Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis

Abstract

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

FIG. 1

Separation of PCR products from different Bifidobacterium species with genus-specific primers in 45 to 55% DGGE (increasing gradient of denaturant from top to bottom).

FIG. 2

DGGE of bifidobacterial PCR products of fecal samples from adult individuals (lanes I to V) and mixed PCR products from pure cultures (lanes m1 and m2). Single-stranded DNA and presumed heteroduplexes are above the line indicated with arrowheads. Indications 7B to 16F refer to the corresponding clones in Table 4.

FIG. 3

DGGE of bifidobacterial PCR products of fecal samples from two adult individuals (II and V) from a 4-week period (samples from weeks 0, 3, and 4). In samples 0′, 3′, and 4′ 10-fold-diluted DNA was used for PCR. Single-stranded DNA and presumed heteroduplexes are above the line indicated with arrowheads.

FIG. 4

DGGE profiles of B. adolescentis E-981074^T 16S PCR product and its derivative clones. (a) Heteroduplex formation experiment by PCR. Templates used in PCR are as follows: lane 1, plasmid from clone b1; lane 2, plasmid from clone c1; lane 3, plasmids from clones b1 and c2; lane 4, DNA from E-981074^T. (b) Heteroduplex formation experiment by melting PCR products. PCR products are as follows: lane 5, clone b1; lane 6, clone c1; lane 7, clones b1 and c1 heat denaturated together and cooled slowly to allow reannealing of complementary strands; lane 8, E-981074^T. Single-stranded DNA is indicated with an arrowhead.

FIG. 5

Sequence alignment of B. adolescentis E-981074^T clones with the B. adolescentis sequence from GenBank (M58729), showing sequence differences that were found (in boldface type). b1, b2, c1, nru-1, and nru-5 are double-stranded sequences, and c2 is a single-stranded sequence.

FIG. 6

Southern blot analysis of rrn operons of B. adolescentis E-981074^T. The genomic DNA cleaved with EcoRI (lane 1), EcoRV (lane 2), and NruI (lane 3) and hybridized with 16S rDNA probe.