ESTIMATION OF THE RELATIVE ABUNDANCE OF DIFFERENT BACTEROIDES AND PREVOTELLA RIBOTYPES IN GUT SAMPLES BY RESTRICTION ENZYME PROFILING OF PCR-AMPLIFIED 16S RIBOSOMAL-RNA GENE-SEQUENCES

We describe an approach for determining the genetic composition of Bac teroides 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 contr ibutions of different ribotypes to total Bacteroides and Prevotella 16 S rDNA are estimated after end labelling of one of the PCR primers, an d the contribution of Bacteroides and Prevotella sequences to total eu bacterial 16S rDNA is estimated by measuring the binding of oligonucle otide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA acc ounted 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 acc ounted for between 20 and 86% of the total amplified Bacteroides and P revotella rDNA in these samples. The most abundant Bacteroides or Prev otella ribotype in four animals, however, was ribotype 8, for which th ere 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 Prevotel la isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.