Putative azithromycin resistance mutations in <i>Chlamydia trachomatis</i> are globally distributed but arose before azithromycin was discovered

Azithromycin is widely used to treat <i>Chlamydia trachomatis</i> infections, yet the extent of resistance to the drug across the species has not been addressed. We surveyed mutations and substitutions linked to putative azithromycin resistance across 1,349 high-quality <i>C. trachomatis</i> genomes. Mutations in the <i>rpl</i>V gene encoding three non-synonymous substitutions, compared with the canonical <i>C. trachomatis</i> reference strain D//TW-3/Cx sequence, were found to be common but largely conserved within phylogenetic lineages causing prevalent urogenital and anorectal infections and lymphogranuloma venereum. However, no mutations were identified in the ocular lineage. Time-scaled phylogenetic analysis suggested that these mutations predate the clinical introduction of azithromycin. In contrast, no consistent resistance-associated patterns were observed in <i>23S</i> rRNA or <i>rpl</i>D genes. This large-scale genomic surveillance provides critical insights into the evolutionary trends of putative azithromycin resistance in <i>C. trachomatis</i> and underscores the importance of integrating genomic monitoring with phenotypic susceptibility testing to accurately assess and manage antimicrobial resistance.