Prevalence and Distribution of Antimicrobial Resistance-Associated Mutations in Mycoplasma genitalium Identified Through Routine Molecular Diagnostics in Korea

<i>Mycoplasma genitalium</i> is a significant sexually transmitted pathogen, and its clinical management is increasingly complicated by the global distribution of mutations associated with macrolide and fluoroquinolone resistance. To characterize the molecular resistance landscape in a routine diagnostic setting, we retrospectively analyzed residual clinical specimens collected during routine sexually transmitted infection testing between January and December 2024. Among 374,021 specimens screened, we included 4019 <i>M. genitalium</i>-positive samples containing sufficient residual material. Using multiplex polymerase chain reaction assays, we detected mutations associated with macrolide and fluoroquinolone resistance in the 23S rRNA and <i>parC</i> genes, respectively. Frequent substitutions included A2059G and A2058G in the 23S rRNA gene (1253 samples) and substitutions at positions 248 and 259 in the <i>parC</i> gene (1306 samples). Mutations in the predefined 23S rRNA and/or <i>parC</i> targets were identified in approximately 44% of the analyzed samples, with distinct patterns of mutation distribution and co-occurrence. Although phenotypic susceptibility and clinical outcomes were not assessed, this large-scale, assay-based analysis provides a comprehensive overview of resistance-associated mutation patterns in <i>M. genitalium</i> derived from routine diagnostics, supporting molecular surveillance for monitoring antimicrobial resistance trends.