Restriction fragment length polymorphism analysis of PCR‐amplified 16S ribosomal DNA of human Capnocytophaga

Abstract

The confusion in the taxonomic status of the genus Capnocytophaga has made identification of strains and studies on the role of this genus in infectious diseases equivocal. In this study 33 strains of Capnocytophaga including reference strains and various clinical isolates, were studied using RFLP analysis of 16S ribosomal RNA genes. The 16S ribosomal RNA (rRNA) gene sequences from whole cell suspensions and isolated genomic DNA samples were amplified by the polymerase chain reaction (PCR) using eubacterial specific primers. PCR products were purified and characterized by single digestions with 12 restriction endonucleases. Five of these, BanI, CfoI, HaeIII, HphI and RsaII were found to discriminate reproducibly between strains, and restriction patterns (ribotypes) produced by these were analysed to clarify the classification of Capnocytophaga strains. Dendrograms inferring similarities were derived from these data by the UPGMA method. This analysis produced three major clusters of strains, each of which was associated with a previously proposed species type strain: C. gingivalis, C. sputigena and C. ochracea. The results support the division of Capnocytophaga into three species and demonstrate that, despite the heterogeneity of this genus, the modified ribotyping method provides a simple, rapid and reproducible way to identify Capnocytophaga strains.