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Aims: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. Methods and results: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20–80 gene copies g−1, recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. Conclusions: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. Significance and impact of study: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches. [ABSTRACT FROM AUTHOR]

Microbial source tracking ( MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters that rely on the association of certain fecal microorganisms with a particular host. MST is used to assess recreational water quality and associated human health risk, and total maximum daily load allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microorganisms. Human sewage pollution is among the greatest concerns for human health due to (1) the known risk of exposure to human waste and (2) the public and regulatory will to reduce sewage pollution; however, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves. Here, we review the performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR ( qPCR). Relationships among human-associated MST markers, fecal indicator bacteria, pathogens, and human health outcomes are presented along with recommendations for future research. An integrated understanding of the advantages and drawbacks of the many MST methods targeting human sources advanced over the past several decades will benefit managers, regulators, researchers, and other users of this rapidly growing area of environmental microbiology. [ABSTRACT FROM AUTHOR]