Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland

It has been estimated that 75% of emerging infectious diseases comprise zoonoses, whose majority have freeliving animals as reservoirs and are mainly transmitted by arthropod vectors. Although rodents represent important Bartonella reservoirs, there are few studies on the genotypic characterization of Bartonella species commonly found in this taxon and from different Brazilian biomes. Therefore, the present study aimed to investigate the occurrence, isolate and molecularly, morphologically and phenotypically characterize a new Bartonella species infecting free-living rodents sampled in the Brazilian Pantanal, the largest wetland in South America. For this purpose, 129 free-living rodents (79 Thrichomys fosteri, 4 Clyomys laticeps, and Oecomys mamorae) were captured. While blood samples were collected from 57 T. fosteri, 4 degrees C. laticeps and 32 O. mamorae; spleen samples were collected from 22 T. fosteri and 14 O. mamorae. Blood and spleen samples were submitted to a qPCR for Bartonella spp. targeting the nuoG gene, using DNA samples extracted directly from blood/spleen, after passage in pre-enrichment liquid culture, and from colonies obtained from solid culture on chocolate agar. Combining all techniques, occurrence of 24.8% for Bartonella sp. was found among the sampled rodents. One Bartonella isolate (strain 56A) obtained from a T. fosteri's blood sample was closely related to the Bartonella vinsonii complex and selected for Whole Genome Sequencing (WGS) hybrid approach using Illumina NovaSeq and Nanopore sequencing platforms. This strain exhibits a circular 2.7 Mbp genome with an average C+G content of 39% and encoding to 2239 genes. In the phylogenomics based on 291 shared protein-coding genes, this strain was positioned in a unique clade, closely related to Bartonella vinsonii subsp. vinsonii, B. vinsonii subsp. berkhoffii and B. visonii subsp. arupensis. An Average Nucleotide Identity of 85% was found between the obtained isolate and Bartonella species belonging to B. vinsonii complex. These findings supported the separation of this strain, now formally named as Bartonella machadoae sp. nov., from the Bartonella vinsonii complex. In addition, Bartonella machadoae sp. nov. was characterized by capnophilic, microaerophilic and aerobic small rods with absence of pili and flagella. Phylogenetic and distance analyses based on five concatenated molecular markers suggest that Bartonella machadoae may parasite rodents from different Brazilian biomes. In conclusion, we described biochemical, phenotypic and genomic characteristics of Bartonella machadoae nov. sp. isolated from blood samples of T. fosteri rodents from the Brazilian Pantanal.