| Contamination of recreational and shellfish waters with fecal pollution is a major water quality issue with associated economic impacts and human health risks. Reliable fecal source identification and rapid, quantitative analyses are essential components of risk assessment. Enteric bacteria that are endemic to specific hosts have a potential role as public health indicators of fecal pollution. Building on previous work to discriminate ruminant and human fecal contamination, we cloned class Bacteroidetes 16S rRNA genes from pig, elk, dog, cat, and seagull fecal DNAs. Unique restriction patterns were identified among clones from each of the host species using Terminal Restriction Fragment Length Polymorphisms (T-RFLP). Clones exhibiting unique patterns were sequenced and analyzed phylogenetically, along with human, horse, and cattle sequences recovered from previous work. The analysis revealed both endemic and cosmopolitan (global) host distributions. The sequence data were used to identify host-specific genetic markers for pig and horse feces, and to design PCR primers that identify these sources of fecal pollution in water. There was a high degree of sequence overlap among the fecal Bacteroidetes of wild and domestic ruminants, and among human, domestic pet, and seagull Bacteroidetes. We compared fecal Bacteroidetes rRNA genes from these hosts using subtractive hybridization, a method that identifies differences between closely related genomes or gene sequences. A Bacteroidetes rDNA marker that distinguishes elk and cow feces was identified, as well as a host-specific marker for dog fecal Bacteroidetes. The four newly designed PCR primers were tested for specificity and sensitivity, and the dog primer was successfully used, along with the human and ruminant-specific primers, in a collaborative study comparing fecal source tracking methods. We also developed a real time Taq nuclease assay for quantification of fecal Bacteroidetes 16S rDNA, and compared it with an EPA-approved enumeration method for the current standard public health indicator, Escherichia coli , in serial dilutions of sewage primary influent. There was a strong, positive correlation between the methods, and the Taq nuclease assay was sensitive and much more rapid than the E. coli assay. PCR source identification and enumeration of fecal Bacteroidetes 16S rDNA show promise for application in a health risk-based analysis of fecal pollution. |
