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 Course, April 20th-22nd 2009 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 French Food 2 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 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 French Food 3 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 French Food 4 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 I.1.2) Criteria Regulation EC 2073/2005 microbiological criteria for foodstuffs : Matrix n c m M when Action 10 4 cfu/g 10 5 cfu/g Cheese from raw milk during process improve hygiene rippened when CPS high 100 cfu/g 1000 cfu/g - unpasteurisedcheese if >10 5 SET fresh unpasteurisedcheese 10 cfu/g 100 cfu/g 5 2 detection milk and whey powder 10 cfu/g 100 cfu/g end of process cooked crutaceans and 100 cfu/g 1000 cfu/g improve hygiene molluscan shellfish milk & milk products SET not detected in 5 0 end of process improve hygiene 25g If CPS > 10 5 French Food 5 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 I.1.3) CRL-CPS location : AFSSA-LERQAP • 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 French Food 6 6 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 I.2) CRL organizational chart Acting Director Laurent LALOUX Co-ordinator Bertrand LOMBARD Administrative affairs Deputy Co-ordinator Akpéné PLACCA Adrien ASSERE Bacteriology Enterotoxins Scientific Leader Scientific Leader Marie-Laure DE BUYSER Jacques-Antoine HENNEKINNE CPS detection/ Risk Typing Investigation enumeration Molecular Assessment TOP-BAC food-poisoning typing team CEB HMPA/MB MQER CEB French Food 7 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 I.3) Bacterial characterization and epidemiology Unit (CEB) staff involved in the CRL-CPS activities Unit leader Anne BRISABOIS Team manager Scientific leader Team manager Marie-Laure DE BUYSER Sophie ROUSSEL Sophie GRANIER Investigation food- Typing Antibioresistance poisoning Molecular typing team CEB CEB Project leader Benjamin FELIX Technician Technician Joël GROUT Trinh Tam DAO French Food 8 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 I.4) CRL-CPS activities of the Typing Unit 2009 2010 - 2011 SE gene detection - Training - In house method diffusion - Inter-laboratory trial - PFGE Optimisation following - In house method diffusion PFGE Harmony the Harmony protocol - Training PFGE Harmony MLST - In house method diffusion spa typing - In house method diffusion - spa / PFGE comparison French Food 9 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 II) CRL CPS skills, tasks and perspectives French Food 10 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 II.1.1) se gene detection for food poisonning investigations Background: 21 21 Staphylococal Enterotoxin Staphylococal Enterotoxin (SE) types have (SE) types have been identified identified been SE SE-like Non emetic or not Emetic effect described tested A J Classical SEs B K SEA is the main SE C : C1,C2,C3 L involved in food poisoning D M E N F � TSST (Toxique Shock Syndrom Toxin) O G P H Q I U and and U2 U2 U R V V S Recently described (Ono et al. 2008) T French Food 11 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 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 Multiplex 2 Primers from Primers and procedure from Mehrotra et al. , 2000 ( sea-e ) Bania et al. , 2006 Chiang et al. , 2008 ( ser ) French Food 12 CRL-CPS 2009 Activity Update Safety Agency
ser (123 pb) seb (164 pb) sed (278 pb) 13 sec (451 pb) II.1.3 Results see (209 pb) sea (102 pb 05-102 STA ( sea ) 05-80 STA ( seb ) 06-83 STA ( sec ) Multiplex 1 06-71 STA ( sed ) FRI 326 ( see ) FRI 137 ( sec-sed-ser ) 08 CEB 325 STA CRL-CPS 2009 Activity Update 08 CEB 326 STA 08 CEB 327 STA FRI S6 ( sea-seb ) FRI 137 ( sec-sed-ser ) T- ladder 100 pb sep (396 pb) seh (173 pb) sej (131 pb) seg (198 pb) sei (328 pb) MRSA Training Course, April 20th-22nd 2009 ladder 100 pb Multiplex 2 FRI 361 ( seg-sei-sej ) 05-102 STA ( seh ) 367 F ( seg-sei-sep ) 08 CEB 325 STA 08 CEB 326 STA Safety Agency French Food 08 CEB 327 STA T-
MRSA Training Course, April 20th-22nd 2009 II.2 PFGE subtyping 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 French Food 14 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 II.3 Other molecular typing methods : spa typing “ spa typing technology is based on the sequencing of protein A gene X region " � Wall protein, specific of S. aureus . Protein A � This protein interact with the immunoglobuline Fc region � Virulence factor 1113 f 1514 r 2150 bp Fc – binding region X - region C-terminus � 3 to15 different repeats of 24 bp spa type depend of the number of repeats � and their position inside the X region spa type spa type All spa types are available on: www.seqnet.org or www.spa.ridom.de French Food 15 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 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 “ Number of STs Number of alleles (Sequence Types) in the 109 arcC MLST database 151 aroE For Staphyloccoci the glpF 118 genes are : gmk 1354 84 pta 117 tpi 112 yqiL 108 All Sequence Types and isolates data are available on public databases : www.mlst.org French Food 16 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 II.3 Other molecular typing methods : SCC mec « SCC mec 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 5 SCC mec profils Type III Type IV Type V French Food 17 CRL-CPS 2009 Activity Update Safety Agency
MRSA Training Course, April 20th-22nd 2009 III) MRSA in French fattening pigs _________ A collaboration between the Afssa Ploufragan and Afssa Maisons Alfort for the French AMR NRL French Food 18 CRL-CPS 2009 Activity Update Safety Agency
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