1. Recovery, detection and sanitizer susceptibility of Listeria spp. from meat processing environments -by- Jovana Kovačević June 15 th 2010

2. Outline Listeria spp. background Listeria spp. background Project Overview Project Overview  Comparison of sampling methods  Evaluation of detection methods  Occurrence and distribution of Listeria spp. in meat processing environments  Sanitizer susceptibility Summary Summary 2

3. 3 Project Members Food Safety Division, Alberta Agriculture, Food and Rural Development Food Safety Division, Alberta Agriculture, Food and Rural Development  Dr. Valerie Bohaychuk – Project Manager  Gary Gensler – Supervisor (Food Microbiology)  Robin King – Supervisor (Molecular Biology)  Deana Rolheiser – Technician (Food Microbiology)  Sonja Marshall – Technician (PFGE)  Dr. Pablo Romero Barrios – Epidemiologist University of Alberta, Department of Agricultural, Food and Nutritional Science, University of Alberta University of Alberta, Department of Agricultural, Food and Nutritional Science, University of Alberta  Dr. Lynn McMullen – Food Microbiology Professor, Graduate Student Supervisor  Jovana Kovacevic – Graduate Student

4. Six species: Six species: L. monocytogenesL. monocytogenes L. ivanoviiL. ivanovii L. seeligeriL. seeligeri L. innocuaL. innocua L. welshimeriL. welshimeri L. grayiL. grayi –Subspecies L. murray Genus Listeria 4

5. Gram positive, facultative anaerobic rods Gram positive, facultative anaerobic rods Motile at 10 to 25°C Motile at 10 to 25°C Grow between -0.4ºC and 50ºC Grow between -0.4ºC and 50ºC Isolated from: Isolated from:  Environmental sources: soil, water, effluents  Food processing environments  Variety of foods  Feces of humans and animals 5 Clin. Micorbiol. Rev. 14(3):584-640.

6. Project Goals Phase 1: To compare 3 methods for recovery of Listeria spp. in the environment of meat processing facilities Phase 2: To evaluate 4 methods for detection of Listeria spp. Phase 3: To assess the occurrence and distribution of Listeria spp. in meat processing environments Phase 4: To determine the susceptibility to sanitizers of persistent and nonpersistent strains of L. monocytogenes 6

7. 7 Project Overview Two Federally inspected meat processing facilities Edmonton, Alberta Two time points: After cleaning and sanitation but prior to processing (ACS) During or after processing but before sanitation (PRO) Sampling in 2005 and 2006 for 19 months, 3 week intervals 1) Comparison of environmental sampling methods: – –5 months – –Ten sampling locations: * 5 where RAW meat was processed * 5 where COOKED meat was handled – –Total of 720 samples

8. 8 Project Overview 2) Comparison of detection methods: – –Until 100 positive and 100 negative samples collected per method – –14 sampling locations: *7 where RAW meat was processed *7 where COOKED meat was handled – –Criteria for 3 methods reached after 11 samplings; 1 method required 14 samplings 3) Occurrence and Distribution of Listeria spp. – – All the samples collected by SS method and analyzed by culture method (n=820) – –19 months period

9. Phase 1: Comparison of Environmental Sampling Methods 9  soaked in 1 ml of neutralizing buffer neutralizing buffer  pre-wetted  soaked in 10 ml of neutralizing buffer neutralizing buffer 1) Cotton Swab (CS) 2) Sterile Sponge (SS) 3) Composite tissue (CT)

10. Phase 1: Sample Collection Sampling every 3 weeks, for 5 months in 2005/2006: 6 visits 120 samples / method / facility 240 samples for each method Total = 720 samples collected 10

11. 11 Phase 1: Sample Collection Table 1. Areas sampled for recovery of Listeria spp. in two federally inspected meat processing facilities. a Sampling locations added for the comparison of detection methods.

12. Sample Processing: ISO 11290-1 Confirmation Environmental Sample + Demi-Fraser Broth PALCAMOxford FB Confirmation 35°C for 24 to 48 h PALCAMOxford 12

13. Phase 1: Results 13 Figure 1. Number of samples that tested positive for presence of Listeria spp. obtained using a cotton swab (CS), a sterile sponge (SS) and a composite tissue (CT) sampling method. *Represents the total number of times samples were positive for Listeria spp.

14. Evaluated sensitivity and specificity of detection methods using culture method as “gold standard” Evaluated sensitivity and specificity of detection methods using culture method as “gold standard”  Petrifilm™ Environmental Listeria Plates (EL Petrifilm™)  Lateral flow immunoprecipitation (LFI) device - Reveal ® Listeria Test System Reveal ® Listeria Test System  Automated polymerase chain reaction (PCR) BAX ® BAX ®  Conventional culture ISO 11290-1 100 positive100 negative Samples collected until 100 positive and 100 negative samples were obtained Phase 2: Detection Methods 14

15. Phase 2: Materials and Methods 1)Petrifilm TM Environmental Listeria Plates (3M™ Microbiology, London, ON)   Tests were performed according to the Health Canada MFLP-11 method 15 Listeria spp. A

16. 2)Lateral flow immunoprecipitation - Reveal ® Listeria Test System (Neogen Corporation, Lansing, Mich.)  Lateral flow device combining immunoassay with chromatography (45 h) 16

17. 17 3)Polymerase chain reaction (BAX ®, DuPont Qualicon, Wilmington, DE)  Tests were performed according to the Health Canada MFLP-15 method http://www2.dupont.com/Qualicon/en_US/assets/downloads/baxproc.pdf

18. 18 Phase 2: Results Culture (n=440) Culture (n=440) –156/440 positives  35.5% – 110/328 positives  33.5% Petrifilm  (n=440) Petrifilm  (n=440) –103 positives  23.4% Reveal ® (n=328) Reveal ® (n=328) –105 positives  32.0% BAX ® (n=328) BAX ® (n=328) –109 positives  33.2%

19. Phase 2: Results Table 3. Sensitivity, specificity, and kappa values of the EL Petrifilm™, LFI and PCR methods relative to the culture method for identification of Listeria spp. in samples obtained from the environment of two federally inspected meat processing facilities. EL Petrifilm™ LFIPCR Sensitivity (%)50.695.599.1 Specificity (%)91.5100 Kappa0.4570.9580.993 Kappa (95% CI)0.370 – 0.5440.935 – 0.9950.980 – 1.00 19

20. 20 Definitions Sensitivity Sensitivity  Probability that a sample which is truly positive is detected as positive (Doho et al., 2003). Specificity Specificity  Probability that a sample which is truly negative is detected as negative (Doho et al., 2003). Test A PositiveNegative Test B PositiveabN1 NegativecdN2 N3N4NT

21. 21 Phase 3: Occurrence and Distribution Environmental samples collected for 19 months in 2005 and 2006 Environmental samples collected for 19 months in 2005 and 2006  Sampling method: SS only  Detection method: ISO culture method only  Total: 820 samples tested  249 (30.4%) positive for Listeria spp.

22. 22 Phase 3: Results Table 4. The number of samples collected from two meat processing facilities either ACS or PRO from either an area where raw meat was processed or an area where cooked products were handled that tested positive for Listeria spp. No. of positive samples/total number collected ACS*PRO † Raw Product Area Cooked Product Area Raw Product Area Cooked Product Area Facility A (n=652) 95/16322/163117/16314/163 Facility B (n=168) 0/42 1/420/42 Total95/20522/205118/20514/205 *ACS, After Cleaning and Sanitation and prior to processing †PRO, During or after Processing

23. 23 Phase 3: Results Figure 2. The number of samples that tested positive for different species of Listeria collected either after cleaning and sanitation (ACS) or during or after processing (PRO), from the areas where raw and cooked products were processed in two meat processing facilities.*Represents the total number of times samples were positive for Listeria spp. n= 410

24. 24 Figure 3. The distribution and number of the samples that tested positive for species of Listeria and the total number of times samples were positive for Listeria spp. in meat processing Facility A. Raw food areasCooked food areas

25. 25 Phase 3: Results Facility A Facility A  Overall contamination with Listeria spp. was 38.0% (248/652)  L. monocytogenes recovered from an area where raw meat was processed (157/326) and an area where cooked products were handled (25/326) Facility B Facility B  Only one sample (n=168) was positive for L. innocua

26. Phase 4: Susceptibility to Sanitizers Persistent and nonpersistent strains identified using Pulsed Field Gel Electrophoresis (PFGE) as part of a concurrent study Persistent and nonpersistent strains identified using Pulsed Field Gel Electrophoresis (PFGE) as part of a concurrent study Sanitizer susceptibility of L. monocytogenes Sanitizer susceptibility of L. monocytogenes  Planktonic cells in tryptic soy broth (24 h)  Growth on stainless steel (48 h)  Growth in the MBEC™ HTP assay (4 days) Sanitizers Sanitizers  E-San ® 10% (5% N-alkyl dimethyl benzyl ammonium chloride and 5% N-alkyl dimethyl ethyl benzyl ammonium chloride)  Perox-E ® (hydrogen peroxide and acetic acid) 26

27. 27 Phase 4: Materials and Methods L. monocytogenes strains L. monocytogenes strains  Two persistent  Two nonpersistent  L. monocytogenes ATCC 19115 (American Type Culture Collection, Manassas, VA) Stainless steel coupons Stainless steel coupons  Type 304, No. 4 finish, 12 mm diameter MBEC™ assay MBEC™ assay  High-throughput device (Innovotech Incorporated, Edmonton, AB)

28. 28 1) Susceptibility to Sanitizers: Planktonic Cells Centrifuged and decanted 1.9 ml of sanitizer or 0.85% saline for controls in each well 1 x 10 9 CFU/ml 30 s exposure time Serially diluted TSB 100 µl DEB 100 µl 35°C for 24 – 48 h Resuspended in 10 ml of saline 1 x 10 7 CFU/ml Sanitizer concentrations: E-San ® : 50, 100, 200, 300, 400, 800 ppm Perox-E ® : 70, 200, 500, 800, 1100 ppm

29. 29 2) Susceptibility to Sanitizers: Cells Grown on Stainless Steel Coupons 1 x 10 9 CFU/ml Sanitizer concentrations: E-San ® : 100, 200, 300, 400, 500, 600 ppm Perox-E ® : 900, 1100, 1300 ppm 5 min exposure time DEB 35°C for 48 h TSB 2 ml into each well Serially diluted 1 x 10 5 CFU/ml TSB 2 ml of sanitizer or 0.85% saline for controls in each well 35°C for 24 – 48 h

30. 30 3) Susceptibility to Sanitizers: Biofilms Grown on MBEC™ Devices Sanitizer concentrations: E-San ® : 5,000  39 ppm Perox-E ® : 19,200  150 ppm Two-fold dilution series Set up the sanitizer challenge plate Expose biofilms to sanitizers (10 min) Use challenge plates to obtain MIC (planktonic cultures)  streak contents of each well onto TSA Incubate recovery plate (DEB) at 35°C for 24 – 48 h

31. 31 Phase 4: Results

32. 32 Summary Sterile sponge and composite tissue superior environmental sampling methods than cotton swab Sterile sponge and composite tissue superior environmental sampling methods than cotton swab LFI and PCR-based methods excellent alternatives to conventional culture method for detection of Listeria spp., while EL Petrifilm™ is easy to use but less efficient LFI and PCR-based methods excellent alternatives to conventional culture method for detection of Listeria spp., while EL Petrifilm™ is easy to use but less efficient Perox-E ® is more efficient in inactivation of cells of L. monocytogenes adhered to stainless steel than E-San ® Perox-E ® is more efficient in inactivation of cells of L. monocytogenes adhered to stainless steel than E-San ® Concentrations far greater than the MRC for both E-San ® and Perox-E ® required for inactivation of biofilms grown for four days on MBEC™ devices Concentrations far greater than the MRC for both E-San ® and Perox-E ® required for inactivation of biofilms grown for four days on MBEC™ devices NO DIFFERENCE in susceptibility to sanitizers among persistent, nonpersistent and control strains of L. monocytogenes NO DIFFERENCE in susceptibility to sanitizers among persistent, nonpersistent and control strains of L. monocytogenes

33. 33 Acknowledgements Dr. Lynn McMullenDr. Lynn McMullen Dr. Valerie Bohaychuk and Listeria in Meat Plants (LMP) teamDr. Valerie Bohaychuk and Listeria in Meat Plants (LMP) team The financial support from The financial support from The Alberta Agricultural Research Institute,The Alberta Agricultural Research Institute, The Alberta Chicken Producers, andThe Alberta Chicken Producers, and The Food Safety Division, Alberta Agriculture and FoodThe Food Safety Division, Alberta Agriculture and Food The participation of two meat processing facilitiesThe participation of two meat processing facilities Phases 1 and 2 of the project were published in the Journal of Food Protection:Phases 1 and 2 of the project were published in the Journal of Food Protection: Kovačević, J., V. M. Bohaychuk, P. Romero Barrios, G. E. Gensler, D. L. Rolheiser and L. M. McMullen. 2009. Evaluation of environmental sampling methods and rapid detection assays for recovery and identification of Listeria spp. from meat processing facilities. J. Food Prot. 72(4): 696-701.

34. 34 http://www.umanitoba.ca/faculties/afs/soil_science/MSSS/Ecology/Cartoons/Cartoons %20Images/Microbes%20multiplying.jpg