Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences.
We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.
Wood
J
J
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom.
Scott
K P
KP
Avgustin
G
G
Newbold
C J
CJ
Flint
H J
HJ
D000818:Animals; D001439:Bacteroides / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D018720:Prevotella / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012417:Rumen / Q000382:microbiology*; D012756:Sheep; D013552:Swine
D017931:DNA Primers; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S