Application of Whole Genome Sequencing and Pan-Family Multi-Locus Sequence Analysis to Characterize Relationships Within the Family Brucellaceae .

The bacterial familyBrucellaceaeis currently composed of seven genera, including species of the genusBrucella, a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenicBrucellaspp. may be hindered by their close phenotypic similarity to other members of theBrucellaceae, particularly of the genusOchrobactrum. Additionally, a number of novel atypicalBrucellataxa 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 genusOchrobactrumis polyphyletic, raising further questions regarding the relationship between the genusBrucellaand widerBrucellaceae. We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel ofBrucellaceaetype 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-Brucellaspp. type strains from within the family, alongside type strains of twelveBrucellaspecies. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypicalBrucellaisolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genusOchrobactrumand wider family. Both WGS and MLSA methods closely groupedBrucellaspp. with a sub-set ofOchrobactrumspecies. However, results also confirmed that the genusOchrobactrumis 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 familyBrucellaceae, isolated from a wide variety of sources. This analysis confirmed the utility of the pan-BrucellaceaeMLSA 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 of additional taxonomic diversity within some members of theBrucellaceae. The WGS and pan-family MLSA approaches applied here provide valuable tools for resolving the identity and phylogenetic relationships of isolates from an expanding bacterial family containing a number of important pathogens.