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Evaluation of Methods of Enrichment and Compositing of Environmental Samples for Detection of Listeria monocytogenes.

Various methods exist for the enrichment and detection of Listeria spp. and L. monocytogenes from environmental samples. Procedures for the compositing of environmental samples are not as well-defined. In this study, different enrichment procedures involving Buffered Listeria Enrichment Broth (BLEB), University of Vermont Medium (UVM), and Fraser Broth (FB) were evaluated to determine the limits of detection (LOD) for L. monocytogenes from culture and from swabs of stainless steel and to assess the efficacy of composite sampling by wet (pooling of primary enrichments) and dry (pooling of swabs) procedures. For detection of cells in pure culture, the computed LOD95% values using a single-step BLEB or two-step UVM-FB enrichment were 0.33 and 0.49 CFU per 225 mL enrichment, respectively. No significant differences in detection were observed for procedures using either two-step BLEB-FB or UVM-FB enrichments for swabs of stainless steel when L. monocytogenes was inoculated at 2-6 log CFU; LOD95% values were 3.82 and 3.62 log CFU per 4 in2 area, respectively. Wet compositing of L. monocytogenes from culture with and without Romaine lettuce wash (RLW) resident microbiota was conducted using BLEB-FB and UVM-FB enrichment methods; both allowed detection of the pathogen at ratios of 1:1, 1:2, 1:4 and 1:7 (1 positive : x negative samples) with no loss in sensitivity. From swabs of stainless steel, L. monocytogenes was detected similarly for both wet and dry composites of up to eight samples (1:7) with RLW. However, the BLEB-FB method allowed for significantly faster detection (after 24 h of FB incubation) in composites of 1:4 and 1:7 compared to the UVM-FB method under the conditions tested. The results of this study provide data to evaluate the efficacies of the different enrichment procedures and aid in assessing the use of wet and dry compositing of environmental samples for use in food production and processing facilities as part of a Listeria control plan.