (164 pb)

sec (451 pb)

sed (278 pb)

see (209 pb)

ladd

er10

0 pb

FR

I 361

(seg

-sei

-sej

)

05-1

02 S

TA

(seh

)

367

F (s

eg-s

ei-s

ep)

08 C

EB

325

ST

A

08 C

EB

326

ST

A

08 C

EB

327

ST

A

T-

sep (396 pb)

sei (328 pb)

seg (198 pb)

sej (131 pb)

seh(173 pb)

sea (102 pb

II.1.3 Results

Multiplex 1 Multiplex 2

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Safety Agency

II.2.1 Skills :

• We have a PFGE procedure for S. aureus strains• Database (Bionumerics Applied Math) of 558 entries

II.2.2 Task and perspectives : Optimisation of PFGE

• Join the « Harmony group » : new reference standard (NCTC 8325) + new migration and digestion protocole

• Optimisation on progress to provide recommandations to NRLs

• New database including PFGE, spa and MLST data

Expected date : end of 2009

II.2 PFGE subtyping

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Safety Agency

II.3 Other molecular typing methods : spa typing

2150 bp

Fc – binding region X - region C-terminus

“ spa typing technology is based on the sequencing of protein A gene X region "

Protein A

1113 f 1514 r

spa typespa type

� 3 to15 different repeats of 24 bp

� spa type depend of the number of repeats and their position inside the X region

� Wall protein, specific of S. aureus. � This protein interact with the immunoglobuline Fc region � Virulence factor

All spa types are available on: www.seqnet.org or www.spa.ridom.de

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Safety Agency

II.3 Other molecular typing methods : MLST (Multi Locus Sequence Typing)

“ MLST technology is based on the polymorphism analys is of 7 loci located on a polymorphic region of Staphylococcus aureus

housekeeping genes “

All Sequence Types and isolates data are available on public databases : www.mlst.org

For Staphyloccoci the genes are :

arcC

aroEglpF

gmk

ptatpi

yqiL

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Number of STs (Sequence Types) in the

MLST database

1354

151

11884117112

108

Number of alleles

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Safety Agency

II.3 Other molecular typing methods : SCCmec

« SCCmec typing is based on the analysis by PCR of the mobil e genetic element that carries the mecA genes and its adjacent regulation elements as recombinase ( ccr) gene, and insertion

sequence structure (IS) »

Type I

Type II

Type III

Type IV

Type V

5 SCCmec profils

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Safety Agency

III) MRSA in French fattening pigs_________

A collaboration between the Afssa Ploufragan and Afssa

Maisons Alfort for the French AMR NRL

CRL - Coagulase Positive Staphylococci (CRL-CPS ... › ... ›...

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