Morphological, biochemical, molecular and phenotypic characterization of BARTONELLA spp. of Rodents and marsupials in the Brazilian Pantanal, with description of BARTONELLA MARCHADOAE sp. nov and BARTONELLA HARRUSI sp. nov.

The genus Bartonella comprises facultative and fastidious intracellular Gramnegative alphaproteobacteria capable of infecting a wide variety of mammals and transmitted by arthropod vectors. Although rodents represent important reservoirs of Bartonella around the world, there are few studies on the genomic characterization of Bartonella species commonly found in this taxon. On the other hand, Bartonella spp. has been detected in marsupials and/or associated ectoparasites only in Australia and the United States. The present study aimed to characterize molecularly, morphologically, biochemically, and phenotypically Bartonella isolated from blood samples of free-living rodents and marsupials sampled in the Brazilian Pantanal, the largest wetland in South America. For this purpose, 61 animals (59 rodents and 2 marsupials) were captured in December 2018 and 76 (70 rodents and 6 marsupials) in February 2019, totaling 137 small mammals sampled, of which 129 (94.4%) were rodents and eight were (5.6%) marsupials. Among 129 rodents sampled animals, 79 (61.2%) were Thrichomys fosteri, four (3.1%) Clyomys laticeps, 46 (35.7%) were Oecomys mamorae. Among the eight marsupials, 5 (62,5%) were Thylamys macrurus and 3 (37,5%) were Monodelphis domestica. Biological samples were submitted to a qPCR for Bartonella based on the nuoG gene, pre-enrichment liquid culture and solid culture on chocolate agar. Bartonella spp. detected in 24% of sampled rodents and in 4 out of 8 sampled marsupials. A strain isolated from a blood sample of T. fosteri and a strain isolated from a blood sample of T. macrurus, which shown to be closely related to the Bartonella vinsonii complex by phylogenetic (Maximum Likelihood) and distance (SplitsTree) analyzes based on several molecular markers, were selected for whole genome sequencing, using for such a hybrid approach in Illumina NovaSeq and Nanopore sequencing platforms. The isolated strains of T. fosteri (strain 56A) and T. macrurus (117A) exhibited a circular genome of 2.7 Mbp and 2.35 Mbp, with an average C + G content of 39% and 38.8%, and encoding 2.239 and 2.013 genes, respectively. In addition, they exhibited a mean nucleotide identity (ANI) of 85% with Bartonella species of the vinsonii group and 91% with each other. Phylogenomic analysis based on 291 protein-coding genes shared between genomes of 53 Bartonella species positioned both strains in a clade closely related to Bartonella vinsonii subsp. vinsonii, B. vinsonii subsp. berkhoffii and B. vinsonii subsp. arupensis, with high clade support value, supporting the separation of these new strains into two different species, now formally named Bartonella machadoae nov. sp. (isolated from T. fosteri) and Bartonella harrusi nov. sp. (isolated from T. macrurus). Bartonella machadoae sp. nov. and Bartonella harrusi sp. nov. were characterized as small capnophilic, microaerophilic and aerobic rods with the absence of pili and flagella. In conclusion, we describe the biochemical, morphological, phenotypic and genomic characteristics of Bartonella machadoae, a new species isolated from T. fosteri rodent blood samples, and Bartonella harrusi, a new species isolated from T. macrurus marsupial blood samples in the Brazilian Pantanal. (AU)