Introduction

Bartonella quintana is a fastidious Gram-negative bacterium transmitted among humans by the body louse Pediculosis humanus humanus and known to cause trench fever, chronic bacteremia, bacillary angiomatosis and endocarditis. Trench fever was first described in Europe in 1915, during World War I [1], and later during World War II [2, 3]. However, recent paleomicrobiological studies have provided evidence that B. quintana has been associated with human infection for 4000 years [4]. After an interval of several decades where trench fever almost disappeared, small outbreaks of B. quintana chronic bacteremia have reemerged in HIV-uninfected people living in urban areas. Homelessness, alcoholism and louse infestation were identified as risk factors [1, 3, 5, 6].

For years, humans have been considered as the only reservoir of B. quintana, however, recent studies have identified the non-human primate macaques as natural hosts of B. quintana [7, 8]. Based on multi-locus sequence typing (MLST), B. quintana strains have been classified into 22 sequence types (STs): STs 1–7 exclusively found in humans [9], STs 8–14 and STs 15–21 in cynomolgus macaque and in rhesus macaque strains from China [7], respectively, and ST22 in Japanese macaque strains in Japan [8].

Bartonella species is recognized as an important cause of culture-negative endocarditis, accounting for 2–40% of cases, with B. quintana responsible for the majority of Bartonella endocarditis cases [10,11,12,13,14,15,16]. Data regarding the molecular epidemiology of B. quintana strains causing endocarditis, however, is limited to the description of only 3 STs (STs 1, 2, and 7) identified in 4 endocarditis patients from Europe and Australia [9].

In the present study, we applied B. quintana-specific MLST technique to 11 Bartonella endocarditis patients diagnosed in Israel, who have presumably acquired the infection in Eastern Africa or in Israel, to investigate the genetic diversity and clinical relatedness of B. quintana strains identified in endocarditis patients from distinct geographic regions.

Materials and methods

Patients and clinical specimens

Eleven patients with definite endocarditis, as defined in the modified Duke criteria [17], were included in this study. All patients have undergone valve surgery for endocarditis-associated valve dysfunction or the presence of intracardiac abscess; 5 patients arrived from Eastern Africa for cardiothoracic care at Wolfson Medical Center in Israel through the Save a Child’s Heart fund activity, 1 patient immigrated from Ethiopia to Israel while being severely ill with endocarditis, and 5 patients have been Israeli residents for several years prior to diagnosis. PCR of cardiac specimens (10 patients) and of a B. quintana blood culture isolate (1 patient) followed by DNA sequencing was performed as previously described [18]. Relevant patient characteristics are summarized in Table 1. Patients no. 1–5 have been described before [19, 20].

Table 1 Characteristics of 11 patients with B. quintana endocarditis
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MLST analysis of B. quintana DNA

Genomic DNA was extracted from cardiac tissues of 10 patients using the QIAamp DNA minikit (Qiagen, Valencia, CA) and from a B. quintana blood culture isolate of 1 patient using E.Z.N.A. bacterial DNA kit (Omega Bio-Tek), and then kept at − 20 °C until further analysis. All DNA samples were subsequently analyzed by B. quintana-specific MLST scheme which has been developed based on 9 genetic loci (atpF, bqtR, ftsZ, gap, gltA, groEL, nlpD, ribE, rpoB) [9]. PCR conditions for all loci were as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles (94 °C for 1 min, 48 °C for 30 s, and 72 °C for 45 s), and a final extension step at 72 °C for 6 min. Genomic DNA from B. quintana Toulouse strain (CIP 103739) and nuclease-free water were used for positive and negative controls, respectively. Nucleotide sequences of the PCR products were determined for both strands and repeated as necessary (DNA Sequencing Unit at the G.S. Wise Faculty of Life Sciences, Tel Aviv University, Israel). The nucleotide sequences of each locus were compared with MLST data previously reported [7,8,9] using a genomic analysis software GENETYX ver.15 (GENETYX Corp., Tokyo, Japan) and were assigned allelic numbers. New allelic combinations identified in the present study were assigned to new STs.

An evolutionary relationship between the new STs and the previously reported STs (1 to 22) was visualized by a minimum spanning tree constructed based on goeBURST algorithm implemented in PHYLOViZ 2.0 [21], which allows phylogenetic inference and data visualization. A lineage was defined as the same group of STs sharing identical alleles at 7 or more of the 9 loci. A phylogenetic tree was constructed with the concatenated sequences (4271 bp) of the 9 loci in each ST by using the maximum-likelihood method in MEGA X [22]. B. henselae Houston-1T was set as an outgroup in the tree. Substitution model was Tamura 3-parameter model (T92) and gap/missing data were treated by complete deletion. Bootstrap analysis was conducted with 1000 replicates.

Results

The 9 genetic loci of B. quintana were detected in all patient-derived specimens. Strains EA1, EA3, and EA6, strain IS3, and strains IS1/Bq-TA7 and IS2 were classified into the previously described STs 2, 4, and 6, respectively. Strains EA2, EA4, EA5, ISEA, and EA7 were identified for the first time and classified into 5 new STs 23–27, resulting in discrimination of human strains into a total of 12 STs, including 8 endocarditis-related STs (Table 2). In a previous study by Arvand et al. [9], 4 of the 16 B. quintana strains were identified in clinical specimens of patients with endocarditis: ST1 in one strain (Jouhanneau), ST2 in two strains (strains UR.BQ.TIE 326 and HROEH), and ST7 in one strain (Adelaide 1300/02). Together with the current study, there are 15 patients with B. quintana endocarditis analyzed by MLST, with ST2, being the most prevalent, found in 5 patients (33.3%). The new endocarditis-related STs 23–27 clustered with the previously reported STs 1–7 from humans infected with B. quintana as supported by the minimum spanning tree and the phylogenetic tree, constructed based on the 9 loci (Figs. 1, 2). Moreover, ST26, found in strain ISEA, appears to be a primary founder in the human lineage, as it is surrounded by STs 3, 4, 5, and 27, each differs from ST26 by only a single locus (Fig. 1). Strain ISEA was detected in 2003 in the aortic valve of a 32-year-old male (patient no. 8, Table 1), who was born in Ethiopia and immigrated to Israel at the age of 13. Therefore, B. quintana strain ISEA showing ST26 has most likely been acquired in Israel.

Table 2 Allelic profiles and sequence types (ST) identified in this study
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Fig. 1
figure 1

Minimum spanning tree constructed from allelic profiles of STs 1 to 27 of B. quintana strains by goeBURST in PHYLOViZ 2.0. A lineage was defined as the same group of STs sharing identical alleles at 7 or more of the 9 loci. Black line and black dashed line indicate 1 and 2 differences of the locus between two STs, respectively. Green color square shows a founder in each lineage. Color circles show 4 lineages classified by host species

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Fig. 2
figure 2

Phylogenetic tree constructed from the 9 loci-concatenated sequences of STs 1 to 27 of B. quintana strains. The tree was constructed from the concatenated sequences (4271 bp) of the 9 loci used for MLST analysis by using the maximum-likelihood method based on the Tamura 3-parameter model in MEGA X [22]. The B. quintana strains from humans (STs 1 to 7 and STs 23 to 27), cynomolgus macaques (STs 8 to 14), rhesus macaques (STs 15 to 21), and Japanese macaques (ST22) were included in the tree. Colored rectangles show 4 groups classified by host species. The scale bar indicates estimated evolutionary distance. Bootstrap values were obtained with 1000 replicates. Only bootstrap replicates > 50% are noted. B. henselae Houston-1T was set as an outgroup in the tree

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Discussion

In the current study, we have analyzed genomic DNA of 11 B. quintana stains from patients with endocarditis by using MLST and identified 5 new STs which clustered with the previously reported STs 1–7 from humans infected with B. quintana. Although Arvand et al. [9] presented a phylogenetic relationship among these 7 STs determined by eBURST, the STs were divided into two clonal complexes and a singleton, which was not qualified for a definition of a single group. The new STs described herein were found to provide the missing links to form a single human lineage, clearly separated from the other 3 lineages of B. quintana strains of cynomolgus macaque, rhesus macaque, and Japanese macaque. From evolutionary perspectives, these findings further support the assumption that B. quintana strains have co-evolved with host species to form a host-speciation pattern.

Among the new STs, ST26 found in strain ISEA is suggested herein as a primary founder of the human lineage, rather than ST2 suggested previously [9]. Of note, in previous reports [7,8,9], ST5, identified in strain FullerT isolated in 1945 from a trench fever patient in the former Yugoslavia, was assigned as a singleton without any phylogenetic link to other STs since it differed in 2 or more alleles from all other STs. The phylogenetic analyses of the present study showed that of all the STs identified in Australia, France, Germany, Russia, and the USA, ST26 found in strain ISEA is the most closely related to ST5 (Figs. 1, 2), a finding that further supports ST26 as being a group founder. In addition, we have shown that ST2 is the most frequent ST causing endocarditis in humans. Of interest is the fact that the 15 B. quintana strains detected and analyzed by MLST hitherto in patients with endocarditis were identified in geographically diverse countries in 5 different continents, Europe, America and Australia previously reported [9] as well as Africa and Asia (Middle East) firstly reported here, without indication of geographical structuring.

To clarify whether ST26 is a veritable primary founder in human-derived B. quintana strains, substantiate an epidemiological association between ST2 and endocarditis, and explore where B. quintana had first originated, more molecular epidemiological studies are needed to be conducted worldwide.