Genomic Characterization of Clinical Brucella melitensis Isolates from Tunisia: Integration into the Global Population Structure

Brucellosis represents a significant global health concern that is endemic in many regions of the world. It is most prevalent in the Mediterranean region, especially in Maghreb (Tunisia, Morocco and Algeria). In Tunisia the diagnosis of human brucellosis is primarily based on serological tests and cultivation of the causative pathogen, without the knowledge of the underlying Brucella species or biovar. In addition, the scarcity of laboratories with adequate biosecurity measures to handle suspected specimen constitutes a significant challenge. Furthermore, the absence of full genome data limits our understanding of the genetic diversity of Brucella in Tunisia in comparison to the strains circulating in the North African region and the world. In the present study, a total of 36 bacterial isolates derived from human patients diagnosed with brucellosis in Tunisia were subjected to examination. Real-time PCR confirmed all isolates as B. melitensis. In subsequent core genome-based MLST analysis (cgMLST) based on 2706 target genes, the isolates formed two separate but closely related clusters with a distance of 140 alleles. Intra-cluster diversity was 1 to 6 alleles. In the larger geographical context and in comparison to almost 1000 other Brucella genomes, the isolates showed the highest genetic relationship to B. melitensis isolates from Italy and Egypt with distances of 130 and 150 alleles, respectively. All isolates were most similar to biovar 3 genotype. Markedly, strains from a reported brucellosis outbreak in Austria were grouped closely (26 and 27 alleles respectively) together with strains from Tunisia, suggesting that this country may represent their geographical origin. This research represents a significant advancement in our understanding of B. melitensis strains circulating in the Maghreb region, as it is the first study to elucidate the molecular characterization of strains isolated from humans in Tunisia. The cgMLST analysis of the strains provided information on the regional distribution of the strains and the association with neighboring countries and significant outbreaks in the region. The data will form the basis of a future reference framework for strains circulating in the Mediterranean region.