The use of Bacteroides specific molecular markers to identify sources of fecal contamination in natural waters

Knowing the origin of fecal pollution of water is paramount in assessing the safety of surface waters and in developing effective pollution control strategies. Bacteroides-specific molecular markers have been widely used to differentiate human sources of fecal bacteria from other sources in waters. However, recent work in our lab indicated that many assays/methods currently used to detect human-specific Bacteroides produce false positive results in the presence of fish (tilapia, catfish, trout, salmon) fecal DNA, which is widely prevalent in surface waters. Evaluation of five protocols previously reported as human-specific showed that in 80% of cases fecal DNA from at least one fish species was amplified providing false positive results. This situation highlights the need for accurate molecular assays for the identification of fecal sources from human origin. It was hypothesized that sequencing of the Bacteroides genome from pure culture isolates from fish and human feces may help in identifying unique molecular markers, which can potentially be used to develop a more stringent assay that can be utilized to differentiate between human and fish fecal pollution of surface water. Total Bacteroides from human and fish feces were isolated using blood agar and each isolate was further streaked on BBE agar to obtain pure cultures. The pure cultures were enriched in Chopped Meat Broth Medium and identified using rapid ID 32A API strip. In human feces, B. vulgatus were the dominant species; whereas, B. eggerthii were the dominant species in tilapia feces. Bacteroides from grass carp, channel catfish and blue catfish were either B. uniformis, B. ovatus, or B. sterocoris. The pure cultures of Bacteroides species from human and fish were sequenced and genomic data was analyzed to assess relatedness of Bacteroides isolates. Sequences from cultured human Bacteroides, and sequences from each of the cultured fish Bacteroides (tilapia, grass carp, blue catfish, channel cat fish and trout) were aligned using Multi-align interface software. Phylogenic analyses showed distinct Bacteroides groupings from each fish species, while human sequences clustered with B. vulgatus. Bioinformatics analyses of genomic data identified the regions of 16S rRNA with the highest variability. These variable regions will provide a basis for developing a new microbial source tracking method targeting a human-specific Bacteroides molecular marker. 2011