Development, validation, and application of molecular microbial source tracking methods to be used in the assessment of environmental waterways.

Abstract

The fecal loading of aquatic environments by various animal hosts is of concern for public and environmental health. The microbiological contamination of waterways is amongst the most commonly listed water quality impairment in the U. S. The federal Clean Water Act (CWA) requires individual states to provide the U. S. Environmental Protection Agency (USEPA) with an aggregate quality assessment of its waterways every biennium, in effort to identify waters that do not meet state and federal quality standards. Once waterways are deemed impaired, states must conduct a Total Maximum Daily Load (TMDL) assessment in order to mitigate the impairment and restore the water body to acceptable quality. Successful bacterial TMDL implementation strategies require the use of microbial source tracking (MST) technologies that accurately and efficiently characterize the host-specific source of bacterial loading of waterways, and the relative quantities of each bacterium. Here I report that suppression subtraction hybridization (SSH) was found useful to identify gene markers specific to E. coli derived from swine fecal sources. The ability of this marker gene set to identify 62.3% of E. coli isolated from swine hosts suggests it may be useful in determining their fecal contribution to impaired waterways. I also investigated the influence of cattle grazing operations on the microbiological impairment of a small stream system in Southeastern Minnesota. Impairment by fecal indicator bacteria (FIB) was assessed by using plate count analyses and a quantitative PCR (q-PCR) was developed to estimate the presence of a bovinespecific Bacteroides marker gene in the waterway. The q-PCR data were compared to E.coli plate count data, revealing a lack of correlation between the two methods. Several physical and environmental factors likely influenced the level of E. coli found in the stream, confirming the hypothesis that other information will be needed to supplement current efforts to monitor fecal indicator bacteria in order to determine accurate sourcespecific fecal impacts. Lastly, spatial and temporal variation in the population structure of a fecal pathogen (Salmonella) in association with an alternate host and habitats, including the green alga Cladophora found in stream and lake water, aquatic plants, beach sand, and sediments, was evaluated by use of horizontal fluorophore-enhanced rep-PCR (HFERP) DNA fingerprinting. It was revealed that Salmonella populations associated with Cladophora varied both spatially and temporally, suggesting potentially different input sources of Salmonella over space and time. In addition, differing environmental stressors may play a role in selecting particular Salmonella genotypes that are best suited for growth in these environments The use of rapid molecular-based assays to determine the presence and source-specific loading of fecal indicator bacteria and pathogens has the potential to improve the accuracy and efficacy of TMDL studies, and to expedite implementation strategies to remediate impaired waterways.