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

Abstract

A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. 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 14 Bacteroides spp. 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 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 corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species like B. pyogenes, B. salyersiae, and B. nordii were detected only collectively using a primer that targeted the B. fragilis subgroup (corresponding to ϳ0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) 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 of Bacteroides spp. predominant in fecal samples.

Hierarchical Oligonucleotide Primer Extension as a Time- and Cost-Effective Approach for Quantitative Determination of Bifidobacterium spp. in Infant Feces

“…The lower detection sensitivity of the Bifidobacterium-targeting primers than those obtained in previous studies (5,19) may be due to the high GC content of DNA templates. As Bifidobacterium spp.…”

“…A subsequent study demonstrated that HOPE can be used for rapid and specific determination of Bacteroides spp. present in feces and wastewaters at different taxonomical levels (5). It also has the versatility to be expanded to include other bacterial groups.…”

“…For HOPE, a total of 12 primers specifically targeting six Bifidobacterium spp. at different taxonomic levels were designed based on a previously described protocol (5). The specificity of the designed primers was verified in silico against entries in RDP II (2), and the sensitivity of the primers was determined as described previously (19).…”

Pathogenic Potential of Campylobacter ureolyticus

“…High levels of toxin lyses cells as the pores formed by the toxin allow cytoplasmic contents to leak out (24). Of interest was the identification of a hypothetical protein belonging to Bacteroides eggerthii, a member of the Bacteroides genus that has been isolated from human feces (3,13). The relevance of identifying this protein from C. ureolyticus was that the bacterium was previously a member of the Bacteroides genus.…”

Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities

“…This primer was included in all reaction mixtures and was used as an internal standard for comparison of Bacteroides populations in a wastewater treatment plant, in stools (10), and in wastewater samples (41).…”