Relative Abundance ofBacteroidesspp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension

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Abstract
A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominantBacteroidesspp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14Bacteroidesspp. at different hierarchical levels (domain, order, cluster, and species) and were arranged into and used in six multiplex HOPE reaction mixtures. Results showed that species likeB. vulgatus,B. thetaiotaomicron,B. caccae,B. uniformis,B. fragilis,B. eggerthii, andB. massiliensiscould be individually detected in human feces at abundances corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species likeB. pyogenes,B. salyersiae, andB. nordiiwere detected only collectively using a primer that targeted theB. fragilissubgroup (corresponding to ∼0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., mostBacteroidales) were observed to account for 28 to 44% of PCR-amplified 16S rRNA genes from human fecal microbiota, and their abundances were higher than those detected in the bovine and swine fecal microbiota and in wastewaters by factors of five and two, respectively. These results were comparable to those obtained by quantitative PCR and to those reported previously from studies using whole-cell fluorescence hybridization and 16S rRNA clone library methods, supporting the conclusion that HOPE can be a sensitive, specific, and rapid method to determine the relative abundances ofBacteroidesspp. predominant in fecal samples.

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