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

Abstract

We describe an approach for determining the genetic composition ofBacteroides and Prevotellapopulations 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 andPrevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution ofBacteroides and Prevotellasequences 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 Prevotellastrains, together accounted for between 20 and 86% of the total amplified Bacteroides andPrevotella rDNA in these samples. The most abundantBacteroides 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 abundantBacteroides and Prevotella groups in the rumen are underrepresented among cultured rumenPrevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

PCR-DGGE-based quantification of stability of the microbial community in a simulator of the human intestinal microbial ecosystem

“…Taking into account the fact that, when using bacterial primers, only the most abundant species are detected, two sets of group specific primers were applied to monitor the stabilisation period. The Bacteroides/Prevotella group comprises the most dominant anaerobic bacteria present in the intestinal tract [34]. Lactobacillus sp.…”

“…(2) 1 0 ; 94°C/1 0 ; 56°C/2 0 ; 72°C (35Â) (3) 10 0 ; 72°C FD1 [34] (1) 5 0 ; 94°C Bacteroides/Prevotella RbacPre (2) 2 0 ; 94°C/30 00 ; 60°C/2 0 ; 72°C (30Â) (3) 10 0 72°C Fig. 1.…”

Genetic diversity and diet specificity of ruminal Prevotella revealed by 16S rRNA gene-based analysis

“…Culture-based studies showed that Prevotella strains account for 60% of total cultivable bacteria from the rumen of cows (Van Gylswyk, 1990). Based on restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences from rumen samples, Wood et al (1998) reported that the relative abundance of rumen Prevotella/Bacteroides ribotypes in the total eubacterial 16S rRNA gene could range from 12% to 62%. They also demonstrated that the most abundant Prevotella ribotype was related to very few cultured strains, suggesting underrepresentation of certain members of the genus by cultured strains.…”

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

“…Several studies have suggested that fecal members of the phylum Bacteroidetes are ecologically diverse, numerically important members of the intestinal microbial communities and exhibit some host specificity (Salyers, 1984;Gherna & Woese, 1992;Wood et al, 1998;Boone & Castenholz, 2001;Daly et al, 2001;Leser et al, 2001;Hold et al, 2002). Recently, Bernhard & Field (2000a, b) developed PCR-based, 16S rRNA gene assays targeting Bacteroidetes which were capable of distinguishing between human and ruminant sources (Bernhard & Field, 2000a, b;Bernhard et al, 2003;Field et al, 2003).…”