An extended multilocus sequence analysis (MLSA) scheme applicable to the Brucella, an expanding genus that includes zoonotic pathogens that severely impact animal and human health across large parts of the globe, was developed. The scheme, which extends a previously described nine locus scheme by examining sequences at 21 independent genetic loci in order to increase discriminatory power, was applied to a globally and temporally diverse collection of over 500 isolates representing all 12 known Brucella species providing an expanded and detailed understanding of the population genetic structure of the group. Over 100 sequence types (STs) were identified and analysis of data provided insights into both the global evolutionary history of the genus, suggesting that early emerging Brucella abortus lineages might be confined to Africa while some later lineages have spread worldwide, and further evidence of the existence of lineages with restricted host or geographical ranges. The relationship between biovar, long used as a crude epidemiological marker, and genotype was also examined and showed decreasing congruence in the order Brucella suis > B. abortus > Brucella melitensis. Both the previously described nine locus scheme and the extended 21 locus scheme have been made available at http://pubmlst.org/brucella/ to allow the community to interrogate existing data and compare with newly generated data.

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Introduction: Brucellosis is a zoonotic disease that has a significant economic, social and public health impact in many parts of the world. The causative agents are members of the genus Brucella currently comprising 11 species and with an expanding known host range in recent years. Case presentation: One of a pair of White’s tree frogs (Litoria caerulea) developed skin lesions from which a pure growth of a haemolytic organism was obtained. The isolate was identified as Brucella melitensis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, although the colony morphology was inconsistent with this identification. Applying the classical biotyping approach used to subdivide members of the genus Brucella, the isolate did not correspond to any known Brucella sp. However, PCR targeting of genes specific for members of the genus Brucella was strongly positive and 16S rRNA gene sequencing revealed a close relationship with extant Brucella spp. In order to place the isolate more accurately, a multilocus sequencing approach was applied, which confirmed that the isolate represented a novel member of the emerging ‘atypical’ Brucella group, which includes isolates from human disease, from rodents and, more recently, reported isolations from frogs in Germany. Conclusion: This case represents the first report of isolation of a Brucella sp. from frogs outside Germany and suggests that these isolates may be widespread. Whilst there is no evidence to date that these isolates represent a zoonotic threat, the association of other ‘atypical’ Brucella sp. with human disease suggests that appropriate measures should be taken to avoid unnecessary contact with potentially infected amphibians until the zoonotic potential of this group is better understood.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

The bacterial family Brucellaceae is currently composed of seven genera, including species of the genus Brucella, a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenic Brucella spp. may be hindered by their close phenotypic similarity to other members of the Brucellaceae, particularly of the genus Ochrobactrum. Additionally, a number of novel atypical Brucella taxa have recently been identified, which exhibit greater genetic diversity than observed within the previously described species, and which share genomic features with organisms outside of the genus. Furthermore, previous work has indicated that the genus Ochrobactrum is polyphyletic, raising further questions regarding the relationship between the genus Brucella and wider Brucellaceae. We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel of Brucellaceae type strains, in order to characterize relationships within the family. Phylogenies based on WGS core genome alignments were able to resolve phylogenetic relationships of 31 non-Brucella spp. type strains from within the family, alongside type strains of twelve Brucella species. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypical Brucella isolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genus Ochrobactrum and wider family. Both WGS and MLSA methods closely grouped Brucella spp. with a subset of Ochrobactrum species. However, results also confirmed that the genus Ochrobactrum is polyphyletic, with seven species forming a separate grouping. The pan-family MLSA scheme was subsequently applied to a panel of 50 field strains of the family Brucellaceae, isolated from a wide variety of sources. This analysis confirmed the utility of the pan-Brucellaceae MLSA scheme in placing field isolates in relation to recognized type strains. However, a significant number of these isolates did not cluster with currently identified type strains, suggesting the existence