Subtyping of uncultured bartonellae using sequence comparison of 16 S/23 SrRNA intergenic spacer regions amplified directly from infected blood

This study aimed to assess the usefulness of a PCR-based approach to the de tection and differentiation of Bartonella strains in infected blood. The co nservation of potential genus-specific PCR primer hybridisation sites withi n the 16 S/23 S rRNA gene intragenic spacer regions of Bartonella species w as confirmed following sequence analysis of the intragenic spacer regions o f four previously untested species. The extent of intra-species variation w ithin the specific amplicons was assessed by comparison of sequences obtain ed from 17 strains of four Bartonella species. Eight sequence variants were obtained. Each species for which multiple strains were tested possessed at least two intragenic spacer regions variants, but the differences between these strains were markedly less than those observed inter-species. Sequenc e analysis was performed on 60 amplicons obtained from blood pellets collec ted from woodland rodent communities in which bartonella infections were kn own to be highly prevalent. Twelve variants were encountered, only five of which had been found among the studied isolates. Partial intragenic spacer region amplification followed by product sequence analysis offers a potenti ally sensitive and totally transferable means of inter- and intra-species d ifferentiation of Bartonella strains, and its use in this study has broaden ed our knowledge of the genotypic spectrum of bartonellae associated with n atural infections among UK woodland rodents. (C) 2000 Academic Press.