In vitro selection and characterization of resistance to pristinamycin in Mycoplasma genitalium

ABSTRACT Pristinamycin, a streptogramin combination, is recommended as a third-line treatment for Mycoplasma genitalium infections according to European guidelines. However, the molecular mechanisms underlying pristinamycin resistance have not yet been elucidated. We investigated the in vitro development of pristinamycin-resistant M. genitalium mutants under exposure to subinhibitory concentrations of pristinamycin or josamycin. Resistant mutants were characterized by determining the MICs of seven antibiotics, performing Sanger sequencing of 23S rRNA and the ribosomal protein L4 and L22 genes, and conducting whole-genome sequencing in comparison with the parental, susceptible G37 reference strain. Mutants selected in the presence of pristinamycin harbored either an A2062C or A2062G mutation in 23S rRNA, both associated with a marked increase in the MICs of pristinamycin and josamycin. By contrast, selection with josamycin produced a mutant carrying the A2059G mutation, which exhibited elevated MICs for erythromycin, azithromycin, josamycin, and clindamycin, but not for pristinamycin. None of the three resistant mutants showed any detectable growth defect in culture. To mimic pristinamycin treatment in a macrolide-resistant background, further selection was performed using subinhibitory concentrations of pristinamycin on the azithromycin-resistant A2059G mutant. This led to the emergence of an additional C2611T substitution in 23S rRNA, resulting in a pristinamycin MIC of 8 mg/L. No stable mutations were observed in the ribosomal protein L4 or L22 genes during the selection process. In conclusion, this study demonstrates that pristinamycin resistance in M. genitalium is mediated by mutations in the 23S rRNA gene. These laboratory-derived mutations may foreshadow resistance mechanisms emerging in clinical isolates.