The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel Brucella Species in Exotic Frogs

Abstract

The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer.

Fig 1. Phylogenetic tree from maximum likelihood analysis of the recA gene alignment of Brucella isolates from exotic frogs including classical and ‘atypical’ Brucella species.

The tree was calculated with 100 bootstrap repetitions. Ochrobactrum served as outgroup. Bar: 0.08 substitutions per site. Accession numbers are given in brackets.

Fig 2. Core-genome-based phylogenetic neighbor-joining tree with 200 repetitions.

Bar: 0.002 substitutions per site. Isolate 10RB9215 is indicated in bold letters. Accession numbers are given in brackets.