Evaluation of microbial source tracking alternative indicators in comparison with conventional indicators in human and cow wastes

Sewage or fecal input into waterbodies results is a serious concern. Direct monitoring of waters for the presence of these waterborne pathogens is expensive, laborious, and time consuming. Therefore, fecal indicators such as E. coli and enterococci are used as regulatory tools to monitor water as presence of these indicators indicate potential presence of enteric pathogen. But critical uncertainties such as proliferation of bacteria in places where there is no fecal contamination and strong evidence for their extended survival and re-growth in sediments have furthered water pollution science towards new approaches. Recently, members of Bacteroides genus are found to have the features that will enable them to be used as alternative Microbial Source Tracking (MST) fecal indicators. But before they are used for the routine monitoring for fecal contamination, they need to be evaluated and validated. The overall aim of this research was to investigate the prevalence and concentrations of recently identified human and cow specific fecal markers in comparison with E. coli and enterococci in both the human and cow waste environments and to understand the variation expected when cultivation methods are replaced with qPCR methods in these environmental samples. Monitoring of raw sewage, septage samples and their treated effluents for human specific MST marker, Bacteroides thetatiataomicron during human waste treatment processes in comparison with conventional indicators such as E. coli and enterococci showed that B. thetaiotaomicron qPCR equivalent cells were present in significantly higher concentrations than that of E. coli or enterococci in raw sewage and septage and fate of these target qPCR signals were similar to E. coli and enterococci DNA during the treatment of these wastes. It was observed that around 102 to 103 qPCR equivalent cells/100mL of these targets were being discharged through the final effluents into surface waters. Another specific aim of this research was to study the distribution of different cow specific markers in cow manure environment and use these markers to characterize contamination of water bodies due to runoff from manure farms. Three different specific MST targets, a Bacteroides bovine cluster, an M2 cow specific marker, and an M3 cow specific marker were evaluated in comparison with E. coli and enterococci qPCR target signals using samples from manure pits just prior to the pumping of this waste for land application. It was found that these manure samples had E. coli and enterococci at concentrations of 105 -10 7 CFUs or cells/100 mL and cow specific markers in the concentrations of 107-109 qPCR target copies per 100 mL. This study suggested that M3 cow specific marker could be used as MST tools to monitor water bodies for contamination with agricultural wastes than M2 marker. This study explored the application of qPCR tools that targeted E. coli O157 specific eae and rfbE genes and detected these genes in human and cow waste environments. However, these assays were further inspected to evaluate their cross-specificity with other serotypes of E. coli that arose after some speculation about the concentration detected in environmental samples. In conclusion, qPCR is a viable alternative and it is recommended that E. coli, enterococci, B. thetaiotaomicron, and M3 marker be used for studies in future.