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

This study aimed to assess the usefulness of a PCR-based approach to the detection and differentiation of Bartonella strains in infected blood. The conservation of potential genus-specific PCR primer hybridisation sites within the 16 S/23 S rRNA gene intragenic spacer regions of Bartonella species was confirmed following sequence analysis of the intragenic spacer regions of four previously untested species. The extent of intra-species variation within the specific amplicons was assessed by comparison of sequences obtained 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. Sequence analysis was performed on 60 amplicons obtained from blood pellets collected from woodland rodent communities in which bartonella infections were known 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 potentially sensitive and totally transferable means of inter- and intra-species differentiation of Bartonella strains, and its use in this study has broadened our knowledge of the genotypic spectrum of bartonellae associated with natural infections among UK woodland rodents.