Comparison of Methods for Detection of Listeria on Wooden Shelves used for Cheese Aging: Challenges Associated with Sampling Porous Surfaces

This thesis examined the efficacy of various sampling and detection methods used for environmental monitoring of Listeria species on wooden surfaces used for cheese aging. Government agencies including the Food and Drug Administration (FDA) and United States Department of Agriculture (USDA) recommend enrichment methods coupled with use of environmental sponges and swabs. Our study compared efficacy of sponge swabs manufactured by 3MTM and World Bioproducts. There is a lack of research validating the best performing swab type and enrichment method combination that is sensitive when used on rough, porous surfaces. The sensitivity of these environmental sampling tools and methods are critical considerations to effectively monitor the presence of Listeria species on wooden boards used during aging of artisan cheese. Seasoned spruce wooden shelves, cut into 100cm2 replicates, were spot inoculated with varying concentrations of Listeria species inocula, the Listeria species strains consisted of two L. monocytogenes strains and a Green Florescence Protein (GFP) expressing strain of L. innocua. The inoculated wooden surface was swabbed with three environmental sampling sponge/swab formats (World Bioproducts© EZ ReachTM environmental swabs (WBEZ) with HiCap (WBHC) and Dey-Engley (WBDE) neutralizing broths; and 3MTM environmental swabs (3MTM) with Dey-Engley neutralizing broth). Enumeration methods were used to determine the low target limits of detection. Once the low target concentrations were identified, five enrichment methods consisting of 3MTM Listeria Environmental Plate, FDA, Dual Enrichment, modified USDA, and modified FDA were challenged against low concentrations of Listeria species inocula (0.01 cfu/cm2, 0.1 cfu/cm2, 1 cfu/cm2) and the three environmental sponge swab formats. Performance of the swab formats was assessed by collection of naturally contaminated environmental samples (n=405) from dairy farm environments, swabbing where wooden surfaces existed, and analyzed using the most effective enrichment methods found from previous experiments. Lastly, the wooden surfaces and sponge swabs were observed under a Florescent Microscope using GFP L. innocua to visually determine how each sponge material of the 3MTM and World Bioproducts recovered the inocula. When wood surfaces were inoculated at high concentration levels of Listeria spp. , all swab formats performed equally for detecting Listeria. Success of positive recovery at low concentrations was variable, where enrichment methods and swabs were not dependent on each other. The swab format that worked best for detecting low levels of Listeria species was the WBDE sponge swab. The WBDE swab also performed the best in dairy farm environmental sampling. The m-USDA enrichment method was found to be most effective in recovery and repair of low and potentially injured Listeria spp. Wooden surfaces are rough and porous and should be taken into consideration when creating an environmental sampling plan for these food contact surfaces. All swabs and methods performed with only slight variation, but the variation could be significant when monitoring wooden shelves with low level contamination of Listeria species. Artisan cheesemakers who use wooden shelves during the aging of their cheese, should ensure use of the most sensitive detection method