Aims: To determine phylogenetic groups of clostridia inhabiting the mouse intestine that are essential for normalization of germfree (GF) mice. Methods and Results: Using both the culture method and cloning, clostridia inhabiting the mouse intestine were isolated, and phylogenetic analysis based on 16S rRNA gene sequences was carried out. As a result, the isolates were found to have novel sequences, and no isolate was determined to be identical to previously known identified clostridia. Although the taxonomy of mouse intestinal clostridia was complex, many of them belonged to Clostridium clusters XIVa and IV in conventional (CV) and limited flora mice and ex‐germfree mice administered chloroform‐treated CV mouse faeces. The clostridia that belonged to cluster XIVa were most often present and showed the highest diversity. Conclusions: Clostridia belonging clusters XIVa and IV are dominant in the mouse intestine as in other gut ecosystems. The novel groups in these clusters are essential for normalization of GF mice. Significance and Impact of the Study: The results of this study can be applied in the strict control of mouse intestinal microbiota and will provide important information for normalization of GF mice and also for research on microbiology of the mouse intestine.
Clostridia dominate the rodent intestinal bacterial community and play an important role in physiological functions of the host. However, their ecology and diversity are still unclear. In our previous report, we showed that phylogenetically novel groups of clostridia inhabit the mouse intestine and contribute to the normalization of germfree mice. In this study, five new oligonucleotide probes were designed and applied to detect these clostridial groups that are essential for the normalization of germfree mice. Faecal microbiota of conventional mouse strains and specific pathogen-free mice from different breeding colonies were analysed by fluorescence in situ hybridization using these five probes. Our results showed that the composition of clostridia differed among mouse strains and also among mouse groups of the same inbred strain from different breeding colonies. These five new probes for mouse clostridia were able to detect the difference in clostridial diversity in each mouse group. In addition to Clostridium, we also analysed Bacteroides and Lactobacillus using previously described probes and the number or the frequency of occurrence of Bacteroides was shown to be different among mouse groups analysed. The oligonucleotide probe set including our newly developed and previously described probes used in this study can be applied to monitoring of significant groups of mouse intestinal microbiota.
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