Great Lakes WATER Institute, School of Freshwater Sciences, 600 E. Greenfield Ave., Milwaukee, WI 53204, USA.
Appl Environ Microbiol. 2011 Oct;77(19):6972-81. doi: 10.1128/AEM.05480-11. Epub 2011 Jul 29.
The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. 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-associated 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 the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated 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 indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.
粪便微生物群落的复杂性以及人与其他动物来源之间的重叠，使得确定特定来源的粪便指示菌变得困难。然而，下一代测序技术的出现为解决环境内和环境间微生物群落组成提供了更大的测序能力。这些数据可用于挖掘特定来源的生物型和/或生物型组合（即微生物特征）的信息。我们报告了一种新的人类粪便污染遗传标记的开发，该标记是通过对 16S rRNA 基因 V6 区的微生物 pyrotag 序列分析确定的。对 37 个污水样本的序列分析与数据库序列比较显示，lachnospiraceae 科内存在一个与 blautia 属密切相关的人类相关生物型。该生物型称为lachno2，在威斯康星州密尔沃基的污水进水样本中平均是第二丰富的粪便细菌生物型。我们开发了一种针对lachno2 的定量 PCR（qPCR）检测方法，并与基于 qPCR 的人类 bacteroidales（基于 HF183 遗传标记）、总 bacteroidales spp.、肠球菌和传统的大肠杆菌和肠球菌平板计数检测方法一起，检测密尔沃基港的粪便和人类粪便污染的流行情况。传统的粪便指示物和与人类相关的指示物都显示出该港存在慢性粪便污染，大雨事件和合流污水溢流后会显著增加。该港的两种与人类相关的遗传标记丰度紧密相关，这强烈表明它们针对的是同一来源（即人类污水）。在该港的所有条件下都经常检测到人类腺病毒，当人类指示物浓度增加 10 倍时，其出现的概率增加 154%。lachno2 和人类 bacteroidales 与一般指示物相比，检测污水的特异性更高，与人类病原体群的关系表明，使用这些替代指示物将提高对城市水域中人类健康风险的评估。
