In this paper we present the identification of an atypical Brucella-like strain (BO2) isolated from the lung biopsy of a 52-year-old patient. As a young adult he lived in Oregon on two occasions (1981 and 1985-1987), and experienced an unexplained 'liver failure' and then severe pneumonia (with pleurisy) from which he recovered with multiple courses of antimicrobial therapy as reported by the patient to his physicians in Australia. This patient was originally misdiagnosed because of the misidentification of the BO2 strain as O. anthropi on an AP1 20NE system. It is a common practice for clinical labs to attempt rapid identification of gram-negative coccobacillus organisms like Brucella spp. from blood culture using automated systems. However, the Brucella spp. are often misidentified due to their similar phenotypic characteristics to closely related organisms such as Ochrobactrum spp. [34, 35]. Though the patient was initially treated for both Ochrobactrum and Brucella infections due to the difficulties in diagnosis, he recovered with an extended course of combination oral antimicrobial therapy.
This BO2 strain is phenotypically and molecularly similar to the recently identified B. inopinata type strain (BO1T) recovered from a patient from Oregon, which was also originally misidentified as O. anthropi by the API 20E and API 20NE [7, 8]. Both these strains share common colony morphology and biochemical characteristics including rapid urease and positive H2S production, inability or very weak agglutination with Brucella specific antisera for the lipopolysaccharide-O-antigens or acriflavin. Neither the BO1T or BO2 strains supports gel formation or exhibits growth inhibition to the dye media as shown by common members of the genus Brucella. BO2 also exhibited incomplete lysis by Tbilisi phage and had very similar antimicrobial susceptibility profiles to BO1T in comparison to other Brucella reference strains.
Insertion sequence (IS) fingerprinting in the Brucella species has shown that the genomic localization and copy number of the IS711 insertion element (also called IS6501) is species-specific and could have an association with specific pathogenicity for a preferred host [36–38]. The presence of multiple copies of BO1T-like IS711 insertion sequences suggest not only that BO2 is a member of the Brucella genus (Figure 1) but that the BO2-IS711 amplification pattern specifically resembles that of the newly described B. inopinata species . Positive identification of the BO2 strain as a member of B. inopinata by our real-time BO1 PCR assay was significant. Both BO1T and BO2 strains were the cause of distinct and unusual forms of human brucellosis. Atypical clinical isolates of this nature can often be misdiagnosed by automated systems as was the case with BO1T and the BO2 strain described here [8, 35]. The availability of the real-time TaqMan assay served as a reliable first-line tool for determining B. inopinata-like species.
These initial findings led to further characterization and sequence-based typing which provided additional supporting evidence that this new BO2 strain most resembles the B. inopinata sp. within the Brucella genus. Using broad-range eubacterial primers, Gee et. al. effectively demonstrated the advantage of 16S rRNA gene sequencing to identify Brucella isolates reporting 100% identity in all the strains examined . Interestingly, the full-length 16S rRNA gene sequence of BO2 was 100% identical to that of BO1T and 99.6% identical to the Brucella spp. consensus 16S rRNA gene sequence. The high sequence identity of the BO2 16S rRNA sequence to the recently described B. inopinata sp. is remarkable and represents the first recognized Brucella species to have a divergent 16S rRNA sequence .
The recA gene has been investigated as an alternative phylogenetic marker for several bacterial genera due to its highly conserved nature and ubiquity in prokaryotes [33, 39, 40]. Unlike the high sequence homology of the recA gene within the Brucella genus , we identified unique variability in the recA gene sequences of BO2 and BO1T. Sequence analysis revealed that the recA nucleotide sequence of the BO2 strain shared greater similarity with the Brucella spp. recA consensus sequence than to BO1T. Both BO2 and BO1T
recA sequences are distanced by 8 and 11 unique SNPs, respectively, from the Brucella spp. recA consensus sequence, and share only one common transversion at the 517 nucleotide position. Translation of the recA gene sequences of BO1T, BO2 and the Brucella spp. consensus sequence shows that all base pair changes were synonymous substitutions having no effect on protein structure or function. The Brucella outer membrane proteins have been studied extensively for their function in virulence, pathogenicity, bacteriophage reception, antigenic factors and antibacterial evasion [42–45]. The genetic variability among the omp genes within the Brucella spp. has proven effective at characterizing Brucella spp. and strain types and is often used for higher resolution molecular typing [4, 32, 43, 45]. The omp2a/2b genetic analysis we report here is very interesting in that BO2 consistently associates with not only BO1T but the atypical B. suis 83-210 strain that was isolated from a rodent in Australia ; and thus further investigation may be warranted into rodents as a possible natural reservoir for these novel Brucella species.
Investigation of the nine housekeeping genes by multi locus sequencing analysis demonstrates that BO2 is genetically distinct from BO1T yet exhibits remarkably similar divergence (1.5%) from the classical Brucella sequence types as shown in Figure 4. The relative similarity of the nucleotide sequences of BO1T and BO2 by MLSA demonstrates uniquely distant sequence types within the currently characterized Brucella spp. and should be considered as a new group of STs within the Brucella genus. They also exhibit distinct allelic profiles by MLVA although all alleles in both the BO1T and BO2 allelic profiles have been observed in other Brucella spp. Furthermore, the phylogenetic analysis shown in Figure 5 demonstrates that these strains form a single separate cluster from the classical Brucella spp. .
The molecular and microbiological characteristics presented here provide supporting evidence that strain BO2 is most closely associated with the BO1T strain and should be considered as a novel lineage of B. inopinata sp. Attempting to understand the evolutionary origin of these two strains is somewhat confounded by the interesting and disparate medical histories of the case patients (who both happened to have lived in Portland, Oregon) from whom these strains were isolated and suggests that there are new and emerging Brucella strains capable of causing unusual presentation of human brucellosis.