Hybrid Whole Genomes of Brucella melitensis from Tunisian Animal Isolates: Virulence Factors, Antimicrobial Susceptibility, and Phylogeny.

Brucellosis remains endemic in Tunisia, causing abortions in small ruminants, and represents a public health threat through occupational exposure and the consumption of contaminated animal products. The aims of this study are to assess the antibiotic susceptibility of two Brucella melitensis isolates (TATA and SBZ) from aborted sheep, to analyze their genomes using hybrid whole-genome sequencing, and to investigate their antimicrobial resistance (AMR), potential virulence factors (VFs), and phylogenetic relationships. Both isolates were phenotypically confirmed to be susceptible to doxycycline, gentamicin, rifampicin, streptomycin, and trimethoprim-sulfamethoxazole, and no corresponding classical AMR genes were identified. However, several potential AMR-related genes (mprF, bepCDEFG, qacG, and adeF) and a mutation in the parC gene were detected. The analysis of the genotypes revealed 74 potential virulence genes, primarily involved in lipopolysaccharide synthesis and type IV secretion systems. Genomic comparison showed over 99% nucleotide identity between the Tunisian strains, B. melitensis bv. 1 16M and B. melitensis bv. 3 Ether. Five gene clusters, including three hypothetical proteins with 100% identity, were detected exclusively in the TATA and SBZ strains. Additionally, two unique gene clusters were identified in SBZ: a rhodocoxin reductase and another hypothetical protein. Both isolates were assigned to sequence types ST11 and ST89. Core-genome-based phylogenetic analysis clustered both strains with biovar 3 and ordered the Tunisian strains into two distinct groups: TATA within Tunisian Cluster 1 is closely related to strains from Egypt and Italy, while SBZ near MST Cluster 4 is more related to isolates from Austria and two outliers from Italy and Tunisia. This study provides the first genomic characterization of B. melitensis from aborted sheep in Tunisia and offers valuable insights into AMR, virulence, and phylogenetic distribution.