Department of Agriculture and. Food Laboratory. Cynthia M. Mangione

Transcription

1 Department of Agriculture and Markets Food Laboratory Cynthia M. Mangione

2 Administered i d by Microbiological Data Program (MDP) United States Department of Agriculture (USDA) Agriculture Marketing Services (AMS) Monitoring Programs Office (MPO) Martha Lamont, Director

3 Microbiological Data Program Pesticide Data Program (PDP) President s Food Safety Initiative (1989) Creation of a National Pesticide Residue Program (1991) Microbiological Data Program (MDP) President s Food Safety Initiative (2001)

4 MDP - Goals Provide statistically valid information about foodborne pathogens on fresh fruit and vegetables and develop baseline data. Voluntary data gathering not a regulatory or enforcement effort.

5 MPD Participating States State Agriculture and Consumer Services Laboratories California (sampling only) Colorado Florida Maryland (sampling only) Michigan Minnesota New York Ohio Texas (sampling only) Washington Wisconsin USDA National Science Laboratory (analytical services only)

6 MDP Participants Map

7 MDP - Current Analytes and Commodities Salmonella E. coli O157:H7 STEC and ETEC

8 MDP Peanut Butter In Feb/March 2009 asked by the CDC to test PB being sold in retail stores for Salmonella. ll The program has analyzed approximately 1300 samples of PB. To date no Salmonella has been detected. Four (4) isolates of Enterobacter sakazakii have been found.

9 MDP - Future Commodities October 1st Cantaloupe Cilantro

10 MDP Sample Collection Samples collected at Terminal Markets and Distribution Centers

11 MDP Sample Collection

12 MDP Sample Processing and Analysis Weighed out Round Tomatoes 3 whole tomatoes Roma Tomatoes 5 whole tomatoes Cantaloupe one whole cantaloupe Lettuce 375 g Green Onions 200 g Hot Peppers 200 g Alfalfa sprouts 50 g Spinach 375 g Peanut Butter g Cilantro 375 g

13 MDP Sample Processing and Analysis Sample Preparation Surface wash/soak (except spouts) Pre-enrichment UPB (PB - Lactose Broth), Postenrichment (O157:H7) - mec+n Samples Pooled (except sprouts) DNA Extraction Maxwell 16

14 MDP Sample Processing and Analysis Screening PCR DuPont Qualicon BAX Q7 mpcr using BioRad Laboratories I-Cycler Thermal Cycler

15 MDP Sample Processing and Analysis Confirmations Immunomagnetic Separation (IMS) Procedure Dynal BeadRetriever Biochemicals biomerieux VITEK 2

16 MDP Sample Processing and Analysis Flowchart Produce Sample + UPB Surface Wash (Stomach for SR, PB) All commodities except SR: Soak/incubate in wash eluent SR: Remove from wash eluent Add 25ml of each UPB sample to 225ml mec+n for LT, SP, and GO. Incubate 35 o for hours Incubate 35 o for Hours Pool 10ml of each UPB sample from same sample site (3) Add 25ml of each UPB sample to 225 mec+n for SR. Incubate 35 o for hours Pool 10ml of each mec+n sample from same sample site (3) Perform DNA extraction Perform BAX PCR for Salmonella (all commodities) Perform BAX PCR for E. coli O157:H7 (LT, SP, GO, SR) Perform mpcr for ETEC/STEC (all commodities) If pooled sample is positive, culturally confirm pool and individual samples OR if individual SR sample is positive, culturally confirm

17 MDP - Confirmations Salmonella E. coli O157:H7 RV Incubate at 42 C hrs UPB TT Incubate at 42 C hrs mec+n UPB mec+n Dynal BeadRetriever 10 typical colonies to TSI and LIA One set of plates incubated at 35 o C the other at 42 o C Suspicious Reactions to SBA Isolated colonies to VITEK and Serology 10 typical colonies to respective plates VITEK - Biochemical Confirmation Incubate at 35 C hrs 5 colonies on SPA and full confirmation VITEK - Biochemical Confirmation Serology Confirmation

18 MDP ETEC MDP - Confirmations STEC UPB Incubate at 44 ±2 C for hrs UPB TP Incubate at 35 ±1 C for hrs Incubate at 42 ±2 C for hrs Incubate at 35 ±1 C for hrs mec+n mec+n TP Incubate at t44 ±2 C for hrs Incubate at 42 ±2 C for hrs Incubate at 35 ±1 C for hrs Incubate at 44 ±2 C for hrs 20 typical colonies to LST broth 20 typical colonies to LST broth Perform DNA extraction on 20 individual LST broths mpcr for 20 isolates mpcr POS LST broths streak for isolation on selective media Incubate at 35 ±1 C for hrs Perform DNA extraction on 20 individual LST broths mpcr for 20 isolates mpcr POS LST broths streak for isolation on selective media Incubate at 35 ±1 C for hrs 5 typical colonies re-streak in selective media and SBA 5 typical colonies re-streak in selective media and SBA Incubate at 35 ±1 C for hrs Incubate at 35 ±1 C for hrs 5 different isolates from SBA to Biochemical confirmation 5 different isolates from SBA to Biochemical confirmation 3 E.coli isolates to mpcr re-confirmation 3 E.coli isolates to mpcr re-confirmation

19 Salmonella Timeframe Sun Mon Tues Wed Thur Fri Sat Samples Taken Samples Rec in lab Wt out, eluted & Incubated DNA Extracted Maxwell 16 BAX PCR Screen If Positive Streaked to Selective agars LIA TSI Set up SBA Streaked BioChemicals VITEK II Serotyping PFGE Plugs Made Gel Run Posted on PulseNet

20 E. coli O157:H7 Timeframe Sun Mon Tues Wed Thur Fri Sat Samples Taken Samples Rec in Lab Wt out, eluted & Incubated Secondary Enrichment mec+n overnight DNA Extracted Maxwell 16 BAX PCR Screen If Positive Secondary Enrichment at 42 o C IMS Dynal BeadRetriever Streaked to Selective agars Re-streaked to selective agars Streaked to SBA and NA Slant BioChemicals VITEK II Serology PFGE Plugs Made Gel Run Posted on PulseNet

21 STEC/ETEC Timeframes Sun Mon Tues Wed Thur Fri Sat Samples Taken Samples Rec in Lab Wt out, eluted & Incubated DNA Extracted Maxwell 16 DNA Amplified icycler mpcr Gels Run If Positive Secondary Enrichment Streaked to Selective agars Pick colonies to LST DNA mpcr Gels Re-Streaked Re-Streaked BioChemicals PFGE Plugs Gel Run Extracted Maxwell 16 Run to Selective to Selective VITEK II Made DNA agars agars DNA Amplified Extracted icycler Maxwell 16 DNA Amplified icycler Posted on PulseNet

22 MDP Data Commodity Total Cantaloupe , , , , , , ,037 12,430 Celery 2,175 2,190 1,113 5,478 Cilantro 572 1, ,913 Green Onions 1,128 2,294 1,536 1, ,166 Hot Peppers Lettuce leaf 2,180 2,202 3,340 2,298 1,530 11,550 Lettuce Romaine 2,177 2,195 4,372 Lettuce, Bagged 1,039 2,078 3,117 Onions Parsley 588 1,118 1,706 Spinach 2,078 2,078 Sprouts, Alfalfa 72 1,512 1,047 2,039 4,670 Tomatoes 2,706 2,194 2,237 2,304 1,535 1,064 2,702 14,742 Annual Totals 10,315 10,965 11,211 11,512 7,646 5,279 11,669 68,597

23 MDP Salmonella Data Year Number of Confirmed Isolates * Total 44 * January through April Data

24 MDP Salmonella Data Number of Commodity Isolates Cantaloupe 7 Celery 0 Cilantro 2 Green onions 2 Hot peppers 1 Lettuce 6 Bagged Lettuce 1 Onions 0 Parsley 5 Spinach 1 Alfalfa Sprouts 12 Tomatoes 2

25 MDP Pathogenic E. coli Data Number of Year Isolates TOTAL 37

26 MDP Pathogenic E. coli Data Commodity Number of isolates Cantaloupe 2 Celery 0 Cilantro 5 Green onions 3 Hot peppers 0 Lettuce 9 Bagged Lettuce 3 Onions 0 Parsley 5 Spinach 8 Alfalfa Sprouts 2 Tomatoes 0

27 MDP Good Fit with PulseNet? The majority of food safety laboratory testing takes place at the state level > 328,000 food samples tested/yr. for food safety parameters nationally by state labs. Significantly more samples than the FDA. Most testing is performed in labs located in state departments of agriculture.

28 For Example: NYS Dept. of Agriculture and Markets Food and Dairy Microbiology; analyzes over 15,000 food samples/yr. ~ 2000 of these for foodborne pathogens and the remainder for other microbial parameters. Food and Dairy Chemistry; analyzes over 3000 food samples/yr. ~ 1500 for chemical hazards. Pesticide residues; analyzes over 2500 produce and water samples/yr. for > 200 different pesticide residues and a variety pharmaceutical products.

29 MDP Good Fit with PulseNet? PulseNet membership by Ag sector labs allows for more timely submission of food origin isolates. Helps foster better relationships and integration ti of allied food safety programs in health and agriculture agencies. Supports national agendas for a fully integrated food safety system.

30 MDP and PulseNet

31 Microbiological Data Program Special Thanks to the NYS MDP staff: Gary Barnard April Vrba Jennifer Conaway Michele Pickett

32 Thank you! Cynthia M. Mangione