Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor

Great Lakes WATER Institute, School of Freshwater Sciences, 600 E. Greenfield Ave., Milwaukee, WI 53204, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 07/2011; 77(19):6972-81. DOI: 10.1128/AEM.05480-11
Source: PubMed


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.

Download full-text


Available from: Ryan J Newton,
  • Source
    • "McLellan and Eren , 2014 ) . For example , development of qPCR probes based on Lachnospiraceae sequences enriched in human sewage were used to evaluate sewage contamination in an urban freshwater harbor where occurrence of this new indicator group was highly correlated with a more established marker of human fecal contamination ( i . e . , HF183 ; Newton et al . , 2011 ) . Analysis of profiles of microbial SSU rRNA genes ( V4 / V6 regions ) with a source estimation program employing Bayesian statistics ( SourceTracker ) allowed identification of human fecal and sewage signatures which correlated to the distribution of human - source markers Lachno2 and HF183 along the coastline of Lake Michigan ( Newt"
    [Show abstract] [Hide abstract]
    ABSTRACT: Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable regions have allowed identification of signature microbial species that serve as bioindicators for sewage contamination in these environments. Beyond amplicon sequencing, metagenomic and metatranscriptomic analyses of microbial communities in fresh water environments reveal the genetic capabilities and interplay of waterborne microorganisms, shedding light on the mechanisms for production and biodegradation of toxins and other contaminants. This review discusses the challenges and benefits of applying NGS-based methods to water quality research and assessment. We will consider the suitability and biases inherent in the application of NGS as a screening tool for assessment of biological risks and discuss the potential and limitations for direct quantitative interpretation of NGS data. Secondly, we will examine case studies from recent literature where NGS based methods have been applied to topics in water quality assessment, including development of bioindicators for sewage pollution and microbial source tracking, characterizing the distribution of toxin and antibiotic resistance genes in water samples, and investigating mechanisms of biodegradation of harmful pollutants that threaten water quality. Finally, we provide a short review of emerging NGS platforms and their potential applications to the next generation of water quality assessment tools.
    Frontiers in Microbiology 10/2015; 6(1027). DOI:10.3389/fmicb.2015.01027 · 3.99 Impact Factor
  • Source
    • "Sample processing and filtering methods are described in Newton et al. (2011b). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Water quality is impacted significantly by urbanization. The delivery of increased nutrient loads to waterways is a primary characteristic of this land use change. Despite the recognized effects of nutrient loading on aquatic systems, the influence of urbanization on the bacterial community composition of these systems is not understood. We used massively-parallel sequencing of bacterial 16S rRNA genes to examine the bacterial assemblages in transect samples spanning the heavily urbanized estuary of Milwaukee, WI to the relatively un-impacted waters of Lake Michigan. With this approach, we found that genera and lineages common to freshwater lake epilimnia were common and abundant in both the high nutrient, urban-impacted waterways, and the low nutrient Lake Michigan. Although the two environments harbored many taxa in common, we identified a significant change in the community assemblage across the urban-influence gradient, and three distinct community features drove this change. First, we found the urban-influenced waterways harbored significantly greater bacterial richness and diversity than Lake Michigan (i.e., taxa augmentation). Second, we identified a shift in the relative abundance among common freshwater lineages, where acI, acTH1, Algoriphagus and LD12, had decreased representation and Limnohabitans, Polynucleobacter, and Rhodobacter had increased representation in the urban estuary. Third, by oligotyping 18 common freshwater genera/lineages, we found that oligotypes (highly resolved sequence clusters) within many of these genera/lineages had opposite preferences for the two environments. With these data, we suggest many of the defined cosmopolitan freshwater genera/lineages contain both oligotroph and more copiotroph species or populations, promoting the idea that within-genus lifestyle specialization, in addition to shifts in the dominance among core taxa and taxa augmentation, drive bacterial community change in urbanized waters.
    Frontiers in Microbiology 09/2015; 6. DOI:10.3389/fmicb.2015.01028 · 3.99 Impact Factor
  • Source
    • "The latter are not well known in conventional testing regimes because they are anaerobes that do not grow in plate culture. Many of the recently proposed indicator bacteria have been identified in the Human Microbiome Project as the dominant bacteria in feces (e. g., Arumugam et al., 2011) and typically belong to the Bacteroidales, Bifidobacterium, Clostridiaceae, Lachnospiraceae or Ruminococcaceae (Mieszkin et al. 2009; Silkie and Nelson 2009; McLellan et al. 2010, 2013; Newton et al. 2011, 2013; McQuaig et al. 2012; Harwood et al. 2013). The use of multiple species and techniques to obtain several lines of evidence is now considered an important component of effective microbial source tracking (Harwood et al. 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microbial source tracking is an area of research in which multiple approaches are used to identify the sources of elevated bacterial concentrations in recreational lakes and beaches. At our study location in Darwin, northern Australia, water quality in the harbor is generally good, however dry-season beach closures due to elevated Escherichia coli and enterococci counts are a cause for concern. The sources of these high bacteria counts are currently unknown. To address this, we sampled sewage outfalls, other potential inputs, such as urban rivers and drains, and surrounding beaches, and used genetic fingerprints from E. coli and enterococci communities, fecal markers and 454 pyrosequencing to track contamination sources. A sewage effluent outfall (Larrakeyah discharge) was a source of bacteria, including fecal bacteria that impacted nearby beaches. Two other treated effluent discharges did not appear to influence sites other than those directly adjacent. Several beaches contained fecal indicator bacteria that likely originated from urban rivers and creeks within the catchment. Generally, connectivity between the sites was observed within distinct geographical locations and it appeared that most of the bacterial contamination on Darwin beaches was confined to local sources.
    MicrobiologyOpen 12/2014; 3(6). DOI:10.1002/mbo3.209 · 2.21 Impact Factor
Show more