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Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules.

ABSTRACT: An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 10(2) to 10(4) cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 10(8) cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log(10)(6.3 +/- 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

SUBMITTER: Matsuda K

PROVIDER: S-EPMC2663197 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules.

Matsuda Kazunori K Tsuji Hirokazu H Asahara Takashi T Matsumoto Kazumasa K Takada Toshihiko T Nomoto Koji K

Applied and environmental microbiology 20090205 7

An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with ...[more]

PMID: 19201979

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Project description:In broiler chicken production, microbial populations on the eggshell surface following oviposition are still poorly characterized, though they may significantly impact both poultry and public health. The aim of this study was to describe the microbiota of both broiler breeder hens' feces and the surface of their eggs to assess the contribution of the parental fecal microbiota to the eggshell microbiota. A total of twelve breeder flocks in Quebec, Canada, were sampled at two different times, and a total of 940 feces and 16,400 egg surface samples were recovered. Using 16S rRNA gene sequencing, we showed that even if the microbiota of both feces and eggshells were mainly composed of the phyla Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes, the bacterial community compositions and structures differed between both types of samples. Our results also showed that both the sampling time and the flock identity significantly influenced the alpha- and the beta-diversities of the studied microbiomes. Using a Venn diagram, we showed that 1790 operational taxonomic units (OTUs) were shared between feces and eggshell samples. Sequences associated with genera of potentially pathogenic and spoilage bacteria, Acinetobacter, Campylobacter, Escherichia/Shigella, Helicobacter, Listeria, Proteus, Pseudomonas, Salmonella, and Staphylococcus, were shared between sample types. Some OTUs highly represented in the fecal microbiota and associated with Lactobacillus and Streptococcus genera, were absent from eggshells, suggesting a selection during the microbiota transfer and/or the potential role of environmental contamination. To the best of our knowledge, this is the first study using 16S rRNA sequencing to describe the contribution of the transfer from the fecal microbial ecosystem of laying breeder hens to the establishment of the microbiota on the surface of laid eggs, as well as the bacterial communities at both the broiler breeder feces and the eggshell levels.

Dataset Information

Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules.

Publications

Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules.

Similar Datasets

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