Candidatus Bartonella mayotimonensis and Endocarditis

We describe a new Bartonella species for which we propose the name Candidatus Bartonella mayotimonensis. It was isolated from native aortic valve tissue of a person with infective endocarditis. The new species was identified by using PCR amplification and sequencing of 5 genes (16S rRNA gene, ftsZ, rpoB, gltA, and internal transcribed spacer region).

The patient lived alone on a farm in Iowa, USA, and had not had recent exposure to animals. However, he had observed murine fecal droppings in his house and mice on the farm. He had had a house cat for 18 years until its death a few years before his hospitalization and had intermittent contact with cats when he visited his daughter.
Serum immunoglobulin G titers were positive for B. henselae and B. quintana (>1,024). Oral doxycycline and rifampin were prescribed for treatment of presumed Bartonella endocarditis. Gentamicin was not administered because of development of the acute renal dysfunction. Two weeks later, he underwent aortic valve and aortic root replacement. Results of gram staining, acid-fast staining, fungal staining, anaerobic bacterial culture, aerobic bacterial culture, mycobacterial culture, and fungal culture on resected aortic valve tissue were negative for Bartonella species.
PCR performed at the Mayo Clinic on resected aortic valve tissue detected part of the citrate synthase gene (gltA) of Bartonella species. However, the melting temperature was not characteristic of B. quintana or B. henselae (7). Oral doxycycline and rifampin were continued for 12 weeks after aortic valve resection. The patient was well and had no signs of relapsing infection at a follow-up visit 11 months after valve surgery.
A Bartonella species was detected in a shell vial by immunofl uorescence after 15 days of culture; identifi cation was confi rmed by PCR. DNA was extracted from valve specimen and injected cells by using the QIAamp Tissue Kit (QIAGEN, Hilden, Germany). DNA was used as a template in a genus Bartonella Lightcycler assay with primers and a Taqman probe specifi c for the internal transcribed spacer (ITS) gene (6) and in standard PCR assays specifi c for the 16S rRNA, ITS, rpoB, gltA, and ftsZ genes (8). Sequences from both DNA strands were determined twice for all PCR products. These products were resolved in an ABI 3100 automated sequencer (PerkinElmer, Waltham, MA, USA). Sterile water was used as a negative control in each assay. Percentages of similarity among sequences were determined by using MEGA 2.1 software (9). Phylogenetic relationships among Bartonella strains were inferred from concatenated sequences by using MEGA 2.1 software (9). Surgically resected aortic valve tissues were fi xed in formalin, embedded in paraffi n, and sectioned to a thickness of 5 μm. Sections were stained with periodic acid-Schiff, Giemsa, Gram, Grocott-Gomori methenamine silver, and Warthin-Starry stains. Immunohistochemical analysis was performed by using a procedure described elsewhere (10) and polyclonal antibody against B. vinsonii at a dilution of 1:1,000.
Serum samples showed immunoglobulin G endpoint titers of 50 against all Bartonella species tested by immunofl uorescent assay. Western blot results were positive and characteristic of Bartonella infection (Figure 1, panel A). Results of PCR (Bartonella genus Lightcycler assay and standard PCR for cardiac valve) and cell culture were positive, and amplifi cation products of the expected size were obtained. Among known validated species, sequences obtained shared 99.1% (1, Figure 2. Sequences of gltA, 16S rDNA, ftsZ, ITS, and rpoB were deposited in GenBank under accession nos. FJ376732-FJ376736. Histologic analysis of resected aortic valve showed infective endocarditis with vegetation containing microorganisms that stained with Warthin-Starry and Giemsa. Warthin-Starry staining showed darkly stained bacilli consistent with Bartonella species (Figure 1, panel B). Results of staining with periodic acid-Schiff, Gram, and Grocott- Gomori methenamine silver were negative. Immunohistochemical analysis detected bacteria in valvular vegetations in a location superimposable with that detected by Warthin-Starry staining ( Figure 1, panels C, D).

Conclusions
We isolated a new Bartonella species and propose that it be named Candidatus Bartonella mayotimonensis to recognize the contributing institutions (Mayo Clinic and Hôpital de la Timone, Marseille, France). This is the seventh Bartonella species documented to cause infective endocarditis in humans.
The reservoir of Candidatus B. mayotimonensis has yet to be determined. Different Bartonella species have been isolated from a variety of mammals, and each species is highly adapted to its reservoir host (11,12). The domestic cat is the primary mammalian reservoir for B. henselae (13). Other Bartonella species have been found in mammalian hosts, including rats (B.  (3)(4)(5)(6)12,14,15). Our patient had direct exposure to mice on his farm and also had intermittent contact with cats while visiting his daughter. Additional investigations are needed to determine the reservoir(s) and vector(s) for this novel bacterium.
The immunofl uorescent assay, the current serologic method for diagnosis of Bartonella infection, does not distinguish among Bartonella species. Only Western blot analysis and cross-adsorption enable serologic identifi cation of species. PCR and culture are critical when a Bartonella species is identifi ed for the fi rst time as a human pathogen. Newly encountered Bartonella strains should be considered a new species if a 327-bp gltA fragment shares <96.0% sequence similarity with those of validated species, and if an 825-bp rpoB fragment shares <95.4% sequence similarity with those of validated species as reported in the current case (8).
This case reinforces the hypothesis that any Bartonella species can cause human infection, including culture-negative endocarditis. Candidatus B. mayotimonensis should be added to the list of human pathogens that can cause culture-negative endocarditis.