Description of Bartonella bennetti sp. nov., a novel rodent-associated species, with comparative genomics of the Bartonella genus
Sean Brierley, Laura Mackenzie, Sandra Telfer, Kevin Bown, Ian Goodhead, Richard Birtles
TL;DR
Scientists discovered a new Bartonella species, Bartonella bennetti, from field voles in the UK, using genomic and ecological data to understand its evolution and host specificity.
Contribution
The discovery of Bartonella bennetti sp. nov., supported by genomic, ecological, and evolutionary evidence, introduces a new species in the Bartonella genus.
Findings
Bartonella bennetti sp. nov. was identified as a new species based on genomic analysis and ecological differences.
The species possesses unique genomic features, including a chromosomally integrated F-type conjugative plasmid.
The study suggests covert host specificity drives diversification in Bartonella lineage 3.
Abstract
The genus Bartonella comprises over 40 species, most of which are haemoparasites of mammals. Herein, we describe Bartonella bennetti sp. nov., a novel member of the genus, isolated from field voles (Microtus agrestis) in Kielder Forest, UK. Polyphasic characterization of three strains (C271T, D105 and J117) of the proposed species indicated that they were closely related to members of phylogenetic lineage 3 (L3) of the genus. The average nucleotide identity (ANI) between C271T and other L3 species ranged between 88.8 and 90.6%, supporting the proposal of a new species. C271T shared ANIs approaching 96% with other members of L3 that are yet to be validly published but exhibited marked genomic, ecological and biogeographical differences from them, further justifying the creation of a new taxon. All three B. bennetti sp. nov. strains were found to possess genes encoding three VirB/D4 type…
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Fig. 4| C271T | D105 | J117 | |||
|---|---|---|---|---|---|
| NCBI assembly/accession | GCA_000706645.1 | GCA_000253015.1 | |||
| Estimated chromosome length (bp) | 1,629,655 | 1,657,696 | 1,643,736 | 1,534,143 | 1,522,743 |
| DNA G+C content (mol%) | 35.9 | 36.0 | 35.9 | 35.7 | 35.7 |
| Protein-coding genes (CDS) | 1,555 | 1,609 | 1,506 | 1,397 | 1,334 |
| tRNA | 41 | 44 | 41 | 42 | 42 |
| rRNA | 6 | 9 | 6 | 6 | 6 |
| Number of contigs (scaffolds) | 1 (circular) | 1 (circular) | 1 (circular) | 19 (3) | 1 (circular) |
| Reference genome (NCBI accession) | C271T | D105 | J117 | RE21 | ||||
|---|---|---|---|---|---|---|---|---|
| OrthoANIu (%) | Coverage (%) | OrthoANIu (%) | Coverage (%) | OrthoANIu (%) | Coverage (%) | OrthoANIu (%) | Coverage (%) | |
| 90.60 | 61.70 | 90.78 | 58.17 | 90.67 | 59.20 | 78.87 | 38.07 | |
| 88.82 | 58.30 | 88.72 | 58.80 | 88.84 | 59.00 | 79.20 | 37.82 | |
| 95.98 |
|
| 62.85 |
|
| 79.11 | 37.32 | |
|
| 61.23 | 95.80 |
| 95.98 | 59.46 | 78.99 | 37.58 | |
| 88.16 | 57.86 | 88.09 | 56.01 | 88.24 | 56.22 | 79.22 | 34.31 | |
| 90.71 | 58.91 | 90.72 | 62.48 | 90.68 | 61.45 | 79.23 | 36.37 | |
| 90.91 | 58.33 | 90.83 | 62.33 | 90.69 | 64.90 | 78.82 | 36.36 | |
| 79.06 | 48.36 | 79.21 | 49.35 | 78.98 | 48.37 |
|
| |
| 79.29 | 47.83 | 79.24 | 46.46 | 79.27 | 47.66 | 87.50 | 50.47 | |
| 79.35 | 48.31 | 79.74 | 46.35 | 79.29 | 49.11 | 88.11 | 51.52 | |
| 79.50 | 48.97 | 79.77 | 46.82 | 79.63 | 48.77 | 79.92 | 36.81 | |
| 78.76 | 45.08 | 79.23 | 41.77 | 78.90 | 43.35 | 79.15 | 33.83 | |
| Reference plasmid | Length (bp) | pRecC271-1 | pRE21-1 | ||
|---|---|---|---|---|---|
| OrthoANIu (%) | Coverage (%) | OrthoANIu (%) | Coverage (%) | ||
| 23,343 | 0.00 | 0.00 | 80.25 | 5.28 | |
| 28,192 | 80.90 | 1.54 | 80.89 | 10.98 | |
| 57,959 | 85.10 | 4.10 | 76.72 | 14.52 | |
| 11,227 | 86.40 | 1.41 | 85.40 | 5.30 | |
| 29,057 | 77.76 | 50.08 | 83.11 | 51.40 | |
| 41,483 | 76.55 | 46.69 | 83.51 | 50.24 | |
| 29,892 | 76.49 | 50.98 | 84.10 | 63.32 | |
| 29,248/30,586 | 78.58 | 48.26 | 78.58 | 49.98 | |
- —http://dx.doi.org/10.13039/100010055 University of Salford Manchester
- —Perry Foundation
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Taxonomy
TopicsBartonella species infections research · Toxoplasma gondii Research Studies · Yersinia bacterium, plague, ectoparasites research
Introduction
The genus Bartonella currently comprises over 40 validly published species together with many Candidatus taxa and partially characterized strains [1]. The evolutionary history of the genus is marked by a remarkable transition from insect symbiosis to mammalian parasitism. The species associated with mammalian parasitism, termed eubartonella, are haemotrophic and arthropod transmitted and form a large monophyletic group that has diverged from taxa considered gut-associated symbionts of social insects [23]. Bartonella species parasitize a wide range of mammals, exploiting them as reservoir hosts through chronic infection of the vascular endothelium and erythrocytes, thereby facilitating their transmission to blood-feeding arthropod vectors [4]. Many bartonellae appear to be adapted to a single mammal species, but some are more generalist, exploiting several, often closely related, mammalian species [5]. Adaptation to reservoir host by bartonellae provides a clear indication of ecological distinction between taxa that is rare amongst bacteria; however, accommodating both ecology and ‘classical’ polyphasic characterization when defining Bartonella species is challenging and has resulted in inconsistency across the genus. The ecological integrity of some species has been challenged [68], and thus, they may well represent a consortium of ecologically distinct organisms that are similar enough in genetic or genomic terms to be considered the same taxon using current thresholds widely applied for bacterial species delineation. Conversely, elsewhere in the genus, ecologically distinct organisms that possess genetic and genomic similarities akin to strains of the same species lie in different taxa [910].
Phylogenetically, the eubartonellae form four deep-branching lineages (L1–L4) [1112], and this radiation is reflected in the evolution of diverse macromolecular systems that mediate host cell interaction and immune evasion. Chief amongst these are type IV secretion systems (T4SSs), which play a central role in host interaction. Three major T4SSs, VirB/D4, Vbh and Trw, have been identified in the genus, each exhibiting distinct structural and functional properties [11]. The VirB/D4 system, present in L3 and L4, secretes bartonella effector proteins (Beps) that modulate host cell physiology and facilitate persistent infection [1213]. The Vbh system, a homologous variant, is variably encoded on plasmids or the chromosome and may function either in interbacterial interactions or as a conjugation system, although many chromosomal versions appear to be degenerate [14]. In contrast, the Trw system is restricted to L4 species and mediates erythrocyte adhesion, replacing ancestral flagella and enabling species-specific host cell binding [1115].
Eubartonella L3 currently contains two validly published species, Bartonella clarridgeiae, which is adapted to cats [16], and Bartonella rochalimae, the ecology of which is currently uncertain [1719]. The lineage also contains a newly described but not yet validly published species ‘Bartonella bilalgolemii’ [20] as well as a number of other partially characterized bartonellae. Amongst these are strains referred to as ‘BGA’ or ‘B. rochalimae-like’, primarily defined on the basis of partial gltA nucleotide sequence similarity, parasitizing various rodent species across Europe [2123]. Given their wide distribution and apparent adaptation to various rodent species, these ‘B. rochalimae-like’ strains may well possess different ecologies and thus their taxonomic integrity is unclear. Here, we report the polyphasic characterization of three Bartonella strains previously identified as ‘BGA’/‘B. rochalimae-like’: C271^T^, D105 and J117 [21], all isolated from field voles (Microtus agrestis) in Kielder Forest, Northumberland, between 2001 and 2004 [21]. Characterization of the isolates supports their accommodation in a novel L3 species, for which we propose the name Bartonella bennetti sp. nov.
Methods
Bacterial isolation and culture
Strains C271^T^, D105 and J117 were isolated from the blood of field voles (M. agrestis) trapped in Kielder Forest (55.2344° N 2.5791° W), which lies on the eastern edge of the border between England and Scotland between 2001 and 2004, as previously described [21]. In addition, we characterized the RE21 strain of Bartonella heixiaziensis, isolated from a field vole in Assynt Forest, North-West Scotland (58.215° N 5.0505° W) in July 2021. We included this isolate as no representative whole-genome sequence for the species was available. All strains were preserved in brain heart infusion broth containing 10% (v/v) glycerol at −80 °C.
Isolates were revived on Columbia agar supplemented with 10% (v/v) whole horse blood and incubated at 35 °C in a 5% CO_2_ atmosphere. We assessed the ability of B. bennetti sp. nov. strain C271^T^ to grow in microaerophilic (8–9% O_2_; 7–8% CO_2_) and anaerobic (<1% O_2_; 9–11% CO_2_) conditions at 35 °C on 10% horse blood-enriched Columbia agar. Atmospheric concentrations were achieved in 2.5 l containers with the ThermoFisher Scientific Oxoid™ CampyGen™ 2.5 l sachet (CN0025A) and the Oxoid™ AnaeroGen™ 2.5 l sachet (AN0025A), respectively. The Oxoid™ Resazurin Anaerobic Indicator strips (BR0055B) were used to verify the presence or absence of oxygen in the sealed containers.
For scanning electron microscopy (SEM), colonies of strains C271^T^, D105 and J117 were sampled by placing sterile glass coverslips directly onto colonies growing the agar surface, followed by gentle removal and rinsing in PBS to eliminate residual media. Subsequent fixation, post-fixation, dehydration, drying and sputter coating were carried out following the protocol described by Fischer (Basic Protocol 1) [24]. Prepared samples were imaged at the Scanning Electron Microscopy Shared Research Facility (SEM SRF), University of Liverpool, UK.
Biochemical characterization
For biochemical and metabolic characterization, strains C271^T^, D105 and J117 were tested with the 20NE (BioMérieux) identification system, following the manufacturers’ protocol. Additionally, all strains were tested for catalase and oxidase activity and subjected to Gram staining.
Genome sequencing and assembly
Genomic DNA extractions were performed on strains C271^T^, D105 and J117 using the Wizard® HMW DNA extraction kit (Promega: A2920) following the manufacturers’ protocol. The genomes were sequenced using both Illumina NovaSeq (2×250 bp paired-end) and Oxford Nanopore Technologies (ONT) MinION (LSK109/R9.4.1) platforms. For Illumina sequencing, 100 ng of DNA in 100 µl was dispatched to MicrobesNG. Genomic libraries were then prepared by MicrobesNG (Birmingham, UK) using the Nextera XT Library Prep Kit following the manufacturers’ protocol with the following modifications: input DNA increased twofold, and PCR elongation time increased to 45 s. ONT libraries were prepared in-house with SQK-LSK109 and barcoded with the native barcoding expansion 1–12 (EXP-NBD104) according to the manufacturers’ protocol. A MinION R9.4.1 flow cell was loaded on the MK1C system running minKNOW 22.08.9 with basecalling set to fast.
Whole-genome sequencing of B. heixiaziensis RE21 was outsourced to MicrobesNG. Short-read sequencing matched that described for the B. bennetti sp. nov. strains. ONT long-read sequencing was performed using 400–500 ng of HMW DNA and the SQK-LSK109 kit with Native Barcoding EXP-NBD104/114 on an R9.4.1 flow cell in a GridION.
All raw short-read and long-read datasets were processed as follows: raw short reads (2×250 bp paired-end) were trimmed with FastP [25] with default parameters and Q-score set for 20. Raw long-read data was concatenated, and adapters were removed with Porechop [26] and filtered to exclude all reads less than 1,000 bp with Filtlong (GitHub rrwick/Filtlong). Assembly of all four strains was executed by the Unicycler (0.5.0) hybrid assembly pipeline [27]. The genomes were ultimately annotated using the RASTtk pipeline [28].
Phylogenetic analysis
A whole-genome phylogenetic tree was reconstructed using RAxML [29] implemented into the BV-BRC [30]. The analysis included all species of the genus Bartonella with validly published names, together with partially characterized strains and all Candidatus bartonellae with complete genomes (Table S1, available in the online Supplementary Material). A total of 500 single-copy genes were identified for the analysis totalling 178,160 aligned amino acids from 53 genomes. Protein sequences were aligned with mafft [31], and the DUMMY2 protein substitution model was used to define amino acid frequencies. Branch support was calculated with the RAxML fast bootstrapping methodology, and Brucella abortus strain NCTC 624 (GCA_900446015.1) was used as the outgroup.
A second whole-genome tree was reconstructed using RAxML implemented into the BV-BRC focusing on L3 Bartonella species and strains. A complete list of strains and species used for the analysis is available (Table S2).
Average nucleotide identity analysis
Whole-genome average nucleotide identity (ANI) was calculated between the closest known relatives of each strain based on whole-genome phylogenetic analyses: for the B. bennetti sp. nov. strains C271^T^, D105 and J117, B. rochalimae, B. clarridgeiae, ‘B. bilalgolemii’, AR15-3, JB15, CDCSkunk and Coyote22sub were chosen. For RE21, Bartonella jaculi, ‘Bartonella gliris’ and Ca. Bartonella washoensis were identified as close relatives. Finally, representatives for each remaining bartonellae lineage, Bartonella bacilliformis (L1) and Bartonella capreoli (L2), were selected. ANI was calculated with OrthoANIu [32] available online (last accessed on 27 March 2025: https://www.ezbiocloud.net/tools/ani).
Plasmid analysis
A complete list of plasmids available on NCBI GenBank was compiled and subjected to ANI analysis with OrthoANIu [32]. The analysis included reconstructed plasmids from chromosomally integrated elements.
Results and discussion
Phenotypic and genotypic characterization
Colonies of all B. bennetti sp. nov. strains (C271^T^, D105 and J117) were indistinguishable from one another and were small, smooth and white after 5 days of growth at 35 °C in a 5% CO_2_ atmosphere, with early growth detectable at 3 days. Strain C271^T^ also grew well at 35 °C under micro-aerophilic (8–9% O_2_; 7–8% CO_2_), but not anaerobic (<1% O_2_; 9–11% CO_2_), conditions. Gram staining of colonies revealed all strains to be indistinguishable Gram-negative rods. Under SEM, strains C271^T^, D105 and J117 were indistinguishable, appearing as small rods 0.8–1.2 µm in length and 0.3–0.4 µm in width with up to four polar lophotrichous flagella measuring up to 2 µm in length visible on a single bacterium (Fig. 1). All three B. bennetti sp. nov. strains were biochemically inert in the tests we performed. They were unable to reduce nitrates, produce indole, ferment glucose or utilize arginine or citrate. They did not have any detectable beta-galactosidase, catalase, gelatinase, oxidase or urease activity. They were unable to assimilate d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetylglucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate or phenylacetic acid. All strains were found to be catalase and oxidase negative. Thus, on the basis of all phenotypical characterizations performed, B. bennetti sp. nov. could not be distinguished from the other L3 species with validly published names, B. clarridgeiae or B. rochalimae. The inability of classical phenotypic testing to distinguish between Bartonella species is widely recognized [33].
SEM of B. bennetti sp. nov. strain C271T. A 500-nm scale bar is shown within the image (80,000×).
Although the B. bennetti sp. nov. strains could not be distinguished from another or from other L3 Bartonella species using phenotypic characterization techniques, there were some genomic variations between the strains (C271^T^, D105 and J117) and between species. Most notably, variation in genome length and predicted number of protein-coding genes (CDS), with the genomes of B. bennetti sp. nov. strains being about 100 kB larger than those of B. clarridgeiae and B. rochalimae and possessing at least 100 extra CDS (Table 1).
Phylogenetic placement and genomic relationships
The phylogenetic tree indicated that B. bennetti sp. nov. was an L3 species closely related to B. rochalimae, B. clarridgeiae and ‘B. bilalgolemii’ (Fig. 2). RE21 was determined to be closely related to B. heixiaziensis CR90HXZ^T^ through comparisons of the 16S rRNA gene (1267 bp) (KJ361623.1) and the gltA gene (326 bp) (KJ175047.1), utilizing the muscle alignment tool [34] integrated into Geneious Prime (11.0.18). RE21 shared 100 and 98.92% similarity at these loci, respectively. Subsequent phylogenetic analysis confirmed the identification of RE21 and placed B. heixiaziensis RE21 into L4 clustering with B. jaculi, Ca. B. washoensis and ‘B. gliris’ (Fig. 2).
Core genome tree of 53 Bartonella taxa and B. abortus (NCTC 642T) as the outgroup. A 500-gene maximum-likelihood tree with 10 allowed duplication and deletion to increase resolution was generated with RAxML from an alignment of 625,854 amino acids and bootstrapped with 100 replicates. The presence and absence of key virulence factors are indicated in columns. P indicates that the Vbh T4SS is present on a plasmid. Species characterized in this study are marked with (); Ca. denotes Candidatus status; taxa in quotation marks are not validly published. NCBI accessions (see Table S1 for more information).*
The topology of the L3-focused tree corroborated the genus tree, identifying the partially characterized strain AR15-3 and ‘B. bilalgolemii’ as the closest relatives of B. bennetti sp. nov. (Fig. 3).
Phylogenetic tree of lineage 3 Bartonella strains including the type strains of B. rochalimae (Br) and B. clarridgeiae (Bc) and ‘B. bilalgolemii’ (Bbi). Strains C271T, D105 and J117 are proposed as a novel lineage 3 species B. bennetti sp. nov. (Bbe). A tree was generated with RAxML using 619,662 amino acid alignments of 500 core single-copy genes for each species with an allowance for 10 duplications and deletions to enhance resolution, bootstrapped with 100 replicates. Strains from this study are marked with * (see Table S2 for more information).
The ANI between B. bennetti sp. nov. C271^T^ and the partially characterized strain Bartonella sp. AR15-3 was 96.04% across 61.23% of the genome, and the ANI between B. bennetti sp. nov., C271^T^ and ‘B. bilalgolemii’ G70 (not validly published under the ICNP) was 95.98% across 67.52% of the genome (Table 2). The ANI values amongst B. bennetti sp. nov. strains were >99.6%.
B. heixiaziensis RE21 shared an ANI of 88.95% across 54.76% of the B. jaculi genome, identifying B. jaculi as the closest known relative of B. heixiaziensis RE21. These results corroborate the whole-genome phylogenetic analysis (Fig. 2).
Virulence factor distribution and analysis
In addition to phylogenetic placement, the presence and absence of genes encoding key bartonellae-associated virulence factors were recorded for all genomes (Fig. 2). These included genes encoding the bartonella gene transfer agent (BaGTA) [35], flagella and three distinct T4SSs: VirB/D4, Trw and Vbh. Genes encoding the Trw T4SS and flagella were mutually exclusive across the genus, with the Trw operon present only in (all) L4 taxa and the divergent species Bartonella australis. Genes encoding the VirB/D4 and Vbh secretion systems were more sporadically distributed across the genus; virB/D4 operons are present in all L3 and L4 species and the L1 species Bartonella ancashensis, indicating at least three separate acquisition events. The vbh operon is present in 23 species either on a plasmid (3 instances) or within the bacterial chromosome (20 instances) (Fig. 2). The distribution of the vbh operon amongst Bartonella species suggests at least seven independent acquisition events; however, given its frequent association with plasmids, it is likely that additional, undetected acquisition events have occurred. Neither B. bennetti sp. nov. nor B. heixiaziensis possessed the vbh operon.
The B. bennetti sp. nov. genome contains three virB/D4 operons (Fig. 4). The first copy of the operon is highly conserved across L3 species and strains (Fig. S1). B. rochalimae is the only species not to possess an associated bep and to have two small uncharacterized CDSs in its place. The second and third copies of the virB/D4 operon are co-located, flanking a central virD4 coupling protein gene and several beps (Fig. S2). The operons themselves can be found between gltA copies and show increased genetic reorganization compared to the first copy. The second and third copies of the virB/D4 operon in C271^T^, J117 and AR15-3 are identical in structure, but in D105, the operon is inverted between the flanking hypothetical CDS and gltA. B. clarridgeiae also contains a second and third virB/D4 operon, but the central bep genes, virD4 and flanking gltA genes are markedly rearranged, and it possesses an additional bep and a duplicate of the small hypothetical CDS present between beps in the B. bennetti sp. nov. strains. More substantial variation was observed in the architecture of the virB/D4 operons in the B. rochalimae genome, with all but virB2 and virB3 of the third operon absent.
Complete chromosome assembly of B. bennetti sp. nov. strain C271T (NCBI accession: GCA_045945445.1). Sites of interest are highlighted, including (1) two copies of the virB/D4 operon, (2) a third copy of the virB/D4 operon and (3) a predicted chromosomally integrated plasmid. The image was generated using the Geneious software package.
Chromosomally integrated F-plasmid and implications for horizontal gene transfer
A putative operon encoding a fourth secretion system, containing the trb and tra conjugative transfer gene clusters, was located in the B. bennetti sp. nov. genomes (Fig. 4). The genes present are indicative of an F-type conjugative plasmid that has been chromosomally integrated. The system was, however, absent from the genome of other L3 taxa or species elsewhere in the genus (Fig. S3), suggesting potentially recent acquisition and enabling the estimation of a plasmid size of 30,586 bp. We have named this chromosomally integrated plasmid pRecC271-1 to indicate it does not exist independently within the bacterium but rather is a reconstruction of a past plasmid that has undergone chromosomal integration.
B. heixiaziensis strain RE21 possesses a 29,248 bp plasmid (pRE21-1) containing 39 putative CDSs (Fig. S4). These include plasmid replication protein repC, plasmid partitioning protein parA and conjugal protein genes (trb, tra), characteristic of a fertility conjugative plasmid (F-plasmid) akin to pTLV-1, pOE11-1 and pBHa, present in Bartonella kosoyi, Bartonella krasnovii and ‘Bartonella harrusi’, respectively [36].
Comparative sequence analysis of pRecC271-1 and pRE21-1 with other (non-cryptic) plasmids associated with Bartonella species indicated between 0–50.98% and 5.28–63.32% sequence coverage, respectively (Table 3). The ANI scores on the covered sequence ranged from 76.49 to 86.40% for pRecC271-1 and from 76.62 to 85.40% for pRE21-1 (Table 3). Examination of the pattern of both the coverage and ANI values amongst bartonellae-associated plasmids revealed two distinct groups; the first of these comprised F-plasmids (pTLV-1, pOE11-1, pBHa, pRE21-1 and pRecC271-1), and the second comprised plasmids (pBT, pBGR3, pML and pNH4) carrying the vbh T4SS operon in Bartonella tribocorum, Bartonella grahamii, Bartonella schoenbuchensis and Bartonella rattaustraliani (Table 3).
Plasmid-mediated gene acquisition plays a pivotal role in the evolutionary divergence of Bartonella species, acting as a major driver of their adaptability and host specificity [3537]. Plasmids, such as pBGR3 in B. grahamii and PML in B. schoenbuchensis, carry vbh operons, illustrating the role plasmids have played in disseminating VirB/Vbh/Trw T4SS across the Bartonella genus [3738]. We encountered the relatively recent (i.e. species-specific) chromosomal integration of a classical F-plasmid into the B. bennetti sp. nov. genome. F-plasmids such as pTLV-1, pOE11-1, pBHa and pRE21-1 are commonly found in the Bartonella genus and represent a well-characterized class of conjugative plasmids, originally described in Escherichia coli [3940]. Their primary function is to facilitate horizontal gene transfer of the plasmid itself into recipient cells via a T4SS encoded by tra and trb genes [41]. Typically, this process involves transfer of the plasmid’s own DNA, including the T4SS operon and any accessory genes it carries. However, when the F-plasmid integrates into the host chromosome, as has occurred in B. bennetti sp. nov., the strain becomes an Hfr (high-frequency recombination) donor [4143]. In this state, gene transfer begins at the oriT site, now embedded in the chromosome and proceeding linearly into adjacent chromosomal DNA [41]. Although conjugation is frequently interrupted before full transfer, studies in Pseudomonas putida have shown that up to 23% of the chromosome could be transferred [44]. Given our discovery of further evidence of F-plasmids amongst Bartonella species and of their chromosomal integration and hence the potential induction of an Hfr state, it is not unreasonable to propose their importance in horizontal gene transfer within the genus. High-frequency recombination could facilitate large-scale exchange of genetic material amongst bartonellae co-infecting a host or vector, potentially spreading traits associated with virulence, antibiotic resistance or host switching. This mechanism could accelerate diversification and adaptation, promoting niche differentiation and host specificity within coexisting populations. Furthermore, Hfr conjugation might help overcome genetic barriers by transferring chromosomal segments beyond the plasmid itself, thereby increasing genomic plasticity and fostering evolutionary innovation within a genus where low levels of genomic plasticity would be expected.
Species delimitation and ecological considerations
Species of bacteria are commonly defined through a combination of phenotypic, ecological and genomic criteria, with ANI widely used as a genomic proxy for species boundaries. A threshold of 95–96% ANI is often applied across bacterial genera [4546]. Although ANI has been proposed as a criterion for the taxonomic review of the Bartonella genus [47], ANI thresholds alone provide only a partial basis for species delineation. In the Bartonella genus, species boundaries are more accurately resolved by incorporating a polyphasic approach that incorporates ecological factors such as host specificity and geographic distribution. For example, B. schoenbuchensis and Bartonella chomelii exhibit borderline ANI values (96.81%) yet are considered distinct species due to differences in host association (deer versus cattle). A similar pattern is observed between B. bennetti sp. nov., ‘B. bilalgolemii’ G70 which is not validly published under the ICNP, and the unclassified strain Bartonella sp. AR15-3, which clusters near the 94–96% ANI threshold but likely exploits distinct host species across separate geographic regions: field voles in the UK [21], Ural field mice (Apodemus uralensis) in Turkey [20] and red squirrels (Tamiasciurus hudsonicus) in the USA, respectively. B. bennetti sp. nov. infections were detected in ~1.2% of nearly 4,000 field voles surveyed between September 2001 and September 2004 [21], but in only 2 of ~1,300 other sympatric rodents captured in the same surveys (Birtles, Bown and Telfer, unpublished observations). Although this suggests that field voles are the likely reservoir for B. bennetti sp. nov. in Kielder Forest, the infection prevalence at which it was detected was markedly lower than that for other co-circulating Bartonella species (18); thus, further work is required to clarify its ecology. The ecologies of ‘B. bilalgolemii’ and AR15-3 are less certain as both were recovered from single individuals [20]. Nonetheless, the distinct host and geographical associations of these strains support the idea that ecological context, particularly host specificity, is an important criterion for resolving species boundaries in the Bartonella genus and that even highly similar genomes may represent distinct evolutionary lineages.
Phylogenetic placement supports host specificity over generalism
The phylogenetic placement of B. bennetti sp. nov. supports the view that host specificity, rather than generalism, may be the dominant ecological strategy within the L3 clade and broadly the Bartonella genus. A maximum-likelihood phylogeny of 52 publicly available Bartonella genomes, constructed using RAxML from 500 single-copy core genes, revealed a deep split between a basal lineage and a well-supported monophyletic clade of eubartonellae composed of 4 major lineages (Fig. 4). Within this framework, B. bennetti sp. nov. clustered within L3, alongside only three other characterized species: ‘B. bilalgolemii’, B. clarridgeiae and B. rochalimae. This clade was highly resolved, with 100% bootstrap support across all nodes. Although B. rochalimae is frequently reported from a wide range of hosts, it is often identified primarily on the basis of partial gltA sequences [48], a low-resolution marker that lacks the discriminatory power to distinguish closely related but ecologically distinct strains. As such, the apparent host generalism of B. rochalimae may reflect methodological artefact rather than true ecological breadth. It remains plausible, and perhaps likely, that what is currently considered B. rochalimae in fact comprises a complex of cryptic, host-specific lineages [49]. In contrast, the other L3 members appear to show specific host associations: B. clarridgeiae with domestic cats (Felis catus) [16] and B. bennetti sp. nov. with field voles (M. agrestis). This pattern is consistent with ecological data indicating low host-switching rates in rodent-associated Bartonella species [212350] and suggests that L3 species have evolved along narrow host-specialist trajectories.
Although the functional drivers of this pattern remain unclear, one explanation may lie in differences in host-adaptation machinery between lineages. Previous work has proposed that both L3 and L4 underwent parallel adaptive radiations, potentially triggered by the acquisition of the VirB/D4 T4SS and its associated effectors (Beps) [12]. However, L4 species, unlike those in L3, also encode the Trw T4SS, a system implicated in mediating host specificity and host switching, which is mutually exclusive with flagellar motility (Fig. 2). The absence of Trw in L3 taxa may limit their capacity for ecological diversification compared to L4. In contrast, some species within L4 have lost functionality in the ancestral Vbh system, which is retained in L2 and B. australis [14]. These patterns suggest a dynamic history of gain and loss in T4SS systems, which may have contributed to the differences in host range and ecological breadth observed between lineages. Together, the phylogenetic and ecological data indicate that B. bennetti sp. nov. and its closest relatives in L3 represent a lineage characterized more by host fidelity than flexibility, with the Trw system potentially facilitating broader host adaptation in L4 species rather than directly causing generalism.
Description of Bartonella bennetti sp. nov.
Bartonella bennetti (ben.net′ti. N.L. gen. n. bennetti, of Bennett, in honour of the veterinary microbiologist Malcolm Bennett, for his contributions to the fields of zoonoses and infectious disease ecology).
B. bennetti sp. nov. was isolated from a field vole (M. agrestis) in Kielder Forest, Northumberland, UK, in September 2004. The bacterium is a rod-shaped bacillus that measures 0.8–1.2 µm in length and 0.3–0.4 µm in width and possesses up to four polar lophotrichous flagella measuring up to 2 µm in length. B. bennetti sp. nov. forms round, small, white–yellow colonies on blood-enriched Columbia agar within 10 days when grown at 35–37 °C with 5% CO_2_ or under microaerophilic, but not anaerobic, conditions. The bacterium is unable to reduce nitrates or produce indole and is unable to ferment glucose or utilize arginine or citrate. It does not have beta-galactosidase, catalase, gelatinase, oxidase or urease activity. It is unable to assimilate d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetylglucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate or phenylacetic acid. The type strain C271^T^ contains a single circular chromosome (GenBank accession number CP174576.1) measuring 1.63 Mbp in length with a G+C content of 35.9 mol%. 16S rDNA data for C271^T^ is available under the accession number OR452865.1. C271^T^ has been deposited in the National Collection of Type Cultures, London, UK (NCTC15117), and in the Collection de Souches de l’Unité des Rickettsies, Marseille, France (CSUR B1113).
The genome of B. heixiaziensis strain RE21 is available under the accession CP176404.2 (RE21 chromosome and plasmid), and the strain has been deposited in the National Collection of Type Cultures, London, UK (NCTC15118), and in the Collection de Souches du Unite des Rickettsies, Marseille, France (CSUR B1114).
Supplementary material
10.1099/ijsem.0.007095Uncited Supplementary Material 1.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Parte AC Sardà Carbasse J Meier-Kolthoff JP Reimer LC Göker M List of prokaryotic names with standing in nomenclature (LPSN) moves to the DSMZ Int J Syst Evol Microbiol 2020705607561210.1099/ijsem.0.00433232701423 PMC 7723251 · doi ↗ · pubmed ↗
- 2Neuvonen M-M Tamarit D Näslund K Liebig J Feldhaar H et al The genome of Rhizobiales bacteria in predatory ants reveals urease gene functions but no genes for nitrogen fixation Sci Rep 201663919710.1038/srep 3919727976703 PMC 5156944 · doi ↗ · pubmed ↗
- 3Bisch G Neuvonen M-M Pierce NE Russell JA Koga R et al Genome evolution of Bartonellaceae symbionts of ants at the opposite ends of the trophic scale Genome Biol Evol 2018101687170410.1093/gbe/evy 12629982531 PMC 6044324 · doi ↗ · pubmed ↗
- 4Kosoy M Goodrich I Comparative ecology of Bartonella and Brucella infections in wild carnivores Front Vet Sci 2019532210.3389/fvets.2018.0032230662899 PMC 6328487 · doi ↗ · pubmed ↗
- 5Buffet J-P Pisanu B Brisse S Roussel S Félix B et al Deciphering Bartonella diversity, recombination, and host specificity in a rodent community P Lo S One 20138 e 6895610.1371/journal.pone.006895623894381 PMC 3722228 · doi ↗ · pubmed ↗
- 6Kosoy M Hayman DTS Chan KS Bartonella bacteria in nature: where does population variability end and a species start?Infect Genet Evol 20121289490410.1016/j.meegid.2012.03.00522449771 · doi ↗ · pubmed ↗
- 7Kosoy M Bai Y Bartonella bacteria in urban rats: a movement from the jungles of Southeast Asia to metropoles around the globe Front Ecol Evol 201978810.3389/fevo.2019.00088 · doi ↗
- 8Xu A Lu L Zhang W Song X Li G et al Microevolution of Bartonella grahamii driven by geographic and host factorsm Systems 20249 e 010892410.1128/msystems.01089-2439345150 PMC 11494883 · doi ↗ · pubmed ↗