Intensive Sampling for E. Coli, Salmonella, Listeria Moncytogenes & Initial Survey & Detection Methods for Norovirus, in the Salinas Valley

Approach: <br/>
ARS will coordinate with scientists at FDA and NASA to plan for the collection of at least 525 samples during this project. In the design, the goal is to achieve optimal “chain of custody” of samples and shorter times from field to lab. Time will be taken during the design phase to assess the benefits of more frequent sampling of a single site, or limited number of sites, to monitor runoff and/or movement during heavy precipitation events. For example, a swab could be introduced into flowing water every day, twice a day, four times a day, for a few days. Sites could be selected based on previous data indicating higher incidence relative to other sites.The sampling sites will be picked from among many that have previously been sampled by ARS. Many of the potential sites are located near produce fields and/or are watersheds that flow through a large number of fields. Type of Work to be completed: We will use “Moore Swabs” for sampling the sites because they pick up much higher levels of bacteria flowing by than can a grab sample. As per plan and schedule, a trained sampler will label, place and extract the “Moore Swabs”, and take the samples to the lab for analysis. The laboratory is approximately 2 to 3.5 hours from the relevant watershed sampling sites in the proposed study (northern Monterey and San Benito counties and Southern Monterey County) based on previous incidence data.The cost of analysis of any environmental sample for STEC and Salmonella are high due to the expensive chromogenic media needed for robust isolation and identification. The STEC method involves enrichment culture, Dynal O157 IMS beads, then subculture on four different media (CT-SMAC, Rainbow O157, Chromagar O157 and Sheep red blood cell agar), and intensive colony picking, RT-PCR for Shigatoxin (Stx) and characterization of each strain by serotyping for the “top 10 serovars,” virulence genes (eae, hlyA, stx1, stx2, fliC, perosamine synthase), PFGE (O157 only), and 11 loci MLVA for O157 STEC strains and 7 loci MLVA-ompA sequence hybrid method for non-O157 STEC strains. Salmonella strains are isolated by non-selective enrichment, then IMS capture and plating on MSRV medium, and colonies are subcultured on XLD and other media. Isolates are confirmed as Salmonella by PCR and fingerprinted by PFGE and MLST. Water samples will be tested by Colilert assay also for MPN of generic E. coli.