Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis.
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.
Satokari
R M
RM
Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen University, 6703 CT Wageningen, The Netherlands. reetta.satokari@algemeen.micr.wau.nl
Vaughan
E E
EE
Akkermans
A D
AD
Saarela
M
M
de Vos
W M
WM
D001483:Base Sequence; D001644:Bifidobacterium / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D020459:Genes, rRNA; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S