Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification

Abstract

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.

FIG. 1.

Schematic representation of host distributions based on a neighbor-joining tree inferred from 16S rRNA gene sequences from Bacteroidales bacteria. Host clades containing cultivated species are depicted as solid wedges. Open wedges represent clusters with no cultivated representatives. Prevotella brevis and Prevotella ruminicola are of ruminant origin; all other cultivated species in the tree are of human origin. Sequences in bold, this study.

FIG.2.

Phylogenetic relationships of partial Bacteroidales 16S rRNA gene sequences (526 positions) with cultivated members of the genus Bacteroides. Sequences from cultivated species are shown in italics. Environmental sequences were recovered from Tillamook Bay on the Oregon coast (3). Trees were inferred using three tree-building algorithms: neighbor joining with a Kimura-2 parameter correction, maximum parsimony, and maximum likelihood. Values at the nodes were obtained by bootstrap analysis based on 1,000 resamplings of both neighbor-joining and parsimony trees (above and below the line, respectively). Bootstrap values over 70% are shown. The closed and open circles at the nodes represent branching orders observed in all treeing methods and those observed in two of the three methods, respectively. The scale bar represents 0.5% estimated sequence divergence. Sequences in bold, this study.

FIG. 3.

Phylogenetic relationships of partial 16S rRNA gene sequences from fecal members of the Bacteroidales with cultivated members of the genus Prevotella. See the legend to Fig. 2 for explanation.

FIG. 4.

Host DNA pools amplified with host-specific Bacteroidales PCR primers. Pools consisted of 4 to 30 individual host fecal DNAs, with each pool normalized to 3 ng/μl total DNA (Picogreen assay). (A) Primer PF163F distinguished pig fecal DNA from other host sources. (B) Primer HoF597F distinguished horse fecal DNA from other host sources.

FIG. 5.

Theoretical sensitivity (detectable copy number) of host-specific primer sets using serial dilutions of template inserted into plasmids. One hundred template copies were detected with primer HoF597F in pure water (A) and in creek water (B). Primer PF163F detected 100 copies in pure water (C) and in seawater (D). Three nanograms of total DNA from seawater or creek water extracts was added to each reaction mixture.