Identification and functional genomic analyses of Bartonella isolates from honey bees, and reassessment of the taxonomy of the genus Bartonella

We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and whole-genome sequence analyses to identify 90 Bartonella isolates from honey bee gut samples in Belgium. While the identification of 62 isolates as Bartonella apihabitans and three as Bartonella choladocola was straightforward, the identification of 25 Bartonella apis-like isolates was challenging. A taxonomic and functional analysis of four B. apis-like genomes and of publicly available B. apis genomes demonstrated that neither OrthoANIu and digital DNA-DNA hybridization analyses, nor functional annotation supported a clear separation of B. apis and B. apis-like genomes. Different phylogenomic analyses showed that B. apis and B. apis-like strains formed a monophyletic clade with an inconsistent internal structure. We therefore considered the remaining 25 isolates identified as B. apis. We subsequently re-addressed an earlier phylogenetic and functional divergence between three major clades of Bartonella species which differed not only in phylogenomic position and ecology, but also in genome size and genomic percentage G + C content, and in many metabolic capabilities. We propose to reclassify the single species of the Bartonella tamiae clade into the novel genus Attibartonella gen. nov., with Attibartonella tamiae comb. nov. as the type species. Similarly, we propose to reclassify species of the honey bee-associated Bartonella clade into the novel genus Ditibartonella gen. nov., with Ditibartonella apis comb. nov. as the type species. The phylogenomic analyses of publicly available genome and metagenome sequences revealed additional Ditibartonella species in honey bee samples, highlighted an evolutionary adaptation of Ditibartonella bacteria to bee hosts and suggested shared transmission routes.