Genotypic and Phenotypic Characterization of Corynebacterium ulcerans Strains Isolated from Domestic Animals in Brazil and Structural Implications of Mutations Conferring Quinolone-Resistance

Corynebacterium ulcerans is an emerging zoonotic pathogen that can cause diphtheria-like infections in humans. In this study, we report the novel detection and comprehensive phenotypic and genomic characterization of three atoxigenic C. ulcerans strains isolated from domestic animals in Brazil. Notably, all isolates belonged to the multilocus sequence type ST-339, which has been previously identified in both human and animal hosts from geographically distant regions, suggesting the potential for international dissemination. Whole-genome sequencing confirmed species identity and revealed high genetic simila-rity among isolates, although distinct phylogenetic subclades were observed. Genomic analyses identified conserved virulence-associated determinants, including incomplete pilus gene clusters, iron acquisition systems, and the pld gene encoding phospholipase D. In contrast, the tox gene was absent in all strains. Notably, one isolate exhibited ciproflo-xacin resistance associated with double mutations (S89L and D93G) in the quinolone- resistance-determining region of GyrA. Molecular modeling and dynamics simulations demonstrated that these mutations impair key interactions within the ciprofloxacin– magnesium–water complex, thereby compromising the stability of drug binding. Additionally, the presence of diverse mobile genetic elements, prophages, and CRISPR-Cas systems highlighted the genomic plasticity of these isolates. Our findings provide new insights into the zoonotic potential, antimicrobial resistance mechanisms, and genomic diversity of C. ulcerans, underscoring the need for strengthened surveillance and mole-cular monitoring of this emerging pathogen in both veterinary and public health contexts.