Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes

Abstract

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made BacteroidesPrevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 ؋ 10؊5 to 2.8 ؋ 10 ؊7 g (dry weight) of feces/liter and 6.8 ؋ 10 ؊7 g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

“…As well as Grampositive cocci, they can develop virulence and became important human pathogens. Different studies have used the microbes of the genus Bacteroides as fecal pollution indicators (Bernhard and Field, 2000;Boehm et al, 2003). Our results show the presence of these microbes in a water environment especially in the area subjected to urbanisation (see Figure 3).…”

Tertiary-Treated Municipal Wastewater is a Significant Point Source of Antibiotic Resistance Genes into Duluth-Superior Harbor

“…as described previously. [29][30][31] The qPCR analysis was conducted using an Eppendorf Mastercycler ep realplex thermal cycler (Eppendorf, Westbury, NY) or an ABI Prism7000 Sequence Detection System (Applied Biosystems). Each qPCR run consisted of initial denaturation for 10 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, and anneal and extension at 60 °C (most targets) or at 56 °C (human-specific Bacteroides) for 1 min.…”

Identification of Bacterial DNA Markers for the Detection of Human Fecal Pollution in Water

“…DNA extract yields were determined with a NanoDrop ND-1000 UV spectrophotometer (NanoDrop Technologies; Wilmington, DE). A general Bacteroides-Prevotella 16S rRNA gene PCR assay was used to verify the presence of fecal bacterial DNA in each extract and to detect PCR inhibition (4).…”