The U.S. Environmental Protection Agency published guidelines in 1986 on recommended water quality criteria for bacteria to protect bathers from gastrointestinal illness in recreational waters. The criteria, based on Escherichia coli and enterococci counts as indicators of the presence of fecal pollution, are still in use today. With the availability of PCR-based methods for the detection and quantification of specific pathogens, it might be possible in the future to base recreational water quality standards on the level of specific pathogens instead of indicator counts. Because most pathogen detection methods were developed for clinical samples, research is needed to adopt such methods to environmental samples where the presence of PCR-inhibitors is a common problem. The purpose of the current project was to determine the best method to process beach water samples for the detection and quantification of Salmonella spp. by qPCR. Although Salmonella is considered a low-grade pathogen that does not persist in the environment, it is one of the most common causes of enteric diseases and several PCR-based methods for its detection have already been developed. Our findings are that filtration through a coarse filter to remove debris followed by centrifugation (5 min at 5,000 x g) was an efficient method to concentrate samples. Detection limit can be lowered to 10 cfu/dL using sample enrichment in either a nonselective medium such as Brain Heart Infusion or a selective medium such as Rappaport Vassiliadis Soya Peptone. DNA purification prior to PCR increases the frequency of false negatives probably as a result of co-precipitation of PCR inhibitors with bacterial DNA. A simple boiling lysis procedure was found to be the most efficient method to prepare samples for PCR. Our conclusion is that it is now feasible to use a PCR-based method to detect and semi-quantitate Salmonella in environmental water samples.
Chandrasekar, Keerthy V.; Ellender, R.D.; and Wang, Shiao Y., "Detection of Salmonella spp. in Coastal Waters" (2006). Presentations. 9.