The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: theBacteroides group, the Clostridium coccoidesgroup, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron,Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.

Intestinal microbial community is involved in the pathogenesis of Crohn's disease, but knowledge of its potential abnormalities has been limited by the impossibility to grow many dominant intestinal bacteria. Using sequence analysis of randomly cloned bacterial 16S ribosomal DNA, the dominant faecal species from four Crohn's disease patients and four controls were compared. Whereas marked inter-individual differences were observed in the faecal microflora of patients, three remained distantly related to controls on the basis of their operational taxonomic unit composition. Bacteroides vulgatus and closely related organisms represented the only molecular species shared by all patients and exhibited an unusually high rate of occurrence. Escherichia coli clones were isolated only in two patients with ileocolonic Crohn's disease. Moreover, numerous clones belonged to phylogenetic groups or species that are commonly not dominant in the faecal microflora of healthy subjects: Pectinatus, Sutterella, Verrucomicrobium, Fusobacterium, Clostridium disporicum, Clostridium glycolicum, Clostridium ramosum, Clostridium innocuum and Clostridium perfringens.

In spite of its shortcoming, analysis of PCR-derived rDNA libraries is being employed increasingly to investigate microbial diversity within many ecosystems. In the present investigation, the effects of the number of PCR cycles (10 vs 25 cycles) on the inferred structure of a 16S rDNA library have been examined. Seventy-five 25-cycle sequences were retrieved and analysed in comparison with 284 10-cycle sequences already described in a previous study. The 359 clones obtained were classified into 94 molecular species (at least 98% sequence similarity). At the level of large phylogenetic groups, the two cloned rDNA libraries were not different. A mathematical model was developed in order to estimate the number of molecular species expected if further sequencing was performed. Coverage-based computing, projections and statistical analysis demonstrated that the structures of the two PCR-derived rDNA libraries were different and that the 25-cycle rDNA library displayed reduced diversity. It is suggested that the number of PCR cycles used for amplification of 16S rDNA genes for phylogenetic diversity studies must therefore be kept as small as possible.

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