Brucella melitensis: Divergence Among Indian Strains and Genetic Characterization of a Strain Isolated from Cattle

Brucella melitensis primarily affects sheep, goats and is associated with brucellosis in humans, which is one of the world's most widespread neglected zoonotic disease. The current study attempted the determination of genetic diversity through comparative genome analysis of B. melitensis strains reported from India with other countries. The study also reports the isolation and identification of B. melitensis BMNDDB8664 from a cow with a history of abortion, whole-genome sequencing (WGS), determination of virulence factors, genotyping, and comparative genome analysis. Multilocus sequence typing, Multiple locus variable number of tandem repeats analysis (MLVA), and WGS based phylogeny revealed the predominance of ST-8 and genotypes (116 and II respectively) that clustered to the East Mediterranean lineage. Identification of hitherto unreported genotypes by MLVA also indicated the existence and circulation of West Mediterranean and American lineages in India. Though the AMOS-PCR results suggest the BMNDDB8664 isolate as Brucella abortus, the outcomes from multiplex PCR, ribosomal multilocus sequence typing, and WGS analysis confirmed it as B. melitensis. The analysis revealed the presence of adeF gene (aids conferring resistance to fluoro-quinolone and tetracyclines). The isolate lacked two important T4SS genes virB2 and virB7 genes (roles in infection and rifampicin resistance respectively) and also lacked the Brucella suis mprF gene that aids intracellular survival. Further, BMNDDB8664 lacked some of the genes associated with LPS synthesis (wbkB, wbkC) and transport (wzm, wzt) and hence, is most likely a rough strain. WGS-based phylogenetic analysis revealed close genetic relatedness of this BMNDDB8664 with a sheep isolate and two human isolates. The results prompt systematic, broad-based epidemiological studies on brucella infection at the species level. For effective control of human brucellosis, a concerted One Health approach with studies encircling the identification of aetiology at species, strain level to find their prevalence, spread, and inter-host transmission patterns need to be understood, for better design and implementation of effective control strategies in India and other endemic regions.