Pan-genome classification of the virulence spectrum of <i>Bartonella</i> and the variation of heme-binding protein virulence factor in isolates from the Tibetan Plateau

This study classifies <i>Bartonella</i> species based on virulence factors and explores the genomic characteristics of <i>Bartonella</i> isolates from the Qinghai-Tibet Plateau to understand their ecological adaptations. PCR detection and isolation culture of <i>Bartonella</i> were performed on 50 captured pikas in their habitats on the Qinghai-Tibet Plateau. The Qinghai-Tibet isolates were identified through <i>gltA</i> and <i>rpoB</i> phylogenetic analysis, ANI analysis, and single-copy homologous gene phylogeny. Reference genomes of <i>Bartonella</i> species were analyzed for virulence classification via pan-genome and virulence factor comparisons. <i>Bartonella</i> species were categorized into five virulence groups, including a high-virulence group containing <i>Bartonella henselae, B. quintana, B. kosoyi, B. koehlerae</i>, and <i>B. tribocorum. B. bacilliformis</i> lacked <i>Trw</i> and <i>VirB/VirD4</i>, while the copy number of <i>flagella</i> was significantly higher. <i>Bartonella grahamii</i> infected up to 12% of plateau pikas from the Qinghai-Tibet Plateau. <i>B. grahamii</i> plateau isolate had the most unique genes (5.6%), with 65.06% of them unknown in function, followed by replication, recombination, and repair (11.64%), and amino acid transport and metabolism (5.48%). The <i>heme-binding protein</i> virulence factor in <i>B. grahamii</i> plateau isolate exhibited 14 amino acid mutations, leading to changes in the protein-binding sites. <i>Bartonella</i> can be classified into five virulence groups. In the high-virulence group, the less common <i>B. tribocorum</i> and <i>B. kosoyi</i> are included. <i>Trw</i> and <i>VirB/VirD4</i> gradually replaced ancestral <i>flagella</i> genes in <i>Bartonella</i> evolution. The isolated strains have more unique genes due to plateau pikas' unique physiological traits, which may be connected to adaptation to the high-altitude host environment. IMPORTANCE <i>Bartonella</i> species are emerging zoonotic pathogens capable of infecting a wide range of mammals, including humans. Understanding their virulence and evolution is critical for assessing potential public health risks. This study classifies <i>Bartonella</i> species based on virulence factors, revealing species with potential human pathogenicity. The Qinghai-Tibet Plateau, with its extreme environment, may drive bacterial adaptation. By analyzing <i>Bartonella</i> isolates from plateau pikas, we identified unique genes and virulence factor variations, providing insights into bacterial evolution in high-altitude hosts. Our findings highlight the significance of pan-genome analysis in identifying pathogenic traits and evaluating zoonotic risks. This study enhances our understanding of <i>Bartonella</i> adaptation, aiding in the clinical identification and characterization of highly pathogenic strains.