Microbiota Assessed by Clone Libraries and Community Profiling Techniques

Two PCR-based profiling strategies have been used to obtain an overall profile of complex bacterial communities—clone libraries and PCR-DGGE [or alternatively PCR-TGGE (temperature gradient gel electrophoresis)]. Both utilize universal PCR primers to amplify the 16S rRNA genes from total DNA isolated from samples.

Suau and colleagues (15) prepared a detailed phylogenetic inventory of the fecal microbiota of a healthy 40-year-old male subject using PCR-cloning. A total of 520 clones were obtained from two transformations of the same ligation product from the 10-cycle PCR amplification (120 from the first and 400 from the second). The 282 clones that were sequenced were classified as belonging to 82 molecular species, 20 of which corresponded to bacteria previously cultivated from human stool samples (i.e., 24% corresponded to sequences available in public databases). Three major monophyletic groups contained 270 (95.7%) of the 282 clones; the Clos. coccoides group (125 clones), the Bacteroides group (88 clones) and the Clos. leptum group (57 clones). The remainder of the clones were distributed among a variety of phylogenetic clusters; two belonged to recognized molecular species (Streptococcus salivarius and Streptococcus parasanguinis), whilst the remainder were potentially novel molecular species. Most interesting was a lack of bifidobacterial sequences amongst the clones analyzed (even though rRNA dot-blot hybridizations indicated the carriage of bifidobacteria). Two possibilities could explain this: (1) lack of amplification of bifidobacteria! rRNA genes, due to DNA extraction protocol, denaturation conditions during PCR, or amplification efficiency; and (2) coverage of the biodiversity provided by the 282 clones was insufficient (coverage was calculated to be 85%; thus, the probability that the 283rd clone was a different molecular species from the 82 already observed was 15%). An investigation of the 25-cycle PCR clone library was performed in parallel to this work, using the same subject (57). Comparison of the 10- and 25-cycle approaches demonstrated that PCR cycle number influences the diversity of the resulting phylogenetic profile. The clonal library obtained from the 25-cycle PCR was less diversified than that from the 10-cycle PCR. However, differences in diversity were seen between the two methods. That is, molecular species or operational taxonomic units (OTUs) were present in the 25-cycle PCR clone library that were not represented in the 10-cycle PCR clone library.

Previous work by Wilson and Blitchington (58) demonstrated somewhat similar results, with 25 of 50 clones (50%) classified as Clos. leptum subgroup, 34% as Bacteroides group and 10% as Clos. coccoides group. The disparity in the clostridial representation of the different clone libraries most probably reflects either inter-individual variations or disparity of the protocols. However, bifidobacteria were again absent from the clone library. In addition, Eubacterium rectale was not covered in the clone library in this earlier study, although Eub. rectale isolates were cultured from the same sample (58). These data highlight the difficulty to approach full coverage of the complex microbiota and further demonstrate that a polyphasic approach is pertinent. However, such work has enabled identification of previously unknown components of the fecal microbiota, and the sequence data can be used to develop new probing strategies to accurately quantify such bacteria.

Work carried out as part of the European Union (EU) human gut microbiota project using PCR clone libraries demonstrated that microbial diversity increased with age (57). In addition, the percentage of OTUs corresponding to known molecular species was highest in infants and lowest in the elderly subjects. Thus, not only was the microbial diversity greater in the elderly subjects, but also 92% of OTUs were undescribed (potentially novel) species.

The alternative to sequencing and subsequent phylogenetic analysis of clone libraries is to employ TGGE or DGGE to separate the 16S rRNA gene clones. Such techniques essentially provide a fingerprint representation of the numerically dominant members of the microbial community and allow rapid profiling of the microbial diversity of different samples (59). In addition, the TGGE/DGGE patterns can be used to selectively identify 16S rRNA amplicons of interest for characterization (which is achieved by sequencing and phylogenetic analysis). Recent years have seen an explosion in the development and application of TGGE and DGGE in human gut microbiology (Table 4). Zoetendal and coworkers (56) demonstrated the use of TGGE for monitoring the bacterial composition of human fecal samples. They compared the PCR-TGGE profiles of 16 healthy adults and identified host-specific patterns reflecting inter-individual variation in the predominant microbiota of stool samples. Some bands were seen in samples from multiple subjects, suggesting that certain members of the predominant human fecal microbiota were common across the volunteers (56). In addition, the study encompassed longer-term surveillance of the microbial community of two subjects. The PCR-TGGE profiles of each individual did not differ greatly with time, demonstrating that the predominant bacterial species were relatively stable. Phylogenetic analysis of the predominant bacteria was performed via cloning and sequencing. PCR-TGGE of each clone enabled mobility comparisons and showed 45 of the 78 clones had similar mobility to one of the 15 prominent bands of the fecal PCR-TGGE profile. This work demonstrated that the majority of predominant bacterial species represented in the fingerprint did not

Table 4 Application of TGGE/DGGE in Human Gut Microbiology

Reference

Target population

Subjects

Investigation

Overall results

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