Simultaneous Enrichment of E. coli O157:H7, Salmonella spp., and Listeria monocytogenes from Environmental Swabs and Detection by Multiplex-qPCR

Introduction: Escherichia coli O157:H7 (EHEC), Salmonella spp.(S), and Listeria monocytogenes (Lm) are the most significant pathogens with respect to FDA regulated food products. Standard methods for environmental swab sample analysis target only one pathogen in a sample by preparation of multiple pre-enrichment media according to the Bacteriological Analytic Manual; this is time consuming, and cost/labor intensive. Current research has identified candidate universal enrichment broths and methods for detection via real-time multiplex qPCR (mqPCR); therefore a rapid and accurate method to simultaneously enrich and detect multiple targets would provide a critical reporting tool.

Purpose: This study is a collaborative effort to identify a universal enrichment broth for the simultaneous enrichment of EHEC, S, and Lm from environmental swab samples.

Methods: A plastic tub, was marked with large (4x4 inch) and small (1x1 inch) grids for swabbing per AOAC. Duplicate grids were spiked with 100ul of high (HS) or low (LS) BHI broth culture concentrations of EHEC (1.82x10^{8 CFU/ml}; 1.82x10^{5 CFU/ml}), S (1.62x10^{8 CFU/ml}; 1.62x10^{5 CFU/ml}), and Lm (2.66x10^{8 CFU/ml}; 2.66x10^{5 CFU/ml}). Large and small surface area swabbing was performed using Dey-Engley broth moistened sponge-tipped swabs and cotton-tipped swabs respectively; followed by two-hour room temperature incubation, twenty-four hour enrichment in LEB broth at 35⁰C, DNA extraction, and mqPCR.

Results: mqPCR analysis detected multiple microorganisms in the HS grids in both the large and small surface areas.  Lm and EHEC were detected with Ct values of 25.74±1.09 and 31.79±1.58, respectively, in LEB broth enrichment from the HS large grid sample (n=2). There was a significant difference for Lm detection in HS of large vs. small surface areas (p<0.02), despite equal amounts of spiked organisms.

Significance: A universal enrichment broth to simultaneously enrich multiple bacterial pathogens would enhance assessment of processing facility sanitation while using fewer resources and reducing reporting time by 24-48 hours.