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Appl. Environ. Microbiol. doi:10.1128/AEM.02568-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension (HOPE)

Division of Environmental Science and Engineering, National University of Singapore, Singapore 117576; Sustainable Environment Research Center, National Cheng Kung University, Taiwan, 701

^* To whom correspondence should be addressed. Email: eseliuwt{at}nus.edu.sg.

	Abstract

A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), is used to determine the relative abundance of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this, genomic DNA in feces of healthy human adults, bovines and swines, and in wastewaters were extracted, and total bacterial 16S rRNA genes were PCR amplified and used as the DNA template of HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at different hierarchical levels (domain, order, cluster, and species), arranged and used in six multiplexing HOPE reactions. Results showed that species like B. vulgatus, B. thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii and B. massiliensis could be individually detected in human feces at abundances down to 0.1 % of PCR-amplified 16S rRNA genes. Minor ones like B. pyogenes, B. salyersiae and B. nordii were only detected collectively using a primer that targets the B. fragilis subgroup ( 0.2 % of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) were observed to account for 28-44 % of PCR-amplified 16S rRNA genes in human fecal microbiota, and their abundances were higher than that observed in the bovine and swine fecal microbiota, and in wastewaters by a factor of five and two, respectively. These results were comparable to that obtained by quantitative PCR or reported previously using whole cell fluorescence hybridization and 16S rRNA clone library methods, supporting that HOPE can be a sensitive, specific and rapid method to determine relative abundance of Bacteroides spp. predominant in fecal samples.