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Recently several human health-related microbiota studies have had partly contradictory results. As some differences may be explained by methodologies applied, we evaluated how different storage conditions and commonly used DNA-extraction kits affect bacterial composition, diversity, and numbers of human fecal microbiota. According to our results, the DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after a week's storage at −20 °C, the numbers of Bacteroides spp. were 1.6–2.5 log units lower (P < 0.05). Furthermore, the numbers of predominant bacteria, Eubacterium rectale (Erec)-group, Clostridium leptum group, bifidobacteria, and Atopobium group were 0.5–4 log units higher (P < 0.05) after mechanical DNA-extraction as detected with qPCR, regardless of storage. Furthermore, the bacterial composition of Erec-group differed significantly after different DNA-extractions; after enzymatic DNA-extraction, the most prevalent genera detected were Roseburia (39% of clones) and Coprococcus (10%), whereas after mechanical DNA-extraction, the most prevalent genera were Blautia (30%), Coprococcus (13%), and Dorea (10%). According to our results, rigorous mechanical lysis enables detection of higher bacterial numbers and diversity from human fecal samples. As it was shown that the results of clostridial and actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may explain the contradictory results previously obtained.

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.