A new member of the novel, non-core Brucella clade: An exotic frog isolate closely related to atypical Brucella isolates from recent human brucellosis cases in Australia.

BACKGROUND: Over the past few decades, the Brucella genus has seen a significant increase in novel strains that deviate from classical Brucella spp. due to their atypical phenotypes. B. inopinata, an atypical Brucella species first isolated from a patient, was recently found in a White's tree frog, raising the question of whether amphibians are reservoirs for these emerging human pathogens. Unfortunately, monitoring atypical Brucella remains challenging because misidentification with Ochrobactrum spp. and Brucella melitensis is common when using routine microbiological tests. RESULTS: In our study, we describe a Brucella strain isolated from White's tree frogs (Litoria caerulea) that were initially examined for chytridiomycosis after they had developed dermal abnormalities. Classical microbiological and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses and a species-specific polymerase chain reaction confirmed that isolate CVUAS_1139.3 is an atypical Brucella strain. This non-fastidious, fast growing, flagellated, and motile bacterium is not susceptible to lysis by the Brucella phages used for typing. Further characterization using the differential metabolic phenotyping approach, revealed that Brucella sp. CVUAS_1139.3 could be differentiated from classical Brucella spp., as well as from Ochrobactrum anthropi and O. intermedium, based on its metabolic activity. The substrate utilization patterns may be suitable for a simple and cost-effective diagnostic assay. Phylogenetic analysis positioned Brucella sp. CVUAS_1139.3 distant from the classical Brucella spp. within the novel, non-core Brucella clade. Within this clade, Brucella sp. CVUAS_1139.3 shares a close phylogenetic relationship with B. inopinata strains and various African bullfrog isolates, and it is most closely related to a recently identified human isolate from Australia. Antimicrobial resistance testing revealed that it is susceptible to antibiotics widely applied in standard treatment regimens. In human THP-1 macrophage-like cells, the replication rate of the novel Brucella frog isolate was comparable to that of B. inopinata. CONCLUSION: In summary, the amphibian-derived strain Brucella sp. CVUAS_1139.3 clusters phylogenetically with and is phenotypically alike to previously reported isolates from amphibian hosts and human brucellosis patients within the novel, non-core clade. Our report and other studies suggest that exotic frogs are potential reservoirs for human pathogenic Brucella spp., which might pose an underestimated zoonotic hazard for exposed individuals.