Genetic Diversity of Bartonella henselae in Human Infection Detected with Multispacer Typing

This techniqueis a suitable tool for evaluating the genetic diversity of B. henselae human isolates.

B artonella henselae, fi rst identifi ed in 1990 and characterized as a new species in 1992, is a gram-negative, fastidious bacterium associated with cats. B. henselae infection in cats is usually asymptomatic, but infected cats may remain bacteremic for long periods, thus playing a major role as a reservoir for the bacterium (1,2). Transmission of B. henselae among cats may be mediated by the cat fl ea, Ctenocephalides felis (3), and to humans by cat scratches or bites (4). B. henselae infection in humans exhibits a variety of clinical syndromes including the most common, catscratch disease (CSD) (5), endocarditis (6), bacillary angiomatosis (7) and peliosis hepatis in immunocompromised patients (8), and other less frequent manifestations (9). B. henselae has also been detected in various domestic or wild animals, including dogs, lions, panthers, and cheetahs (10).
More recently, B. henselae was detected in the porpoise, a marine mammal (11).
Several studies have suggested that human-associated isolates were less genetically heterogeneous than cat isolates (22,27,30), and a small group of cat isolates were hypothesized to be the source of human infection (22,27). However, the limited discriminatory power of the available genotyping methods, with a maximum of only 7 genotypes identifi ed (27), and the small number of human isolates studied prevented the population structure and the genetic relationship between cat and human isolates of B. henselae to be reliably investigated. Recently, we applied multispacer typing (MST), using 9 highly variable intergenic spacers, to 126 B. henselae cat isolates and identifi ed 39 MST genotypes, which made it the highest resolution typing system (available from http://ifr48.timone.univmrs.fr/mst_bhenselae/mst) (31). In this study, we applied MST, based on sequences from the 9 previously described variable intergenic spacers (31), to 75 human-infecting B. henselae isolates to evaluate their genetic diversity and compare it to that of cat isolates.

Study Design
We included in the study, when available, lymph node biopsy specimens and cardiac valve specimens from patients with a clinical diagnosis of CSD or endocarditis who had been referred to our laboratory from 2004 to 2006. All specimens were then frozen at -80°C for further culture and molecular detection of Bartonella species. B. henselae was detected by using PCR targeting the 16S-23S rRNA spacer (ITS) and pap31 gene (24,25). In addition, 3 human B. henselae isolates-type strain Houston-1, isolated from the blood of a patient with bacillary angiomatosis in USA; type strain Marseille, isolated from the lymph node biopsy specimen of a patient with CSD in France; and strain URBH-TOIE49, isolated from the valve biopsy specimen of a patient with endocarditis in France (26)-were incorporated in this study. All B. henselae isolates and PCR amplicons were tested by MST by using 9 variable intergenic spacers named S1-S9, from the most to the least variable among cat isolates (31). When available, the 16S rRNA genotypes of studied strains were indicated (online Appendix Figure, available from www.cdc.gov/EID/content/13/8/1178-appG.htm).

DNA Preparation
Total genomic DNA was extracted from the 3 studied B. henselae isolates by using the Chelex procedure as previously described (32). For patients' specimens, we used the QIAamp Tissue kit (QIAGEN, Hilden, Germany) to extract the total genomic DNA, as described by the manufacturer.

PCR Amplifi cation and Sequencing
ITS and pap31 gene PCR amplifi cations were performed by using the previously described conditions and the 16SF-23S1 (25) and PAPn1-PAPn2 (24) primer pairs, respectively. For amplifi cation and sequencing of the 9 intergenic spacers, we used the same primers as in our previous study (31), except for primers amplifying spacers S1 and S8, which caused unspecifi c amplifi cations from DNA extracted from human specimens. One new set of primers was designed to amplify each of these 2 spacers, as indicated in the online Appendix Table (available from www. cdc.gov/EID/content/13/8/1178-appT.htm).
These new primers also amplifi ed the whole intergenic spacers and thus did not affect genotype comparison between human and cat strains. All primers were obtained from Eurogentec (Seraing, Belgium). PCRs were performed in a PTC-200 automated thermal cycler (MJ Research, Waltham, MA, USA). One nanomolar concentration of each DNA preparation was amplifi ed in a 25-μL reaction mixture containing 50 pmol/L of each primer; 200 μmol/L (each) dATP, dCTP, dGTP, and dTTP (Invitrogen, Gaithersburg, MD, USA); 1.5 U of HotstarTaq DNA polymerase (QIAGEN); 2.5 μL 10× PCR buffer; and 1 μL 25 mmol/L MgCl 2 . The following conditions were used for amplifi cation: an initial 15-min step at 95°C was followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C. Final amplifi cation was completed by holding the reaction mixture for 5 min at 72°C to allow complete extension of the PCR products. PCR products were purifi ed by using the MultiScreen PCR fi lter plate (Millipore, Saint-Quentin-en-Yvelines, France) as recommended by the manufacturer. Amplicons were sequenced in both directions by using the BigDye 1.1 chemistry (Applied Biosystems, Foster City, CA, USA) on an ABI 3130XL automated sequencer (Applied Biosystems) as described by the manufacturer. To avoid contamination, no positive control was used. Sterile water was used as a negative control in each PCR. Sequences from each DNA sample were checked twice in both directions to ensure the reliability of the MST method.

Sequence Analysis
Nucleotide sequences were edited by using the Autoassembler package (PerkinElmer, Waltham, MA, USA). For each intergenic spacer, a genotype was defi ned as a sequence exhibiting unique mutations, which were checked by sequencing the corresponding spacers 3 times. MST genotypes were defi ned as unique combinations of the 9 spacer genotypes.

Phylogenetic Analysis
For the phylogenetic analysis, we concatenated sequences from the 9 studied spacers. We included in the analysis the 39 MST genotypes previously obtained from the MST analysis of cat isolates (31) and those, when different, obtained in the present study. Multiple alignment of sequences was carried out by using CLUSTALW software (33). Phylogenetic relationships were obtained by using the neighbor-joining and maximum parsimony methods within the MEGA 2.1 software (34), and the maximum likelihood method within the BioEdit software (available from www. mbio.ncsu.edu/bioedit/bioedit.html).

Statistical Analysis
The genotypic diversities of human and cat strains of B. henselae were compared by using the Fisher exact test within the Epi Info 6.0 software (Centers for Disease Control and Prevention, Atlanta, GA, USA). A difference was considered statistically signifi cant when p<0.01.

Phylogenetic Analysis
Phylogenetic trees obtained by using alignment of the 9 concatenated spacer sequences and the neighbor-joining, maximum parsimony, and maximum likelihood methods showed similar organizations. The 50 MST genotypes (in- cluding the 39 MST genotypes previously identifi ed among cat isolates [31] and the 11 new MST genotypes identifi ed in the present study) were grouped into 4 clusters (online Appendix Figure). Two clusters were associated with previously described Houston-1 and Marseille serotypes.  Figure). Two genotypes (5, and 22,) were mainly found in the Houston-1 and Marseille clusters, respectively. Genotype 22 contained 37 human strains or amplicons and 16 European cat isolates; genotype 5 included 22 human amplicons, 7 European and 23 American cat isolates. The 3 human strains from patients with endocarditis were classifi ed within 3 different MST genotypes, i.e., genotypes 22, 41, and 50. Type strain Houston-1, obtained from a patient with bacillary angiomatosis, exhibited the unique genotype 50, whereas type strain Marseille shared genotype 16 with an American cat isolate (online Appendix Figure).
The genotypic diversity of human strains was not statistically different from that of cat isolates (16/75 vs. 39/129, p = 0.3), even when restricted to French human strains or cat isolates (14/73 vs. 6/29, p = 0.9). However, when we compared the distribution of human strains among clusters, we found that the Houston-1 and Marseille clusters contained signifi cantly more human strains than did cluster 1 (p<0.01) and also that the Houston-1 cluster contained a signifi cantly higher proportion of human strains than the Marseille cluster (p<0.01).
In addition to type strains Houston-1 and Marseille, the 16S rRNA genotype was known for 67 cat strains. Phylogenetic cluster 1 contained 18 type I strains; cluster Houston-1 contained 21 type I strains and 1 type II strain; and clusters Marseille and 4 contained 20 and 9 type II strains, respectively.

Discussion
We report the successful adaptation of MST to B. henselae detected in human samples. Isolating B. henselae from CSD patients is extremely diffi cult (35). In this context, a reliable and reproducible molecular typing method, using PCR coupled to sequencing, to study the genetic diversity of B. henselae detected in human specimens directly, is a valuable option. Recently, MLST that used 9 housekeeping genes classifi ed 20 cat and 17 human isolates into 7 types, with most of the 17 human isolates belonging to 1 specifi c genotype. That study suggested that human isolates were more homogenous than cat isolates. However, the study was limited by the small number of human isolates studied and the limited discriminatory power of MLST, which hindered in-depth exploration of the genotypic diversity of B. henselae (27). In our present study with MST, using 9 highly variable ITS exhibiting a high resolution for subtyping B. henselae (31), we investigated the genetic diversity of B. henselae detected in humans.
The addition of 11 new MST genotypes to the 39 previously identifi ed did not modify the phylogenetic distribution of 4 main clusters (lineages) described among cat isolates (31). However, the human strains had a specifi c phylogenetic distribution. Clusters 1 and 4 contained signifi cantly fewer human strains than did clusters Houston-1 and Marseille (p<0.01). For cluster 1, this difference may be explained by the fact that this cluster contained only cat isolates from the United States and Asia, whereas we studied mostly human strains from France. The only human strain classifi ed within cluster 1 was detected in 1 patient from the West Indies, but we acknowledge that this single patient, although exhibiting a specifi c genotype, does not allow us to draw any conclusions about the distribution of B. henselae genotypes in this area. Therefore, estimating the MST genotypes of human strains from the West Indies, United States, and Asia and comparing them to MST genotypes classifi ed within cluster 1 might be useful. Regarding the distribution of strains within cluster 4 (4 cat strains from the United States, 10 cat strains from France, and 3 cat strains from Germany, but no human strain), we speculate that these strains may be less pathogenic for humans or that a sampling bias occurred. Thus, additional human and cat B. henselae isolates or amplicons from more countries will be needed to investigate the geographic correlation between human and cat isolates.
Previously, on the basis of the polymorphisms of the pap31 gene, among 107 B. henselae human strains originated from France, Zeaiter et al. identifi ed 4 genotypes grouped into 2 lineages, Marseille, including genotypes Marseille and CAL-1, and Houston-1, including genotypes Houston-1 and ZF-1; this remains the largest genetic study of B. henselae human strains (24). Of the 107 human strains, 63 and 40 hold genotypes CAL-1 and ZF-1, respectively, which were predominant among French human strains (24). In our study, among 73 French human strains, 14 MST genotypes were also identifi ed into 2 lineages, Houston-1, including 38 strains of 2 genotypes, and lineage Marseille, including 35 strains of 12 genotypes; genotypes 22 and 5 within lineages Houston-1 and Marseille, respectively, contained 37 and 22 human strains and were predominant. Thus, the phylogenetic relationships of French human strains identifi ed by pap31 were similar to what was found by using MST, although MST was more discriminatory than pap31-typing (p<0.01). However, MLST based on 9 genes later identifi ed 7 genotypes and 3 lineages (Marseille, Houston-1, and Berlin-2) among 20 cat and 17 human isolates (27). In contrast, our results, which were based on a larger number and a wider distribution of both cat and human strains and the more discriminatory MST, differ from those of these and other authors in that we identifi ed 2 lineages (lineages 1 and 4) besides Marseille and Houston-1 (18,27). Lineage 1 contained only Asian and American strains, which were not included in the study by MLST. Lineages Marseille and Houston-1 thus appear to be the main phylogenetic organization of B. henselae species. However, the phylogenetic organization of B. henselae species, as shown by both MLST and MST, was more complex than the structure of 2 main lineages (27,31). The 4 lineages based on MST provided the most detailed and reasonable illustration of the phylogenetic organization of B. henselae species because of its ability to show the geographic distribution of B. henselae. However, more strains, especially more human strains of various origin, should be studied by using MST to verify and modify this phylogenetic organization.
When we compared the MST classifi cation to classification by 16S rRNA genotypes, we observed that 16SrRNA type I strains were restricted to clusters 1 and 2 (Houston-1), whereas most type II strains were grouped into clusters 3 (Marseille) and 4. However, a discrepancy between MST and 16S rRNA typing was observed within the Houston-1 cluster. One German cat strain classifi ed in MST genotype 22 exhibited the 16S rRNA type II, in contrast to all other strains for which the 16S rRNA type was known, including other German strains. Such a discrepancy is consistent with the fi ndings of Iredell et al., who demonstrated that 16S rRNA typing of B. henselae isolates was not entirely congruent with their lineage allocations (27).
In conclusion, we demonstrated, with 16 genotypes identifi ed among 75 B. henselae human strains, that MST was more discriminatory than previously described methods for investigating B. henselae infection in humans. We did not fi nd any statistically signifi cant difference in genetic diversity between human and cat isolates of B. henselae.
The studied human strains, although geographically limited, were phylogenetically organized into 2 clusters, which matched the origin of cat strains previously described as Houston-1 and Marseille clusters. Further studies incorporating strains from more diverse geographic origins and clinical features will be needed to improve our understanding of the population dynamics of B. henselae. We believe that MST can be a valuable tool for this purpose. Dr Li is a physician and research scientist at the World Health Organization Collaborative Unité des Rickettsies in Marseille, France. His main research interests are the epidemiology and genetic diversity of human pathogenic bacteria.