Genomic analysis of Brucella melitensis isolates recovered from humans in south Tunisia over 35 years between 1988 and 2022.

Brucella melitensis is a zoonotic pathogen that poses a worldwide public health challenge. In recent years, whole-genome sequencing has become a widely accepted molecular typing method for the genomic epidemiology of brucellosis. This study reports the genomic characteristics of 24 B. melitensis strains isolated from human infections in southern Tunisia over 35 years (1988-2022). We utilized WGS to analyze the clonal relationships of these strains, their relatedness to international sequences, their antimicrobial resistance determinants, and their virulence factors. Our findings revealed a high genetic stability over three decades. All isolates were identified as B. melitensis biovar 3 and were assigned to the same sequence type, ST11, using the MLST-9 scheme. Using the MLST-21, Tunisian sequences shared 20 out of 21 alleles and were assigned to 2 closely related STs (ST89 and ST114). Phylogenetic analysis indicated that all Tunisian sequences were grouped into a single subcluster within lineage I, the West Mediterranean clade, and were highly related to other strains from the Maghreb region (Morocco and Algeria). Antimicrobial resistance analysis revealed no classical resistance determinants. However, mprF, bepCDEFG genes, and missense mutations in rpoB, gyrA, gyrB, and parC genes were identified. Virulence analysis identified 67 genes, predominantly involved in lipopolysaccharide biosynthesis and the type IV secretion system. To our knowledge, this study represents the first genomic investigation of B. melitensis strains circulating in Tunisia. Our findings underscore the importance of genomic surveillance in understanding the epidemiology and evolution of brucellosis in North Africa.