Molecular markers from fecal anaerobes to identify nonpoint source pollution in coastal waters

Fecal pollution is a serious environmental problem afflicting many coastal regions in the U.S. and worldwide. Despite efforts to minimize fecal input to coastal waters, the problem persists, partly due to the inability to reliably identify nonpoint sources. We describe a new PCR assay to differentiate sources of fecal contamination in coastal waters. We identified 7 host-specific genetic markers derived from human and cow fecal samples by amplifying 16S rDNA fragments from Bifidobacterium and the Bacteroides-Prevotella group and analyzing them by Length Heterogeneity Polymerase Chain Reaction (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP). Host-specific patterns suggested species composition differences in Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts of the same species. Ease of detection and longer survival in water made Bacteroides-Prevotella better than Bifidobacterium as indicators. Using cloning and sequencing techniques, we recovered fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers, none of which matched published Bacteroides or Prevotella sequences exactly. Phylogenetic analysis of these sequences placed them into three host-specific gene clusters within the Cytophaga-Flavobacter-Bacteroides division. 16S rDNA clones from coastal waters recovered with the same Bacteroides-Prevotella primers clustered with fecal sequences, but did not match them exactly. From the sequence data, we designed PCR primers specific for each gene cluster. To determine host specificity of the markers, we tested fecal samples from other animals for the presence of the genetic markers. Markers derived from human feces were not detected in any non-human sources. Cow-derived fecal markers were detected in all other ruminants tested. Using the source-specific primers, we surveyed water samples from Tillamook Bay, Oregon and its tributaries from June 1998 to April 1999. In most cases, only areas near sewage treatment facilities and outfalls tested positive for human pollution. Fecal pollution from ruminant sources was more widely distributed in the bay and rivers. Although our aim was to identify nonpoint sources of fecal contamination, the method should be widely applicable for following spatial and temporal fluctuations of specific bacterial groups in natural environments.