Phylogenetic Characterization of Clostridium Related Segmented Filamentous Bacteria in Mice Based on 16S Ribosomal RNA Analysis

Abstract

Summary The small intestine of many animals is inhabited by non-pathogenic segmented filamentous bacteria (SFBs). SFBs are strongly attached with a holdfast to intestinal epithelial cells and to cells of Peyer's patches. In the terminal segments of SFBs, spores are formed. Previously, SFBs have been mono-associated with germ-free mice. At present, this is the only available method to obtain mono-bacterial cultures of SFBs. These symbiotic bacteria cannot be cultures in vitro , and have therefore never been characterized phylogenetically. Here, we describe for the first time the phylogenetic relationship of SFBs found in mice with other bacteria. Total DNA was extracted from SFBs, which were isolated from the ileum of these mono-associated mice. Parts of the gene coding for 16S rRNA, corresponding to positions 27 to 1100 and 27 to 1492 in E. coli 16S rRNA, were amplified by PCR using conserved rRNA-targeted oligonucleotide primers. Cloning, sequencing and successive analysis of the amplified 16S rRNA sequences showed that SFBs form a distinct group that is most closely related to Clostridium species. Oligonucleotide probes were developed and directed against parts of the V2, V6 and V9 variable regions of the SFB 16S rRNA. The specificity of these probes was confirmed by southern blot analysis of the PCR-products obtained from total SFB DNA extracts, as well as by in situ hybridization experiments, directly performed on SFB cells in the ileum of mice.