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CRL - Coagulase Positive Staphylococci (CRL-CPS) : Activity Update - - PowerPoint PPT Presentation

Aug 15, 2022 349 likes •547 views

MRSA Training Course, April 20th-22nd 2009 CRL - Coagulase Positive Staphylococci (CRL-CPS) : Activity Update Benjamin FELIX AFSSA Maisons Alfort b.felix@afssa.fr French Food 1 CRL-CPS 2009 Activity Update Safety Agency MRSA Training

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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 FELIX

AFSSA Maisons Alfort b.felix@afssa.fr

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food 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 slaughterhouse

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

I) Introduction I.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 required for the criteria – To train the NRL if needed – To improve the official methods (validation, analytical 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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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

I.1.2) Criteria

Regulation EC 2073/2005 microbiological criteria for foodstuffs : improve hygiene 1000 cfu/g 100 cfu/g cooked crutaceans and molluscan shellfish improve hygiene

  • if >105 SET

detection during process when CPS high 105 cfu/g 104 cfu/g 2 5 Cheese from raw milk improve hygiene end of process SET not detected in 25g 5 milk & milk products If CPS > 105 100 cfu/g 10 cfu/g milk and whey powder end of process 100 cfu/g 10 cfu/g fresh unpasteurisedcheese 1000 cfu/g 100 cfu/g rippened unpasteurisedcheese Action when M m c n Matrix

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food 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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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

I.2) CRL organizational chart

Acting Director Laurent LALOUX Co-ordinator Bertrand LOMBARD Deputy Co-ordinator Adrien ASSERE Bacteriology Scientific Leader Marie-Laure DE BUYSER CPS detection/ enumeration HMPA/MB Typing

Molecular typing team CEB

Investigation food-poisoning

CEB

Risk Assessment MQER Enterotoxins Scientific Leader Jacques-Antoine HENNEKINNE TOP-BAC Administrative affairs Akpéné PLACCA

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

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

Typing

Molecular typing team CEB

Technician

Trinh Tam DAO

Team manager

Sophie ROUSSEL

Technician

Joël GROUT

Team manager

Sophie GRANIER

Antibioresistance

Scientific leader

Marie-Laure DE BUYSER

Unit leader

Anne BRISABOIS

Project leader

Benjamin FELIX

Investigation food- poisoning

CEB

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

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

2010 - 2011 2009

  • In house method diffusion
  • Training PFGE Harmony
  • PFGE Optimisation following

the Harmony protocol

PFGE Harmony

  • In house method diffusion

MLST

  • spa / PFGE comparison
  • In house method diffusion

spa typing

  • Training
  • Inter-laboratory trial
  • In house method diffusion

SE gene detection

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

II) CRL CPS skills, tasks and perspectives

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

II.1.1) se gene detection for food poisonning investigations

Background: 21

21 Staphylococal Enterotoxin Staphylococal Enterotoxin (SE) types have (SE) types have been been identified identified

SE SE-like A J B K C : C1,C2,C3 L D M E N F TSST (Toxique Shock Syndrom Toxin) O G P H Q I U U and and U2 U2 R V V S T 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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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food 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 1 Primers 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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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

05-102 STA (sea) 05-80 STA (seb) 06-83 STA (sec) 06-71 STA (sed) FRI 326 (see) FRI 137 (sec-sed-ser) 08 CEB 325 STA 08 CEB 326 STA 08 CEB 327 STA FRI S6 (sea-seb) FRI 137 (sec-sed-ser) T- ladder 100 pb ser (123 pb) seb (164 pb) sec (451 pb) sed (278 pb) see (209 pb) ladder 100 pb FRI 361 (seg-sei-sej) 05-102 STA (seh) 367 F (seg-sei-sep) 08 CEB 325 STA 08 CEB 326 STA 08 CEB 327 STA 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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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food 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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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food 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 type spa 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

  • r www.spa.ridom.de
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CRL-CPS 2009 Activity Update

French Food Safety Agency

II.3 Other molecular typing methods : MLST (Multi

Locus Sequence Typing)

“ MLST technology is based on the polymorphism analysis 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 aroE glpF gmk pta tpi yqiL

109 Number of STs (Sequence Types) in the MLST database 1354 151 118 84 117 112 108 Number of alleles

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MRSA Training Course, April 20th-22nd 2009

CRL-CPS 2009 Activity Update

French Food Safety Agency

II.3 Other molecular typing methods : SCCmec

« SCCmec typing is based on the analysis by PCR of the mobile 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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CRL-CPS 2009 Activity Update

French Food Safety Agency

III) MRSA in French fattening pigs _________

A collaboration between the Afssa Ploufragan and Afssa Maisons Alfort for the French AMR NRL