16S/23S rRNA intergenic spacer regions for phylogenetic analysis, identification, and subtyping of Bartonella species

Abstract

Species of the genus Bartonella are currently recognized in growing numbers and are involved in an increasing variety of human diseases, mainly trench fever, Carrion's disease, bacillary angiomatosis, endocarditis, cat scratch disease, neuroretinitis, and asymptomatic bacteremia. Such a wide spectrum of infections makes it necessary to develop species and strain identification tools in order to perform phylogenetic and epidemiological studies. The 16S/23S rRNA intergenic spacer region (ITS) was sequenced for four previously untested species, B. vinsonii subsp. arupensis, B. tribocorum, B. alsatica, and B. koehlerae, as well as for 28 human isolates of B. quintana (most of them from French homeless people), six human or cat isolates of B. henselae, five cat isolates of B. clarridgeiae, and four human isolates of B. bacilliformis. Phylogenetic trees inferred from full ITS sequences of the 14 recognized Bartonella species using parsimony and distance methods revealed high statistical support, as bootstrap values were higher than those observed with other tested genes. Five well-supported lineages were identified within the genus and the proposed phylogenetic organization was consistent with that resulting from protein-encoding gene sequence comparisons. The ITS-derived phylogeny appears, therefore, to be a useful tool for investigating the evolutionary relationships of Bartonella species and to identify Bartonella species. Further, partial ITS amplification and sequencing offers a sensitive means of intraspecies differentiation of B. henselae, B. clarridgeiae, and B. bacilliformis isolates, as each strain had a specific sequence. The usefulness of this approach in epidemiological investigations should be highlighted. Among B. quintana strains, however, the genetic heterogeneity was low, as only three ITS genotypes were identified. It was nevertheless sufficient to show that the B. quintana population infecting homeless people in France was not clonal.

FIG. 1

Line diagram showing relative positions of oligonucleotide primers used for PCR amplification and sequencing of the ITS from Bartonella species. The positions of the oligonucleotides are with respect to the sequence of the ITS of B. quintana Fuller (accession number L35100). Nucleotide positions: 16SF, 1 to 20; 23S1, 1316 to 1331; QHVE1 and QHVE2, 274 to 292; QHVE3 and QHVE4, 898 to 915; BABF, 880 to 899; and BABR, 1121 to 1140.

FIG. 2

Comparison of parsimony tree (left side) and neighbor-joining tree (right side) derived from complete ITS sequences for recognized Bartonella species (type strains). The support of each branch, as determined from 100 bootstrap samples, is indicated by the value at the node. The lengths of vertical lines are not significant. For the parsimony tree, the lengths of horizontal lines are also not significant. For the neighbor-joining tree, the scale bar represents evolutionary distance as calculated by using the Kimura two-parameter distance calculation.

FIG. 3

Alignment of QHVE1 and QHVE3 ITS amplicons derived from seven B. henselae strains. ∗, substitution or point insertion or deletion; Rpta and Rptb, repeat regions.

FIG. 3

Alignment of QHVE1 and QHVE3 ITS amplicons derived from seven B. henselae strains. ∗, substitution or point insertion or deletion; Rpta and Rptb, repeat regions.

FIG. 4

Comparison of parsimony trees derived from the gltA gene (left side), from the groEL gene (middle), and from the ITS sequences (right side) for recognized Bartonella species. The support of each branch, as determined from 100 bootstrap samples, is indicated by the value at the node. This analysis provided tree topology only, and the lengths of both vertical lines and horizontal lines are not significant. Boxes contain the clusters which are found consistently in all analyses performed.