Synergistic Application of Molecular Markers and Community-Based Microbial Source Tracking Methods for Identification of Fecal Pollution in River Water During Dry and Wet Seasons.

Hongxia Liang,Fabrice Ndayisenga,Hongxun Zhang,Ruyin Liu,Zhisheng Yu,Gang Wu,Bobo Wang

doi:10.3389/fmicb.2021.660368

Abstract

It is important to track fecal sources from humans and animals that negatively influence the water quality of rural rivers and human health. In this study, microbial source tracking (MST) methods using molecular markers and the community-based FEAST (fast expectation–maximization microbial source tracking) program were synergistically applied to distinguish the fecal contributions of multiple sources in a rural river located in Beijing, China. The performance of eight markers were evaluated using 133 fecal samples based on real-time quantitative (qPCR) technique. Among them, six markers, including universal (BacUni), human-associated (HF183-1 and BacH), swine-associated (Pig-2-Bac), ruminant-associated (Rum-2-Bac), and avian-associated (AV4143) markers, performed well in the study. A total of 96 water samples from the river and outfalls showed a coordinated composition of fecal pollution, which revealed that outfall water might be a potential input of the Fsq River. In the FEAST program, bacterial 16S rRNA genes of 58 fecal and 12 water samples were sequenced to build the “source” library and “sink,” respectively. The relative contribution (<4.01% of sequence reads) of each source (i.e., human, swine, bovine, or sheep) was calculated based on simultaneous screening of the operational taxonomic units (OTUs) of sources and sinks, which indicated that community-based MST methods could be promising tools for identifying fecal sources from a more comprehensive perspective. Results of the qPCR assays indicated that fecal contamination from human was dominant during dry weather and that fecal sources from swine and ruminant were more prevalent in samples during the wet season than in those during the dry season, which were consistent with the findings predicted by the FEAST program using a very small sample size. Information from the study could be valuable for the development of improved regulation policies to reduce the levels of fecal contamination in rural rivers.

Highlights

Fecal contamination of surface water has been recognized as one of the leading causes of the decline of water quality worldwide (Blanch et al, 2006; Reischer et al, 2013)
It is important to identify the sources of microbial contamination for the development of regulatory policies to protect water quality and avoid threats to human health caused by potential pathogenic bacteria from feces
In the AV4143 assay, we found that only one positive sample from equine was classified as moderate cross-reactivity (MCR), whereas the others were no cross-reactivity (NCR) or weak crossreactivity (WCR) (Figure 2B and Supplementary Table 9)

Summary

Introduction

Fecal contamination of surface water has been recognized as one of the leading causes of the decline of water quality worldwide (Blanch et al, 2006; Reischer et al, 2013). Microbial contaminations from human and animal fecal matter are a leading cause of the deterioration of water quality, raising public health concerns (Lee et al, 2013; Reischer et al, 2013). It is important to identify the sources of microbial contamination for the development of regulatory policies to protect water quality and avoid threats to human health caused by potential pathogenic bacteria from feces. The initially developed library-dependent MST methods relied on microbial reference libraries such as the routine collecting, monitoring, and typing of many isolates; it was time-consuming and costly to identify the potential fecal sources (Moore et al, 2005; Stoeckel and Harwood, 2007). Library-independent MST methods, targeting specific gene fragments (e.g., 16S rRNA gene) or taxonomic groups of the fecal sources associated with specific hosts, are considered accurate tools for the detection of fecal contamination and could be applied in diverse geographic settings with less time and effort (Fong et al, 2005; Kildare et al, 2007; Harwood et al, 2014)

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Discussion

Conclusion