Purpose: To investigate the utility of a 16S rDNA Bacteroidalessubtying method for identification of fecal contamination in the fresh produce production environment.
Methods: Initial efforts focused on optimizing a method to detect total Bacteroidales in environmental (lake water) samples using pre-analytical sample processing, DNA extraction and quantitative real-time PCR (qPCR). A combination of filtration and centrifugation was chosen for sample processing, followed by DNA extraction and qPCR amplification using the previously reported AllBac primer/probe combination. A homologous internal amplification control was also developed for inclusion in qPCR to identify those samples having residual matrix-associated inhibitors. The optimized methods were used to detect Bacteroidales contamination in 70 buffer rinsates of fresh produce (cantaloupe, tomatoes, melons), harvester hands, source, and irrigation waters originating from Northern Mexico. The same samples were also screened for the presence of generic E. coli.
Results: The optimized method was able to detect 2 mg feces per 100 ml water sample. Of the 70 samples obtained from Northern Mexico, 59/70 (84%) were successfully screened for Bacteroidales, while 11/70 (16%) were uninterpretable because of excessive levels of PCR inhibitors. Overall, 28/59 (48%) samples showed evidence of Bacteroidales contamination. When analyzed across sample types, positivity was 59%, 25%, and 50% for the produce rinsates, waters, and hand rinses, respectively. Concordance between Bacteroidales and generic E. coli was 46%.
Significance: These results demonstrate that Bacteroidales 16S rDNA can be detected in environmental samples collected from fresh produce production. Additional studies are underway to determine the usefulness of species-specific (e.g., human and ruminant) Bacteroidales assays to delineate fecal contamination source.