Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

Abstract

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.

FIG. 1

Outline of the molecular approaches used to analyze the fecal bacterial communities. PCR 968-1401 represents the RT-PCR with primers U968-GC and L1401, which amplify the V6 to V8 regions. PCR 8-1510 represents the PCR with primers 8f and 1510r, which amplify the complete 16S rDNA.

FIG. 2

Optimization of nucleic acid extraction. Lanes 1 to 5 show the TGGE patterns of amplified V6 to V8 regions of DNA extracted after 1, 2, 3, 4, and 5 min of bead beating, respectively. The arrowhead points to the dominant band in the pattern, which appeared after at least 3 min of bead beating.

FIG. 3

Comparison between TGGE patterns of PCR and RT-PCR products of the V6 to V8 regions from simultaneous rRNA and DNA isolations of fecal samples from individuals A and B (two replicates each). Solid arrowheads indicate bands with higher intensities in DNA- than in RNA-derived patterns. Open arrowheads indicate bands of higher intensities in RNA- than in DNA-derived patterns. Numbers 1 to 3 represent dominant bands found in all TGGE profiles.

FIG. 4

TGGE of PCR products of the V6 to V8 regions of fecal samples from individuals C to Q (from Finland). Numbers 1 to 3 represent dominant bands found in all TGGE profiles.

FIG. 5

(A) TGGE of RT-PCR products of the V6 to V8 regions of four fecal samples from individual A taken at different times during a period of 6 months. (B) TGGE of RT-PCR products of the V6 to V8 regions of two fecal samples from individual B taken after 0 and 7 months.

FIG. 6

Identification of dominant bands in the RT-PCR pattern of the V6 to V8 regions of fecal samples from individual A. Listed are the closest relatives of the clones corresponding to the bands, their percent identity, the number of corresponding clones, and the sequence analysis. #, the V1 to V3 regions of two clones with identical mobility have been sequenced. P, partial 16S rDNA sequence; C, complete sequence. Amplicons encoded with 1, 2, and 3 were found in the TGGE patterns of all subjects.