Lachnospiraceae and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor

Abstract

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.

Fig. 1.

Bar plot and summary box plot of the Lachno2 phylotype relative abundance in 48 human fecal samples and 37 Milwaukee sewage influent samples. Boxes illustrate the 25th-, 50th-, and 75th-percentile data. Whiskers indicate the 10th- and 90th-percentile data. Outliers are depicted with open circles. The samples included in the human fecal content plot are from Dethlefsen et al. (11) (bars 1 to 15) and Turnbaugh et al. (52) (bars 16 to 48). The order of paired Jones Island (JI) and South Shore (SS) treatment plant influent samples included in the Milwaukee sewage influent plot are as follows: 20 April 2005; 18 April, 21 August, 16 October, 20 November, and 11 December 2007; 17 March, 1 April, 8 April, 28 May, 11 June, 10 July, 21 August (JI only), 8 October, and 10 December 2008; and 31 March, 22 April, 13 May, and 5 August 2009.

Fig. 2.

Unrooted consensus phylogram from neighbor-joining phylogenetic analysis depicting clone sequences containing the Lachno2 phylotype and several close relatives. Bacillus subtilis (AB042061) and Bacillus pumilus (AY456263) were used as an outgroup. Only nodes obtaining 50% confidence during bootstrapping are labeled. GenBank accession numbers are listed in parentheses next to the isolate or clone name. The scale bar indicates 1% sequence divergence.

Fig. 3.

Heat map illustrating the in silico-estimated relative abundance of sequences targeted by three qPCR assays, total Bacteroidales spp. (13), human Bacteroidales (7, 21), and Lachno2 (this study) in each sample from the human fecal (11, 52), cow fecal (47), and WI sewage (reference and this study) V6 pyrosequencing data sets. Each square represents a unique sample from the data set. The sequence targets of each qPCR assay were identified by matching the primers/probe (allowing for one mismatch per primer/probe) from each assay to the RDP 16S rRNA gene sequence database filtered by a quality criterion of good and a length criterion of ≥1,200 bp (9). All sequences matching these criteria for each qPCR assay were downloaded as an alignment from RDP, and then the V6 region from these aligned sequences was extracted. Each extracted V6 sequence was used as a query against the V6 data from samples in the three data sets: human fecal, cow fecal, and WI sewage. Exact matches to the query V6 sequences were identified in every sample, and then the occurrences of the matched V6 sequences were summed in each sample. The sum of these exact matches for each assay was divided by the total number of bacterial sequence reads in each sample to obtain a relative abundance for the three qPCR assays. Heat map relative abundance scale bars are depicted next to their intended data sets. The dash symbol in a sample square indicates that no target sequences were identified in the sample.

Fig. 4.

Bar plot of CFU counts per 100 ml for enterococci (yellow) and E. coli (blue) on the left axis and of 16S rRNA gene copies per 100 ml from a total Bacteroidales (Bac) sp. qPCR assay (green) on the right axis. All samples were collected from Milwaukee harbor water in 2007 (A) and 2008 (B). Samples are shown in order and were collected on 17 and 27 July, 9 August, 1 May, 19 June, 6 and 7 August, 11 and 28 September, 2 October, 4 April, 20 August, 5 June, and 21 and 22 August 2007 (A) and 20 May, 24 June, 1 and 8 July, 5 September, 11 April, and 9, 10, 11, 13, 16, and 17 June 2008 (B). Sample dates are color coded (below x axis) by environmental conditions during sampling: blue, <0.5 in. rain over 48-h period prior to sampling; gray, ≥0.5 in. rain over 48-h period prior to sampling; brown, CSO period; and tan, within 5 days post-CSO. nd, not detected.

Fig. 5.

(A) Box plot of the Lachno2 copies per 100 ml of harbor water as determined by qPCR. Boxes indicate the 25th, 50th, and 75th percentiles and whiskers indicate the minimum and maximum data points. Four harbor environmental condition periods are depicted: <0.5 in. rain in 48 h (n = 6), ≥0.5 in. rain in 48 h (n = 9), during CSO (n = 7), and 5 days after CSO (n = 5). (B) Scatter plot of Lachno2 copies per 100 ml versus copies per 100 ml for the qPCR-based assay for human Bacteroidales (Bac) (B) and enterococci (C). One-to-one lines are depicted and all data are plotted on a log scale. Each point is color coded by the environmental conditions represented in the box plot.

Fig. 6.

(A) Scatter plot of the human feces-associated indicators human Bacteroidales (red circles) and Lachno2 (blue circles) versus adenovirus as measured in sewage influent to South Shore WWTP in Milwaukee, WI. Plot point sample dates listed in order from most to least abundant adenovirus are as follows: 13 July, 18 May, 8 June, 14 September, 12 October, 9 November, 10 August, and 14 December 2009 and 11 January 2010. Note that for visualization both axes are log scaled. (B) Scatter plot of human feces-associated indicators (human Bacteroidales plus Lachno2) versus adenovirus genome as measured in Milwaukee's harbor. Sample dates are color coded as follows: blue, <0.5 in. rain; gray, ≥0.5 in. rain; brown, CSO; and tan, ≤5 days post-CSO. Points along the x axis did not contain measurable adenovirus. Note that both axes are log scaled for visualization.