Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers

Abstract

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113-121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.

FIG. 1

PCR products obtained for 10 Bifidobacterium species with their specific primers. Lane M, DNA size markers (sizes [in bases] are indicated on the left); lane 1, B. adolescentis ATCC 15703^T; lane 2, B. angulatum ATCC 27535^T; lane 3, B. bifidum ATCC 29521^T; lane 4, B. breve ATCC 15700^T; lane 5, B. catenulatum ATCC 27539^T; lane 6, B. pseudocatenulatum JCM 1200^T; lane 7, B. longum ATCC 157071^T; lane 8, B. infantis ATCC 15697^T; lane 9, B. dentium ATCC 27534^T; lane 10, B. gallicum JCM 8224^T; lane 11, negative control (PCR performed with primer BiADO and E. coli ATCC 11775^T).

FIG. 2

Detection limits of the species-specific PCR methods, as determined by using DNAs extracted from 10-mg fecal samples mixed with various amounts of B. angulatum ATCC 27535^T. Lane M, DNA size markers (sizes [in bases] are indicated on the left); lane 1, 10⁶ cells per PCR mixture; lane 2, 10⁵ cells; lane 3, 10⁴ cells; lane 4, 10³ cells; lane 5, 10² cells; lane 6, 10 cells; lane 7, 1 cell; lane 8, no cells (negative control).