Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts

Abstract

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.

Taxonomic Organization of the Family Brucellaceae Based on a Phylogenomic Approach

“…The 101 non-Brucella Brucellaceae genomes available in the GenBank assembly summary file as of 07/14/2019 were downloaded from the NCBI FTP repository 1 in fasta format. Since almost 800 Brucella genomes were available at the time of this study and that the phylogenomic organization of the genus was already thoroughly investigated before (Wattam et al, 2014;Al Dahouk et al, 2017), only 12 genomes spanning the diversity of the classical and non-classical Brucella species were used in our analyses ( Supplementary Table S1). A set of 43 diverse non-Brucellaceae Rhizobiales genomes listed as 'reference' or 'representative' genomes in the NCBI database were also downloaded, as well as the genome of two Caulobacteriales (Caulobacter vibrioides and Parvularcula bermudensis) to serve as outgroups.…”

“…One of the striking specificity of the Brucella genus is its extreme homogeneity at the genetic level, with more than 90% DNA-DNA hybridization between species (Verger et al, 1985). Despite this lack of diversity, the phylogenetic relationship between species was extensively investigated using various genetic tools and more recently with the availability of whole-genome sequences, and are now well resolved (Foster et al, 2009;Wattam et al, 2014, Al Dahouk et al, 2017. The current classification includes 12 Brucella species commonly classified as 'classical' or 'atypical' , depending on their phenotypical characteristics and their phylogenetic relationships (Scholz et al, 2018).…”

Brucella microti‐ like prevalence in French farms producing frogs

“…The first strains isolated from anurans of wild origin, kept as pets or in zoological collections, (Eisenberg et al, ; Fischer et al, ; Kimura et al, ; Mühldorfer et al, ; Soler‐Lloréns et al, ; Whatmore et al, ) are phenotypically and genetically related to the B. inopinata clade (Al Dahouk et al, ; Mühldorfer et al, ). In both wild and captive anurans, the distribution of B. inopinata has not been extensively investigated.…”

Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis

“…Brucellae are Gram-negative facultative intracellular coccobacilli causing brucellosis, a major bacterial zoonosis with 500,000 human cases globally reported every year [1]. In the last decade, new species of Brucella, such as Brucella microti, Brucella inopinata and isolates from Australian rodents and amphibians, have been described [2]. These strains are metabolically more active, acidresistant and fast-growing when compared to the wellknown classical, human-pathogenic Brucella species, which include Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis [2][3][4][5][6][7].…”

“…In the last decade, new species of Brucella, such as Brucella microti, Brucella inopinata and isolates from Australian rodents and amphibians, have been described [2]. These strains are metabolically more active, acidresistant and fast-growing when compared to the wellknown classical, human-pathogenic Brucella species, which include Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis [2][3][4][5][6][7]. Their isolation from hitherto unknown wildlife hosts and the environment raised the question whether Brucella may be transmitted from these reservoirs to livestock and humans living in officially brucellosis-free areas of the world.…”