The genus Brucella has gained several species and numerous yet unclassified isolates with atypical phenotypes and genome sequences that differ significantly from the classical, highly pathogenic species such as B. melitensis. Despite the increasing number of these novel, atypical Brucella spp., major information about their reservoirs, pathogenicity, and zoonotic potential are vague. Moreover, monitoring of atypical Brucella remains challenging, since misidentification with environmental Ochrobactrum spp. or B. melitensis occurs frequently. Microbiologists, clinicians and veterinarians are further confused by the recent inclusion of Ochrobactrum into the genus Brucella.

Here, we report a case of two diseased exotic frogs presenting with dermal abnormalities. Tissue samples were tested positive by a Brucella-specific PCR. We also characterized one isolate by standard microbiological methods, species-specific multiplex PCR and WGS for phylogenetic differentiation. Its pathogenic potential was analyzed in an in cellulo infection assay.

The suspected Brucella isolate displayed non-fastidious, rapid growth and showed microbiological characteristics similar to novel, atypical Brucella strains, such as B. inopinata or Brucella sp. from African bullfrogs. Differential metabolic phenotyping with a specific set of growth substrates enabled its discrimination from classical Brucella as well as Ochrobactrum. By genomic and phylogenetic characterization, this atypical Brucella isolate was positioned within the novel, non-core clade including various amphibian and human Brucella isolates distant from Ochrobactrum. Similar to other atypical Brucella, this new isolate rapidly replicated in macrophages.

Here we describe another example of amphibian brucellosis, caused by a new member of the non-core Brucella clade. In addition, we present a phenotyping approach that facilitates the differentiation of Ochrobactrum, atypical and classical Brucella by a cost effective and simple microbiological method. Our and previous reports suggest that exotic frogs are reservoirs for potentially pathogenic Brucella spp., and therefore might pose an underestimated zoonotic hazard.