Classification of Bartonella strains associated with straw-colored fruit bats (Eidolon helvum) across Africa using a multi-locus sequence typing platform

Abstract

Bartonellae are facultative intracellular bacteria and are highly adapted to their mammalian host cell niches. Straw-colored fruit bats (Eidolon helvum) are commonly infected with several bartonella strains. To elucidate the genetic diversity of these bartonella strains, we analyzed 79 bartonella isolates from straw-colored fruit bats in seven countries across Africa (Cameroon, Annobon island of Equatorial Guinea, Ghana, Kenya, Nigeria, Tanzania, and Uganda) using a multi-locus sequencing typing (MLST) approach based on nucleotide sequences of eight loci (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, ITS, and 16S rRNA). The analysis of each locus but ribC demonstrated clustering of the isolates into six genogroups (E1 - E5 and Ew), while ribC was absent in the isolates belonging to the genogroup Ew. In general, grouping of all isolates by each locus was mutually supportive; however, nuoG, gltA, and rpoB showed some incongruity with other loci in several strains, suggesting a possibility of recombination events, which were confirmed by network analyses and recombination/mutation rate ratio (r/m) estimations. The MLST scheme revealed 45 unique sequence types (ST1 - 45) among the analyzed bartonella isolates. Phylogenetic analysis of concatenated sequences supported the discrimination of six phylogenetic lineages (E1 - E5 and Ew) corresponding to separate and unique Bartonella species. One of the defined lineages, Ew, consisted of only two STs (ST1 and ST2), and comprised more than one-quarter of the analyzed isolates, while other lineages contained higher numbers of STs with a smaller number of isolates belonging to each lineage. The low number of allelic polymorphisms of isolates belonging to Ew suggests a more recent origin for this species. Our findings suggest that at least six Bartonella species are associated with straw-colored fruit bats, and that distinct STs can be found across the distribution of this bat species, including in populations of bats which are genetically distinct.

Figure 1. Phylogenetic relationships of the 45 sequence types from 79 bartonella isolates obtained from straw-colored fruit bats (Eidolon helvum) in seven African countries/regions.

The number of isolates belonging to each sequence type is given in parentheses. The phylogenetic tree was constructed from concatenated sequences (4,622bp—5,160bp) of eight loci (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, ITS, and 16S rRNA) using the neighbor-joining method. Bootstrap values were calculated with 1000 replicates. The sequence types are grouped into six phylogenetic lineages (boxed clades) named as E1—E5 and Ew, with each lineage presumably representing a separate and unique Bartonella species.

Figure 2. Phylogenetic relationship of straw-colored fruit bats (E. helvum) with known Bartonella species and other bat-associated Bartonella strains.

The maximum-likelihood tree was inferred using concatenated sequence of seven loci (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, and 16S rRNA) from 31 Bartonella species, other bat-associated Bartonella strains, and 6 sequence types from E. helvum representing the lineages E1—E5 and Ew (bold text). Groups of bat-associated Bartonella strains are indicated with a bat silhouette. The numbers at the nodes correspond to bootstrap values greater than 60% based on 1000 replicates.

Figure 3. Network phylogeny of the 45 bartonella sequence types obtained from Eidolon helvum.

The network was constructed in SplitsTree using the NeighborNet algorithm based on concatenated sequences of eight loci (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, ITS, and 16S rRNA). Clusters of sequence types were named according to phylogenetic lineages (E1—E5, Ew). Individual isolate labels indicate samples with mixed ancestry due to possible recombination.