ARS | Publication request: Lachnospiraceae- and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2011
Publication Date: October 3, 2011
Citation: Newton, R.J., Vandewalle, J., Borchardt, M.A., Gorelick, M.H., Mclellan, S.L. 2011. Lachnospiraceae- and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor. Applied and Environmental Microbiology. 77:6972-6981.

Interpretive Summary: Often times it is difficult to interpret the source of contamination when water, such as a lake or river water, becomes contaminated with fecal wastes. Source identification is important to prevent further contamination. Contamination by human sewage may require repairs to sanitary sewers or wastewater treatment plants, whereas contamination by manure in agricultural runoff would require a different set of prevention measures. We used new technologies to search for bacteria in human sewage and found one type in the family Lachnospiraceae that appears unique to humans. We developed a method for detecting this type, Lachno2, and compared its occurrence and concentrations with other conventional and new bacterial indicators of sanitary quality as well as the group of adenoviruses specific to humans. Lachno2 appears to possess the characteristics that would make it an excellent bacterial indicator. Moreover, as Lachno2 does not occur in cattle manure, it may provide a means of discriminating whether the source of fecally contaminated water was human sewage or cattle manure.

Technical Abstract: The complexity of fecal microbial communities and overlap among human and other animal sources has made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies has provided increased power to resolve microbial community composition within and among environments. Now these data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e. microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human specific phylotype within the Lachnospiraceae family which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci, and also with the traditional Escherichia coli and enterococci plate count assays, to examine the prevalence of fecal and human fecal pollution in Milwaukee’s harbor. Both the traditional fecal indicators and the human-specific indicators revealed chronic fecal pollution in the harbor, with significant indicator increases following heavy rain events and combined sewer overflows. The two human-specific genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e. human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicators concentration. Both Lachno2 and human Bacteroidales increased sensitivity to detect sewage compared with general indicators, and relationship to a human pathogen group suggests use of these alternative indictors will improve assessments for human health risks in urban waters.