TECHNICAL REPORT. Faster Recovery and Detection of Listeria using. with Culture Method. Prepared by

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1 TECHNICAL REPORT Faster Recovery and Detection of Listeria using Elite Listeria Enrichment Media with Culture Method Prepared by Sergiy Olishevskyy, PhD Vice President, Research and Development FoodChek Laboratories Inc. 7755, av Duplessis Saint-Hyacinthe, Quebec J2R 1S5 Canada Phone: This method s performance was reviewed by AOAC RESEARCH INSTITUTE and was found to perform to the manufacturer s specifications. This method s performance was reviewed by AFNOR and was found to perform to the manufacturer s specifications in accordance with the NF Validation general rules and NF102 certification rules. Method has been approved for publication in the HEALTH CANADA Compendium of Analytical Methods.

2 Elite Listeria Enrichment Media competitive advantages for culture detection methods. Single-step enrichment Enhanced recovery of injured Listeria Rapid growth of fastidious species High accuracy Optimized presumptive detection Reduced broth consumption Effective temperatures 29 to 37 C Inhibited or delayed competitors growth Developed and launched by FoodChek Systems Inc., Actero Elite Listeria Enrichment Media (Actero Elite Listeria), a new culture broth that combines a nutritive base with selective agents optimized to efficiently target competing flora without affecting the growth of sublethally injured Listeria being used for the enrichment of food and environmental samples. 2

3 Technical Report Table of Contents Growth of Uninjured and Heat-Injured L. monocytogenes in Different Culture Media Used for the Enrichment of Foods 4 Growth Kinetics Data 5 Growth of Competing Bacteria in Different Culture Media Used for the Enrichment of Foods 6 Growth of Fastidious Species of Listeria in Different Culture Media Used for the Enrichment of Food Samples 7 Co-culture of Injured L. monocytogenes with Competing Bacteria in Different Media Used for the Enrichment of Foods 8 FoodChek s Actero Elite Listeria Enrichment Media Culture Method for the Detection of Listeria 9 Table 1. Performance Parameters for the Detection of Listeria Using a Culture Method in Environmental and Food Samples Enriched with Actero Elite Listeria Enrichment Media (AOAC Method Comparison Validation Studies, ) 10 Recovery of Low Numbers of L. monocytogenes in Food and Environmental Samples Using Actero Elite Listeria Enrichment Media 11 Actero Elite Enrichment Media product list. 12 Appendix 13 Actero Elite Enrichment Media compatible with any diagnostic system. 14 Actero Elite Enrichment Media and Real-Time PCR Assays for Listeria and Salmonella validated matrices. 15 3

4 Growth of Uninjured and Heat-Injured L. monocytogenes in Different Culture Media Used for the Enrichment of Foods 0.6 Uninjured Listeria O D n m O D n m Tim e (H ours ) Heat-Injured Listeria Culture Conditions Initial Inoculum: 20 CFU/well Medium Volume: 200 μl Temperature: 35 C A c te ro T M L is te ria Time: 22 hours A 2 4 c te L is ro T M te ria L is E te n ria ric 2U 4 V ML is te ria E n ric h m e n t Legend UL EV B M O x o id A Actero c ro Elite T Listeria Medium A te ro T M M L ria L is te ria BEL B E B O x(o o id x o id ) 2 BLEB 4 Medium A N S R B L is is te te ria ria E n ric h m e n t B ro L Eth B (O ric (Nx eo oid g) e n ) U UVM V M 1 A N S R (N e o g e n ) L E B O x o id id B L E B (O x o id ) (O id A N S R (N (N e o g e n ) Actero Elite Listeria shows one of the shortest lag phase as well as one of the highest growth rate for L. monocytogenes. In the case of sublethally-injured L. monocytogenes, Actero Elite Listeria demonstrates a strong potential to repair the growing capacities of bacterial cells more efficiently and faster than other media. Tim e (H ours ) T im 1 6 e (H 1 o 8 u rs ) T im ime (H (Ho u rs rs )) 4

5 Growth Kinetics Data Bacterium Kinetics Parameter Actero TM Elite Listeria Primary Enrichment Media Single-Step Enrichment Media# BLEB UVM Medium A Medium B Uninjured Listeria Heat-injured Listeria LPD 9.3 ± ± 0.56** 16.4 ± 0.75*** 12.9 ± 1.40* 11.3 ± 0.95* GR ± ± 0.02* ND ± ± 0.06 LPD 11.1 ± ± 1.26** 18.1 ± 0.88** 13.0 ± ± 0.59** GR ± 0.06 ND ND ND ND Notes: LDP Lag Phase Duration; GR Growth Rate ND Not Determined: Kinetics parameters could not be defined because all growth stages were not observed during the incubation period #Commercially available media allowing single-step enrichment of Listeria *P<0.05, ** P<0.01, *** P<0.001 as compared to the corresponding values obtained for Actero Elite Listeria 5

6 Growth of Competing Bacteria in Different Culture Media Used for the Enrichment of Foods 1.5 Pseudomonas aeruginosa O D n m O D n m Enterococcus faecalis Culture Conditions Initial Inoculum: 2x10 5 CFU/well Medium Volume: 200 μl Temperature: 35 C A c te ro T M L is te ria Time: 22 hours A 2 4 c te L is ro T M te ria L is E te n ria ric 2U 4 V ML is te ria E n ric h m e n t Legend UL EV B M O x o id A Actero c ro Elite T Listeria Medium A te ro T M M L ria L is te ria BEL B E B O x(o o id x o id ) 2 BLEB 4 Medium A N S R B L is is te te ria ria E n ric ric h m e n t B ro L E th B (O (N x eo oid g ) e n ) U UVM V M 1 A N S R (N e o g e n ) L E B O x o id id B L E B (O x o id ) (O id A N S R (N (N e o g e n ) Actero Elite Listeria demonstrates an excellent capacity in controlling the growth of competing bacteria such as E. faecalis and P. aeruginosa T im 1 6 e (H 1 o 8 u rs ) T im ime (H (Ho u rs rs )) 6

7 Growth of Fastidious Species of Listeria in Different Culture Media Used for the Enrichment of Food Samples Listeria grayi O D n m O D n m Listeria ivanovii Culture Conditions Initial Inoculum: 200 CFU/well Medium Volume: 200 μl Temperature: 35 C A c te ro T M L is te ria Time: 22 hours A 2 4 c te L is ro T M te ria L is E te n ria ric 2U 4 V ML is te ria E n ric Legend UL EV B M O x o id A Actero c ro Elite T Listeria Medium A te ro T M M L ria L is te ria BEL B E B O x(o o id x o id ) 2 BLEB 4 Medium A N S R B L is is te te ria ria E n ric h m e n t B ro L Eth B (O ric (Nx eo oid g) e n ) U UVM V M 1 A N S R (N e o g e n ) L E B O x o id id B L E B (O x o id ) (O id A N S R (N (N e o g e n ) Actero Elite Listeria has the strongest capacity to promote the growth of fastidious species of Listeria such as L. ivanovii and L. grayi as compared to the other media tested T im 1 6 e (H 1 o 8 u rs ) T im ime (H (Ho u rs rs )) 7

8 L o g C F U /m L Co-culture of Injured L. monocytogenes with Competing Bacteria in Different Media Used for the Enrichment of Foods Culture Conditions Listeria Starting Inoculum: 10 3 CFU/mL E. faecalis Starting Inoculum: 10 5 CFU/mL Medium Volume: 10 ml Temperature: 35 C Time: 22 hours 4 2 Legend Actero Elite Listeria UVM 0 1 :1 0 0 L is te ria + E. fa e c a lis 1 :1 0 0 L is te ria + E. fa e c a lis 1 :1 0 0 L is te ria + E. fa e c a lis Single-Step Enrichment Broth 1:100 Listeria + E. faecalis In a co-culture model of sublethally heat-injured L. monocytogenes and a high number of the competing bacteria, Actero Elite Listeria advantages are the selective recovery and growth of the target bacterium. 8

9 FoodChek s Actero Elite Listeria Culture Method for the Detection of Listeria 9

10 Table 1. Performance Parameters for the Detection of Listeria Using a Culture Method in Environmental and Food Samples Enriched with Actero Elite Listeria Enrichment Media (AOAC Method Comparison Validation Studies, ) Matrix Total Tested P N FP FN Relative Sensitivity % Relative Specificity % FP Rate % FN Rate % Test Efficacy % Environmental Food TOTAL

11 Recovery of Low Numbers of L. monocytogenes in Food and Environmental Samples Using Actero Elite Listeria Enrichment Media Actero Negative Outcomes Actero Positive Outcomes RM Positive Outcomes RM Negative Outcomes Environmental Samples Food Samples Notes: RM Reference Method * P<0.05; *** P<0.001 Actero Elite Listeria has a strong ability to provide an ideal growing environment for sublethally injured Listeria in foods and environmental samples to allow for accelerated and reliable detection. Enrichment with Actero Elite Listeria results in significantly better recovery of Listeria from environmental samples (38.8% of increase) and foods (15.3% of increase) as compared to the reference methods. Using a culture method for detection of Listeria, Actero Elite Listeria significantly reduces the time-to-results to as short as 48 hours. Actero Elite Listeria is granted AOAC-RI Certification and approved for the detection of Listeria in food and environmental samples using culture method helping to reduce costly false positives and inventory holds. 11

12 Listeria United States Canada Europe Elite Listeria Enrichment Media product list. Our patented Actero Elite Listeria Enrichment Media is a selective bacterial culture medium specifically optimized for the recovery of Listeria spp. from environmental and food samples in a single-step enrichment. We continue to strive to be the food industry s single, trusted provider for pathogen and other microorganism testing supplies and equipment. Actero Elite Listeria Enrichment Media is compatible with RT-PCR and culture methods and represents the result of true food safety innovation. Microorganism FoodChek Cat. # Product Name Format Description FCM-011 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 500G Specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples. 500G dehydrated media (yields 9.29L rehydrated media). FCM-022 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 2KG A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples. Four (4) 2KG of dehydrated Actero Listeria Enrichment Media. FCM-023 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 10KG A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples. 10KG of dehydrated ACTERO Listeria Enrichment Media. FCM-045 FCM-046 ACTERO ELITE LISTERIA ENRICHMENT MEDIABOX 5L 10L A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples in liquid format. A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples in liquid format. FCM-202 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 25KG A selective medium, specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples in dehydrated format. FCM-203 FCM-204 ACTERO ELITE LISTERIA ENRICHMENT MEDIABAG 3x3L 2x5L A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental sample. A box of 3 x 3L liquid Actero Elite Listeria media. A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental sample. A box of 2 x 5L liquid Actero Elite Listeria media. 12

13 APPENDIX 13

14 Elite Enrichment Media compatible with any diagnostic system. Current Listeria spp., Listeria monocytogenes and Salmonella spp. Validations Diagnostic Instrument External Validation License/ Certification # Pathogen Matrix Method Developer AOAC PTM (3) Listeria spp. and Listeria monocytogenes Variety of food and environmental surfaces BAX System RT-PCR (1) AOAC PTM (3) Salmonella spp. Health Canada (5) MFLP-79 Listeria spp. Variety of food and environmental matrices Stainless steel, ceramic, plastic, sealed concrete, and rubber Hygiena, LLC Atlas System (2) AOAC PTM (3) Listeria spp. Stainless steel, PVC and sealed concrete Roka Bioscience, Inc. Agilent AriaMx AFNOR (4) EGS 38/ AOAC PTM (3) , & Listeria spp. and Listeria monocytogenes Listeria spp. and Listeria monocytogenes Variety of food and environmental surfaces Variety of food and environmental surfaces Eurofins GeneScan Bio-Rad CFX96 Touch DeepWell AFNOR (4) EGS 38/ AOAC PTM (3) , & Listeria spp. and Listeria monocytogenes Listeria spp. and Listeria monocytogenes Variety of food and environmental surfaces Variety of food and environmental surfaces Eurofins GeneScan Xpress LM AOAC PTM (3) Listeria monocytogenes Stainless steel, plastic, ceramic tile Crystal Diagnostics MICT Reader Health Canada (5) MFLP-99 Listeria spp. Environmental surfaces AOAC PTM (3) Listeria spp. Environmental surfaces FoodChek Systems Inc. AOAC PTM (3) Salmonella spp. Variety of egg formats and environmental surfaces (1) Formerly an asset of DuPont Nutrition & Health (2) Acquired by IEH Laboratories & Consulting Group (3) This method s performance was reviewed by AOAC RESEARCH INSTITUTE and was found to perform to the manufacturer s specifications. (4) This method s performance was reviewed by AFNOR and was found to perform to the manufacturer s specifications in accordance with the NF Validation general rules and NF102 certification rules. (5) Method has been approved for publication in the HEALTH CANADA Compendium of Analytical Methods. 14

15 Elite Enrichment Media and Real-Time PCR Assays for Listeria and Salmonella validated matrices. ACTERO ELITE LISTERIA ENRICHMENT MEDIA ACTERO ELITE SALMONELLA ENRICHMENT MEDIA AOAC RI-PTM License # AOAC RI-PTM License # (SUITE) AOAC RI-PTM License # AOAC RI-PTM License # (SUITE) Internally Validated Concrete/Sealed Fish/Raw 25 g Stainless Steel Almonds/Raw 375 g Flour/Wheat 25 g Stainless Steel Ham/Cured 125 g Plastic Beef/Ground 25 g Plastic Ice Cream 25 g Cheese/Soft Mexican Style Frankfurters Salmon/Cold Smoked Shrimp/Frozen Cooked Spinach/Fresh Bagged 25 g Milk/Pasteurized 25 g 125 g Turkey/Smoked Breast Chocolate/ Liquor Chocolate/Milk Bar 125 g Cocoa/Powder 25 g 25 g Eggs/Shell 25 g Beef/Ground 375 g 25 g Butter/Peanut 25 g 20 Eggs Chicken/Carcass Rinse Chicken/Ground 30 ml 25 g Pet Food/Dry 25 g Egg/Dried Whole 100 g 25 g Pet Food/Dry 375 g Egg/Liquid Whole* 25 g 100 g Onions/ Dehydrated Pepper/Black Whole 25 g 375 g Parsley/Dried Pepper/Black Whole 25 g 25 g Raisins/Dried 25 g *Pasteurized 15

16 Elite Enrichment Media cutting edge food safety solutions. FOODCHEK SYSTEMS INC. GLOBAL Corporate Head Office: 450, Street SW Calgary, Alberta, T2R 1J6 Canada Terry Macartney Executive Vice President, Operations and Product Development North America Toll Free: x 4 Phone: x 4 tmacartney@foodcheksystems.com UNITED STATES Carl (Chip) Zerr III Vice President, Product Integration Phone: czerr@foodcheksystems.com foodcheksystems.com Contact us today to purchase our products or ask for a free sample.