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. 2000 Apr;66(4):1587-94.
doi: 10.1128/AEM.66.4.1587-1594.2000.

"VSports最新版本" Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes

A E Bernhard  1 , K G Field
Affiliations

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

A E Bernhard et al. Appl Environ Microbiol. 2000 Apr.

Abstract (V体育2025版)

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 VSports手机版. 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. .

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Figures

FIG. 1
FIG. 1
LH-PCR analysis of 16S rDNA gene fragments amplified with Bac32F-FAM and Bac303R (A) and with Bif164F and Bif601R-FAM (B). The solid lines are human fecal DNA; the dotted lines are cow fecal DNA. The samples were mixtures of DNAs from seven or eight individuals. The arrows indicate cow-specific gene fragments.
FIG. 2
FIG. 2
T-RFLP analysis of 16S rDNA gene fragments amplified with Bac32F-FAM and Bac708R and cut with AciI (A) or HaeIII (B). The solid lines are human fecal DNA; the dotted lines are cow fecal DNA. The samples were mixtures of DNAs from seven or eight individuals. The arrows indicate host-specific genetic markers.
FIG. 3
FIG. 3
T-RFLP analysis of 16S rDNA gene fragments amplified with Bif164F and Bif601R-FAM and cut with HaeIII (A) or TaqI (B). The solid lines are community profiles obtained with human fecal DNA; the dotted lines are community profiles obtained with cow fecal DNA. The arrows indicate host-specific genetic markers.
FIG. 4
FIG. 4
Phylogenetic relationships among partial 16S rDNA sequences (558 positions) of human (HF) and cow (CF) host-specific genetic markers identified from fecal clone libraries. The tree was inferred by using the neighbor-joining method. The numbers above the branch points are the percentages of bootstrap replicates that support the branching order. Bootstrap values less than 50% are not shown. Cytophaga fermentans was used to root the tree.
FIG. 5
FIG. 5
T-RFLP analyses of 16S rDNA gene fragments amplified from DNA extracted from Tillamook Bay water samples. DNA was amplified with Bac32F and Bac708R and digested with AciI (A) or HaeIII (B and C). The arrows indicate host-specific markers. (A and B) Cow-specific markers (227 and 222 bp, respectively). (C) Human-specific marker (119 bp).
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