Antimicrobial Susceptibility and Genomic Determinants of Resistance and Virulence in Mycoplasma cynos and Mycoplasma felis

Mycoplasma cynos and Mycoplasma felis are important respiratory pathogens in dogs and cats. Due to the challenges of culturing these fastidious bacteria, little is known about their antimicrobial susceptibility or mechanisms of pathogenicity. Treatment is typically empirical, as in vitro antimicrobial activity has not been evaluated, and therapeutic efficacy remains unclear. This study aimed to assess in vitro susceptibility and identify genetic markers of antimicrobial resistance (AMR) and virulence in M. cynos and M. felis clinical isolates. Minimum inhibitory concentrations (MICs) for doxycycline, tetracycline, minocycline, enrofloxacin, marbofloxacin, and azithromycin were determined using a broth microdilution assay developed for this study. Hybrid genomes were generated using Oxford Nanopore and Illumina sequencing. AMR-associated single-nucleotide polymorphisms (SNPs) in the gyrA gene correlated with high MICs to enrofloxacin and marbofloxacin in both species. Mutations in 23S rRNA were associated with reduced susceptibility to azithromycin. In M. felis , novel variants in gyrA and the 50S ribosomal protein L4 were linked to decreased susceptibility to fluoroquinolones and azithromycin, respectively. The data also suggest potential intrinsic resistance to azithromycin in M. felis . All isolates required low MICs of tetracyclines, and resistance mutations were not identified in the 16S rRNA gene, supporting tetracyclines as effective first-line treatment options. Virulence genes, particularly those associated with adhesion and immune evasion, were detected in both M. cynos and M. felis . This study presents the first comprehensive genomic and phenotypic analysis of AMR and virulence in M. cynos and M. felis , providing new insights into their pathogenicity and informing evidence-based therapeutic strategies.