Simultaneous presence of two different copies of the 16S rRNA gene in Bartonella henselae.

Bartonella henselae is an emerging pathogen of increasing medical significance. Previous investigations have revealed two different 16S rRNA gene variants among B. henselae isolates, resulting in delineation of the B. henselae population into 16S RNA type I and type II isolates. While studying 191 B. henselae isolates by multi-locus sequence typing (MLST) we detected three isolates that could not be assigned to a distinct 16S RNA type upon direct sequencing because of ambiguous nucleotides in a distinct region of the 16S rRNA gene. Cloning and sequencing of the target region of the 16S rRNA gene suggested that these atypical isolates contained different 16S rRNA gene copies. Southern blot and hybridization experiments confirmed the presence of two different 16S RNA gene copies in each isolate. The isolates were further analysed by 16S RNA type-specific PCR, which assigned them to both 16S RNA types I and II. These results suggest that a small percentage of B. henselae isolates may harbour two different 16S rRNA gene copies. These isolates, which accounted for 1.6 % of the isolates in our study, have probably emerged by horizontal gene transfer. The implications of these findings for identification and genotyping studies on B. henselae are discussed.

Prolonged Bartonella henselae bacteremia caused by reinfection in cats.

We analyzed the genetic relatedness of blood culture isolates of Bartonella henselae from 2 cats of patients with cat-scratch disease at admission and after 12 months. Isolates from each cat at different times were clonally unrelated, which suggested reinfection by a second strain.

Multi-locus sequence typing of Bartonella henselae isolates from three continents reveals hypervirulent and feline-associated clones.

Bartonella henselae is a zoonotic pathogen and the causative agent of cat scratch disease and a variety of other disease manifestations in humans. Previous investigations have suggested that a limited subset of B. henselae isolates may be associated with human disease. In the present study, 182 human and feline B. henselae isolates from Europe, North America and Australia were analysed by multi-locus sequence typing (MLST) to detect any associations between sequence type (ST), host species and geographical distribution of the isolates. A total of 14 sequence types were detected, but over 66% (16/24) of the isolates recovered from human disease corresponded to a single genotype, ST1, and this type was detected in all three continents. In contrast, 27.2% (43/158) of the feline isolates corresponded to ST7, but this ST was not recovered from humans and was restricted to Europe. The difference in host association of STs 1 (human) and 7 (feline) was statistically significant (P< or =0.001). eBURST analysis assigned the 14 STs to three clonal lineages, which contained two or more STs, and a singleton comprising ST7. These groups were broadly consistent with a neighbour-joining tree, although splits decomposition analysis was indicative of a history of recombination. These data indicate that B. henselae lineages differ in their virulence properties for humans and contribute to a better understanding of the population structure of B. henselae.

Bartonella henselae exists as a mosaic of different genetic variants in the infected host.

Bartonella henselae is a fastidious bacterium associated with infections in humans and cats. The mechanisms involved in the long-term survival of bartonellae despite vigorous host immune responses are poorly understood. Generation of genetic variants is a possible strategy to circumvent the host specific immune responses. The authors have recently demonstrated the coexistence of different genetic variants within the progeny of three primary B. henselae isolates from Berlin by PFGE analysis. Aims of the present study were to determine whether coexistence of different variants is a common feature of B. henselae isolates worldwide and whether the genetic variants originally emerged in vivo. Thirty-four primary isolates from different geographical regions were analysed by subjecting multiple single-colony-derived cultures to PFGE analysis. Up to three genetic variants were detected within 20 (58.8 %) isolates, indicating that most primary isolates display a mosaic-like structure. The close relatedness of the genetic variants within an isolate was confirmed by multi-locus sequence typing. In contrast to the primary isolates, no genetic variants were detected within the progeny of 20 experimental clones generated in vitro from 20 primary isolates, suggesting that the variants were not induced in vitro during the procedure of PFGE analysis. Hence, the genetic variants within a primary isolate most likely originally emerged in vivo. Consideration of the mosaic structure of primary isolates is essential when interpreting typing studies on B. henselae.

Evaluation of pulsed-field gel electrophoresis and multi-locus sequence typing for the analysis of clonal relatedness among Bartonella henselae isolates.

Pulsed-field gel electrophoresis (PFGE) represents the gold standard among band-based methods for the molecular typing of Bartonella henselae. SmaI and NotI have been frequently used for typing B. henselae by PFGE. However, their appropriateness for the analysis of genetic relatedness among B. henselae isolates has not been assessed systematically hitherto. Aim of the present study was to evaluate SmaI, NotI, and three additional endonucleases for typing B. henselae isolates by PFGE and to compare the PFGE results with multi-locus sequence typing (MLST) data. Twenty B. henselae isolates from different sources and geographic regions were analysed. PFGE analysis upon restriction with SmaI, ApaI, Eco52I, and XmaJI revealed six, five, four, and five different PFGE types, respectively, whereas restriction with NotI revealed 13 PFGE types. Five sequence types (STs) were obtained by MLST. The overall concordance between PFGE types obtained with SmaI, ApaI, Eco52I, XmaJI and STs was high. In contrast, NotI-derived types did not correlate with other PFGE types or STs, indicating that NotI is not an appropriate enzyme for PFGE typing of B. henselae. By combining PFGE results obtained with SmaI, ApaI, Eco52I, XmaJI with STs, the isolates could be assigned to five distinct clonal lineages, including the clones Houston-1, Marseille, CAL-1, and Berlin-2. These data indicate that PFGE and MLST are discriminatory and reliable for molecular typing of B. henselae isolates to the strain level. Combination of PFGE and MLST may be useful for further epidemiological studies on B. henselae.

Emergence of distinct genetic variants in the population of primary Bartonella henselae isolates.

Bartonella henselae isolates from different hosts display a marked genetic heterogeneity, as determined by pulsed-field gel electrophoresis (PFGE). The aim of the present study was to determine whether different genetic variants may coexist within the population of distinct B. henselae isolates and could be detected by PFGE. Three primary B. henselae isolates and the B. henselae reference strains ATCC 49793 and 49882 were subjected as single colony derived cultures in quadruplicate to PFGE analysis upon restriction with SmaI or NotI. Up to 4 fragment differences were found among the cultures obtained from each primary isolate, indicating the coexistence of genetic variants in the population of primary B. henselae isolates. The clonal relatedness of the genetic variants was confirmed by arbitrarily primed PCR and multi-locus sequence typing. In contrast to the primary isolates, no variants were detected among the single colony derived cultures of the high-passage ATCC strains. We hypothesized that the coexistence of different genetic variants may represent a feature that is restricted to primary or low-passage B. henselae isolates. The primary isolates were serially passed in vitro and then subjected as single colony derived cultures to PFGE analysis, which now revealed identical patterns among the quadruplicate cultures of each high-passage isolate. These results suggest that the population of a primary B. henselae isolate is composed of distinct genetic variants, which may disappear upon repeated passages on artificial culture media. Generation of genetic variants by B. henselae may represent an escape mechanism to circumvent the host specific immune responses.