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

Transcription

1 CRL - Coagulase Positive Staphylococci (CRL-CPS) : Activity Update Benjamin FELIX AFSSA Maisons Alfort b.felix@afssa.fr 1

2 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 2

3 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 3

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5 I.1.2) Criteria Regulation EC 2073/2005 microbiological criteria for foodstuffs : Matrix n c m M when Action Cheese from raw milk rippened unpasteurisedcheese 10 4 cfu/g 100 cfu/g 10 5 cfu/g 1000 cfu/g during process when CPS high improve hygiene - fresh unpasteurisedcheese cfu/g 100 cfu/g if >10 5 SET detection milk and whey powder 10 cfu/g 100 cfu/g end of process cooked crutaceans and molluscan shellfish 100 cfu/g 1000 cfu/g improve hygiene milk & milk products If CPS > SET not detected in 25g end of process improve hygiene 5

6 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 for most of its activities in particular analytical sectors involved in CRL activities 6

7 I.2) CRL organizational chart MRSA Training Course, April 20th-22nd 2009 Acting Director Laurent LALOUX Co-ordinator Bertrand LOMBARD Deputy Co-ordinator Adrien ASSERE Administrative affairs Akpéné PLACCA Bacteriology Enterotoxins Scientific Leader Marie-Laure DE BUYSER Scientific Leader Jacques-Antoine HENNEKINNE CPS detection/ enumeration HMPA/MB Investigation food-poisoning CEB Typing Molecular typing team CEB Risk Assessment MQER TOP-BAC 7

8 I.3) Bacterial characterization and epidemiology Unit (CEB) staff involved in the CRL-CPS activities Team manager Sophie GRANIER Antibioresistance Scientific leader Marie-Laure DE BUYSER Investigation foodpoisoning CEB Unit leader Anne BRISABOIS Team manager Sophie ROUSSEL Typing Molecular typing team CEB Project leader Benjamin FELIX Technician Trinh Tam DAO Technician Joël GROUT 8

9 I.4) CRL-CPS activities of the Typing Unit SE gene detection PFGE Harmony MLST spa typing In house method diffusion - PFGE Optimisation following the Harmony protocol - In house method diffusion - In house method diffusion Training - Inter-laboratory trial - In house method diffusion - Training PFGE Harmony - spa / PFGE comparison 9

10 II) CRL CPS skills, tasks and perspectives 10

11 II.1.1) se gene detection for food poisonning investigations Background: 21 Staphylococal Enterotoxin (SE) types have been identified SE Emetic effect described A Classical SEs B C : C1,C2,C3 D E F TSST (Toxique Shock Syndrom Toxin) G H I R S Recently described (Ono et al. 2008) T SEA is the main SE involved in food poisoning SE-like J K L M N O P Q U and U2 V Non emetic or not tested 11

12 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.,

13 13 MRSA Training Course, April 20th-22nd STA (sea) STA (seb) STA (sec) 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 ladder 100 pb FRI 361 (seg-sei-sej) STA (seh) 367 F (seg-sei-sep) 08 CEB 325 STA 08 CEB 326 STA 08 CEB 327 STA T- II.1.3 Results sec (451 pb) sed (278 pb) see (209 pb) seb (164 pb) ser (123 pb) sea (102 pb Multiplex 1 Multiplex 2 sep (396 pb) sei (328 pb) seg (198 pb) seh(173 pb) sej (131 pb)

14 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

15 II.3 Other molecular typing methods : spa typing spa typing technology is based on the sequencing of protein A gene X region " Protein A Wall protein, specific of S. aureus. 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 All spa types are available on: or spa type 15

16 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 For Staphyloccoci the genes are : arcc aroe glpf gmk pta tpi yqil Number of alleles All Sequence Types and isolates data are available on public databases : Number of STs (Sequence Types) in the MLST database

17 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 17

18 III) MRSA in French fattening pigs A collaboration between the Afssa Ploufragan and Afssa Maisons Alfort for the French AMR NRL 18