2012 Annual Report
1a.Objectives (from AD-416):
Acquire E coli and Salmonella contamination data, necessary for parameterizing the predictive geospatial risk assessment model (PGRAM) developed for FDA by NASA.
1b.Approach (from AD-416):
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.
The presence of bacterial foodborne pathogens and Norovirus (NV) in watersheds in the vicinity of raw produce production is a concern. During the initial phase of this project, approximately 25 public access watershed sites within Monterey County have been tested twice a month by planting a swab for 24 hr in the water and then removed and processed in the lab for STEC, Salmonella enterica and Listeria monocytogenes. The isolated strains have been analyzed for O-antigen, virulence factors and genotype. To date, >550 samples have yielded 46, 64, 333 and 233 samples positive for O157 STEC, non-O157 STEC, Salmonella enterica and Listeria monocytogenes, respectively. The preliminary results indicate: (i) sites that are positive more frequently than other sites, (ii) sampling days with high incidence of pathogen compared to other days, especially, for S. enterica and L. monocytogenes, (iii) association with cumulative rainfall preceding and during sampling, (iv) and a correlation of generic E. coli concentration per swab with incidence of all pathogens. NV could be present in watersheds due to leaking septic systems, improper RV waste removal, improperly treated recycled wastewater, or other unknown factors. We have developed a NV detection method for measuring low concentrations of NV in highly diluted watershed samples containing complex microflora, high concentrations of PCR inhibitors and, perhaps, other unknown factors. We have begun preliminary testing of water samples for NV to validate specificity, identify relevant sites for more intensive studies (septic systems) in this region. These incidence data will provide better spatial and temporal resolution for validating a predictive geospatial risk assessment model (PGRAM) developed for FDA by NASA. This work relates to the parent project objectives by providing an extremely relevant collection of Campylobacter, pathogenic E. coli, Listeria monocytogenes and Salmonella enterica strains for DNA sequencing, comparative genomics, mass spectrometry detection and functional fitness studies, and a continuing survey of an important food production region for epidemiology.