First Report of Bartonella spp. in Marsupials from Brazil, with a Description of Bartonella harrusi sp. nov. and a New Proposal for the Taxonomic Reclassification of Species of the Genus Bartonella

Abstract

The genus Bartonella (Rhizobiales: Bartonellaceae) encompasses facultative intracellular Gram-negative alphaproteobacteria that parasitize mainly erythrocytes and endothelial cells, as well as macrophages, monocytes and dendritic cells. Although they can infect numerous mammal species and arthropod vectors worldwide, reports of Bartonella infections in marsupials are scarce. In fact, such agents have only been detected in marsupials and/or associated ectoparasites in Australia and the United States of America until the present moment. The present study aimed to isolate and characterize molecularly, morphologically and phenotypically Bartonella infecting free-living marsupials sampled in the Brazilian Pantanal, the largest wetland in South America. Two marsupials were captured in December 2018 and six marsupials in February 2019, totaling eight small mammals sampled: five (62.5%) Thylamys macrurus and three (37.5%) Monodelphis domestica. All blood samples were submitted to qPCR for Bartonella spp. based on the nuoG gene, a pre-enrichment liquid culture and a chocolate agar solid culture. Bartonella sp. was isolated from 3 T. macrurus and one M. domestica. One Bartonella isolate obtained from a T. macrurus blood sample (strain 117A) that showed to be closely related to the Bartonella vinsonii complex and Bartonella machadoae was selected for whole genome sequencing using a hybrid approach based on Illumina NovaSeq and Nanopore sequencing platforms. This strain showed a genome of 2.35 Mbp, with an average C + G content of 38.8%, coding for 2013 genes, and a 29 kb plasmid with an average C + G content of 34.5%. In addition, this strain exhibited an average nucleotide identity (ANI) of 85% with Bartonella species belonging to the B. vinsonii group and 91% with B. machadoae. Phylogenomic analysis based on 291 protein coding genes shared by the genomes of 53 Bartonella species positioned this strain closely to B. machadoae. This new isolated species was named Bartonella harrusi sp. nov., which was characterized as having small capnophilic, microaerophilic and aerobic rods with an absence of pili and flagella. In conclusion, the present work describes the biochemical, phenotypic and genomic characteristics of Bartonella harrusi, a new species isolated from the T. macrurus blood samples of the Brazilian Pantanal. Finally, a review of the taxonomic classification of members of the genus Bartonella is proposed, based on the ANI values accessed by whole genome sequencing analyses.

Keywords: WGS; bartonelosis; biochemical characterization; marsupialia; phylogenomics; taxonomic classification.

Figure 1

Flowchart of the microbiological and molecular analyses used in this study for the isolation and characterization of Bartonella harrusi sp. nov.

Figure 2

Concatenated phylogenetic analysis within the genus Bartonella based on the 16S rRNA, gltA, ftsZ, groEL, rpoB genes and 16S-23S rRNA intergenic region (ITS). The phylogenetic tree was inferred using the Maximum Likelihood method and the evolutionary model TVM + F + I. The sequences obtained in this study are highlighted in red. The numbers in each branch correspond to the bootstrap values accessed with 1000 replicates. Brucella abortus was used as an outgroup.

Figure 3

Neighbor-Net analysis of DNA sequences from the 16S rRNA, gltA, ftsZ, groEL, rpoB genes and 16S-23S intergenic region (ITS) of Bartonella sp. isolated from wild marsupials (T. macrurus strain 117A, 117B and 117C and M. domestica strain 78A) sampled in the Brazilian Pantanal in the present study when compared to closely related Bartonella sequences previously deposited in GenBank.

Figure 4

Scanning electron microscopy (SEM) of Bartonella strain 117A isolated from a blood sample of T. macrurus (Bartonella harrusi sp. nov.) showing absence of flagellum and pili. A 4 µM scale bar is shown in the image.

Figure 5

Graph of the growth curve of Bartonella harrusi sp. nov. representing the collection hours (X axis) by the quantification of CFU/mL (Y axis).

Figure 6

Heat map generated with OrthoANI values calculated between A: Bartonella harrusi sp. nov. isolated from a blood sample of T. macrurus, B. machadoae, species of the B. vinsonii complex.

Figure 7

Heat map generated with OrthoANI values calculated between the plasmids found in Bartonella species and the plasmid found in Bartonella harussi sp. nov. isolated in the present study using the OAT software Tool version 0.93.1 [46].

Figure 8

Phylogeny based on the complete genome of 53 species of Bartonella, using 293 coding genes shared between these species. Bootstrap values are shown under nodes. Brucella abortus and Brucella ceti were used as an outgroup.

Figure 9

Pie chart showing (A) the completeness of the 217 analyzed genomes of Bartonella spp. and (B) the level of contamination of the genomes used for the analysis of ANI.