Dataset Information

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 Bacteroides-Prevotella 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 x 10(-5) to 2.8 x 10(-7) g (dry weight) of feces/liter and 6.8 x 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.

SUBMITTER: Bernhard AE

PROVIDER: S-EPMC92027 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes.

Bernhard A E AE Field K G KG

Applied and environmental microbiology 20000401 4

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 ...[more]

PMID: 10742246

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Project description:The human microbiome contains many organisms that could potentially be used as indicators of human fecal pollution. Here we report the development of two novel human-associated genetic marker assays that target organisms within the family Lachnospiraceae Next-generation sequencing of the V6 region of the 16S rRNA gene from sewage and animal stool samples identified 40 human-associated marker candidates with a robust signal in sewage and low or no occurrence in samples from nonhuman hosts. Two were chosen for quantitative PCR (qPCR) assay development using longer sequences (the V2 to V9 regions) generated from clone libraries. Validation of these assays with these markers, designated Lachno3 and Lachno12, was performed using fecal samples (n = 55) from cat, dog, pig, cow, deer, and gull sources, and the results were compared with those of established host-associated assays (the Lachno2 marker and two human Bacteroides markers, the HB and HF183/BacR287). Each of the established assays cross-reacted with samples from at least one other animal species, including animals common in urban areas. The Lachno3 and Lachno12 markers were primarily human associated; however, the Lachno12 marker demonstrated low levels of cross-reactivity with samples from select cows and nonspecific amplification with samples from pigs. This limitation may not be problematic when testing urban waters. These novel markers resolved ambiguous results from previous investigations of stormwater-impacted waters, demonstrating their utility. The complexity of the microbiome in humans and animals suggests that no single organism is strictly specific to humans, and the use of multiple complementary markers in combination will provide the highest resolution and specificity for assessing fecal pollution sources.IMPORTANCE Traditional fecal indicator bacteria do not distinguish animal from human fecal pollution, which is necessary to evaluate health risks and mitigate pollution sources. Assessing water in urban areas is challenging, since the water can be impacted by sewage, which has a high likelihood of carrying human pathogens, as well as pet and urban wildlife waste. We demonstrate that the Lachno3 and Lachno12 markers are human associated and highly specific for the detection of human fecal pollution from urban sources, offering reliable identification of fecal pollution sources in urban waters.

Project description:In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

Project description:Intensive agriculture and densely populated areas represent major sources of nutrient pollution for European inland and coastal waters, altering the aquatic ecosystems and affecting their capacity to provide ecosystem services and support economic activities. Ambitious water policies are in place in the European Union (EU) for protecting and restoring aquatic ecosystems under the Water Framework Directive and the Marine Strategy Framework Directive. This research quantified the current pressures of point and diffuse nitrogen and phosphorus emissions to European fresh and coastal waters (2005-2012), and analysed the effects of three policy scenarios of nutrient reduction: 1) the application of measures currently planned in the Rural Development Programmes and under the Urban Waste Water Treatment Directive (UWWTD); 2) the full implementation of the UWWTD and the absence of derogations in the Nitrates Directive; 3) high reduction of nutrient, using best technologies in wastewaters treatment and optimal fertilisation in agriculture. The results of the study show that for the period 2005-2012, the nitrogen load to European seas was 3.3-4.1 TgN/y and the phosphorus load was 0.26-0.30 TgP/y. Policy measures supporting technological improvements (third scenario) could decrease the nutrient export to the seas up to 14% for nitrogen and 20% for phosphorus, improving the ecological status of rivers and lakes, but widening the nutrient imbalance in coastal ecosystems (i.e. increasing nitrogen availability with respect to phosphorus), affecting eutrophication. Further nutrient reductions could be possible by a combination of measures especially in the agricultural sector. However, without tackling current agricultural production and consumption system, the reduction might not be sufficient for achieving the goals of EU water policy in some regions. The study analysed the expected changes and the source contribution in different European regional seas, and highlights the advantages of addressing the land-sea dynamics, checking the coherence of measures taken under different policies.

Dataset Information

Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes.

Publications

Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes.

Similar Datasets

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