The use of Bacteroides Specific Molecular Markers to Identify Sources of Fecal Contamination in Natural Waters

摘要

Knowing the origin of fecal pollution of water is paramount in assessing the safety ofrnsurface waters and in developing effective pollution control strategies. Bacteroides-specificrnmolecular markers have been widely used to differentiate human sources of fecal bacteria fromrnother sources in waters. However, recent work in our lab indicated that many assays/methodsrncurrently used to detect human-specific Bacteroides produce false positive results in the presencernof fish (tilapia, catfish, trout, salmon) fecal DNA, which is widely prevalent in surface waters.rnEvaluation of five protocols previously reported as human-specific showed that in 80% of casesrnfecal DNA from at least one fish species was amplified providing false positive results. Thisrnsituation highlights the need for accurate molecular assays for the identification of fecal sourcesrnfrom human origin. It was hypothesized that sequencing of the Bacteroides genome from purernculture isolates from fish and human feces may help in identifying unique molecular markers,rnwhich can potentially be used to develop a more stringent assay that can be utilized torndifferentiate between human and fish fecal pollution of surface water.rnTotal Bacteroides from human and fish feces were isolated using blood agar and eachrnisolate was further streaked on BBE agar to obtain pure cultures. The pure cultures were enrichedrnin Chopped Meat Broth Medium and identified using rapid ID 32A API strip. In human feces, B.rnvulgatus were the dominant species; whereas, B. eggerthii were the dominant species in tilapiarnfeces. Bacteroides from grass carp, channel catfish and blue catfish were either B. uniformis, B.rnovatus, or B. sterocoris.rnThe pure cultures of Bacteroides species from human and fish were sequenced andrngenomic data was analyzed to assess relatedness of Bacteroides isolates. Sequences from cultured human Bacteroides, and sequences from each of the cultured fish Bacteroides (tilapia,rngrass carp, blue catfish, channel cat fish and trout) were aligned using Multi-align interfacernsoftware. Phylogenic analyses showed distinct Bacteroides groupings from each fish species,rnwhile human sequences clustered with B. vulgatus. Bioinformatics analyses of genomic datarnidentified the regions of 16S rRNA with the highest variability. These variable regions willrnprovide a basis for developing a new microbial source tracking method targeting a humanspecificrnBacteroides molecular marker.