Bartonellae are both Gram-negative alpha-proteobacteria and hemotropic bacteria highly adapted to facultative intracellular lifestyle in a wide variety of mammals, such as rodents, bats, insectivores, carnivores, ungulates, and other vertebrates. During the last two decades, progressively more bacterial species belonging to the genus Bartonella have been recognized with over 30 species described from different mammalian hosts. A number of Bartonella species were found to be associated with human illnesses and are associated with a growing spectrum of emerging diseases, including life-threatening endocarditis [1–9]. Animal reservoirs have been identified for some of the human pathogens, while remain unknown for others. Knowledge of the transmission of bartonella bacteria between mammalian hosts is incomplete. However, hematophagous arthropods such as fleas, flies, lice, mites, and ticks have been found naturally infected and are frequently implicated in transmitting Bartonella species [10–15].

Increasing recognition of bats as natural reservoirs of many emerging pathogens has drawn considerable attentions to study these mammals [16]. Multiple investigations of bartonella in bats have been conducted in different regions of the world [17–23]. These studies reported that bartonella infections are highly prevalent in many bat species and bartonella communities associated with bats are extremely diverse with co-circulation of numerous Bartonella species in the same bat populations. The data on relationships between Bartonella species and bats are quite contradictive depending on the investigated geographic region. Specifically, in Central and South America, multiple bat species may share the same Bartonella species without an evident host-specificity [19,20], while investigations from Asia and Africa demonstrated that a bat population typically harbors one or few Bartonella species specific for a particular bat species [17–18,21–22].

The straw-colored fruit bat (Eidolon helvum) is widely distributed in Africa. Colonies of these bats can be large and often found near human populations, with some roosts containing millions of individuals. Like other bats, straw-colored fruit bats have long life spans, with some individuals reaching at least 14 years of age [24]. Local human residents have a close contact with the bats, as they are frequently hunted for “bush meat”. A previous study in Kenya showed that straw-colored fruit bats are infected with bartonellae with high prevalence reaching 26% [18]. The same study demonstrated that Bartonella associated with the bats were genetically distant and belonged to four distinct genogroups based on sequence variation in the citrate synthase gene (gltA) [18]. Similar observations were reported in a very recent study conducted in Nigeria [22]. Considering the broad distribution and other ecological characteristics of straw-colored fruit bats, it was expected that additional Bartonella genogroups associated with this bat species can be identified in Africa.

In the present study, we aim to expand our knowledge of bartonella infections in straw-colored fruit bats and to better understand how multiple Bartonella species can co-habit populations of one bat species. We compared genetic differences of bartonella isolates obtained from straw-colored fruit bats captured in seven countries across Africa using a multi-locus sequence typing (MLST) approach. Based on comparison of nucleotide sequences derived from multiple loci, MLST has been shown to provide high discriminatory power in epidemiological and genetic analysis of bacterial strain populations, while retaining signatures of longer-term evolutionary relationships or clonal stability [25–28]. In addition, sequencing of multiple loci can detect evidences of micro-evolutionary events, particularly homologous recombination, among identified sequence types [29,30]. The main objective of the current study was to analyze bartonella isolates obtained from naturally infected straw-colored fruit bats from different parts of Africa to determine whether well-defined phylogenetic lineages correspond to currently accepted criteria for discrimination of bacterial species [31]. This, in turn, can help to enhance our understanding of population structure in the bacteria and the relationships between allelic profiles and the animal host. To achieve this goal, we developed a MLST scheme that incorporates eight loci which have been previously proposed for characterization of Bartonella species. In this study, clusters defined by the phylogenetic analysis were called either a “genogroup” or a “lineage”. A genogroup is defined when a single locus sequence was applied for the phylogenetic analysis, while a lineage is defined from concatenated sequences of all loci.

Straw-colored fruit bats have been reported to be commonly infected with multiple Bartonella strains based on gltA sequences [18,22]. In this study, we developed a MLST scheme using eight loci which have been commonly used for Bartonella species description to characterize 79 bartonella isolates obtained from straw-colored fruit bats that were captured in different regions across Africa to better elucidate the genetic diversity among these isolates.

The analyses of selection pattern showed none of the d_N/d_S ratios exceeded 1. This suggested that all of the protein-coding loci in the study are undergoing purifying selection. The analyzed bartonella isolates exhibited a high level of heterogeneity with 9–24 alleles identified by different loci. The MLST scheme resolved 45 STs among the 79 isolates and these isolates/STs clustered into six distinct lineages (E1—E5, and Ew) with genetic distances of 5.1–14% among the lineages. MLST has been regarded as a highly discriminatory tool by a number of previous studies. According to the widely accepted criteria for a new bacterial species based on nucleotide divergence greater than 5% in housekeeping genes [31], the MLST analyses in the present study demonstrated that each lineage identified in straw-colored fruit bats in this study likely represents a new Bartonella species. These Bartonella species have never been found in other bat species, suggesting their specific co-adaptation to Eidolon bats, though future studies are necessary to confirm this. The performed analyses allow us to conclude that there are at least six Bartonella species associated with straw-colored fruit bats. Phylogenetic analysis based on multiple loci demonstrates that four of these lineages, E1, E2, E3, and E5, belong to a phylogenetic lineage that includes other Bartonella strains isolated from Old World bats [18,21–23], while E4 and Ew appear to have evolved independently.

Lineages E3 and Ew, containing 24 and 21 isolates, respectively, and accounting for 57% of all isolates, are likely to be the more common Bartonella species circulating among this bat species. However, because the original bat samples from which the analyzed bartonella isolates obtained were collected in different settings and during different seasons by multiple investigators, there could be some bias in temporal or seasonal variations. Furthermore, the culturing procedure that was necessary for performing such bacterial classification may also contribute to misrepresentation of comparative prevalence of Bartonella species as some of them could hardly be cultured [35]. Due to the limited number of isolates selected from each part of Africa, we did not attempt to investigate any patterns in the geographic distribution of Bartonella species in this study. The presented classification, however, can be used for future studies aimed at describing geographic patterns of bartonella distribution among Eidolon bats.

Most lineages consisted of multiple STs (from 4 to 14). Nevertheless, lineage Ew consisted of only two STs with a large number of isolates, and in fact there is only one nucleotide difference between the two STs. This indicates a lower diversity of allelic polymorphisms in the isolates from this lineage and allows a speculation that Ew has diverged more recently compared to the other Bartonella lineages. Further, our observations of partially missing or non-amplifiable sequences of ribC suggests gene loss, a common evolutionary process in bacteria [36], possibly has occurred and resulted in the appearance of two new Bartonella species (E5 and Ew). As a housekeeping gene, ribC participates in the regulation of riboflavin biosynthesis. The non-amplifiability of the gene might not be true but suggest the gene is in some undetected form. If this gene is truly missing, its loss may be compensated by the fact that bartonellae are intracellular parasites which can perhaps utilize host riboflavin or they have lost the need of it. If this were true, fitness costs may lead to selection against this superfluous genetic material [37]. Overall, little is known about the mechanisms of regulation of bacterial riboflavin genes [38]. Although we cannot explain biological consequences of the loss of ribC, the 'presence/absence' characteristics make ribC a marker for the differentiation and classification of Eidolon-associated Bartonella strains. In addition, the dramatic variation in ribC among the straw-colored fruit bat-associated Bartonella species presents an interesting model for understanding evolutionary trends in developing host-bacterial parasite relations [36].

Another intriguing observation in this study was the set of mismatches between MLST lineages and some genogroups comparing phylograms built based either on entire concatenated sequences or individual locus sequences for four isolates. Particularly, three isolates belonging to E3 based on the ST classification were either Ew (two isolates) or E1 (one isolate) by nuoG or gltA alone and one isolate of E5 by ST classification would be E2 by nuoG or rpoB alone. The loci rpoB and gltA have been shown to be the most potent for species discrimination in other studies [31]. According to our observations in the present study, an analysis limited to two loci may still lead to a misclassification. Thus characterization of multiple loci should be preferably used for Bartonella species identification. The mismatches between lineages and genogroups identified by different loci indicate that recombination and/or lateral gene transfer events between different Bartonella species are ongoing processes.

Our recombination test results indicate that while recombination happens infrequently, an individual recombination event introduces a large number of polymorphisms. Recombination in rpoB, gltA, and other housekeeping loci has been observed in other Bartonella species [29,30,39]. Estimated r/m values for B. grahamii alone (1.7 [38] and 6.81 [30]), B. taylorii alone (3.77 [27]), or B. grahamii and B. taylorii considered together (4.06 [30]) compare favorably with our estimate for recombination among six species of Bartonella in E. helvum (r/m = 1.1). These estimates contrast with results from B. henselae (r/m = 0.1) [40] and the perception that intracellular bacteria have low rates of recombination and horizontal gene transfer [32]. Intermediate to high rates of recombination in these studies suggest that multiple Bartonella species may coexist at some point in the infection cycle, potentially in their mammalian hosts and/or arthropod vectors, thus facilitating gene exchange. Moreover, gene exchange appears to play an important role in generating sequence diversity among co-circulating Bartonella species. Further studies that could measure co-infection of Bartonella species and resulting rates of recombination would help to explain these interesting dynamics.

Our study provides information on the genetic diversity of straw-colored fruit bat-associated Bartonella species that can be used for the discrimination of the lineages at the level corresponding to separate species. Additional information is needed to understand the interaction between these Bartonella species and their bat hosts under natural conditions, as well as their putative ectoparasite vectors [41]. A biological approach to define Bartonella species has been discussed [42] with emphasis on host-association as a phenomenon that promotes biological isolation of Bartonella species. However, such an approach has limitations for situations when several Bartonella species are associated with one animal host. A similar situation was also described among Bartonella strains in grasshopper mice [43] and cotton rats [44]. Nevertheless, among all mammalian species described as a host of one or multiple Bartonella species, the number of presumptive species associated with E. helvum bats is clearly the highest. Although there is no evidence suggesting that Bartonella species associated with African fruit bats may cause illnesses in humans or in bats themselves, a recent study reported the presence of Bartonella mayotimonensis, a reported etiologic agent of endocarditis in humans, in the Daubenton's bat (Myotis daubentonii) and the Northern bat (Eptesicus nilssonii) in Europe [23], suggesting a potential role of bats as reservoirs for human bacterial pathogens. It is important to understand the role of straw-colored fruit bats in biotic communities and their importance as reservoir hosts contributing to the maintenance and transmission of bartonellae to other animals and humans.

The great diversity of Bartonella species associated with E. helvum, however, clearly presents interesting questions from the perspective of microbial evolution. What are the mechanisms that generate and maintain such diversity? How can these bacteria share the same ecological niche? Analyzing such relationships, Chan and Kosoy [45] hypothesized that co-circulation of independent Bartonella species in populations of one host species may represent an escape mechanism to circumvent the host immune responses. We ultimately believe this system will be very informative for understanding the evolution of bacteria in a host-vector-pathogen system.