Background The basis of fluoroquinolone treatment failure for Mycoplasma genitalium is poorly understood. Methods To identify mutations associated with failure we sequenced key regions of the M. genitalium parC and gyrA genes for patients undergoing sequential therapy with doxycycline-moxifloxacin (201 patients, including 21 failures) or doxycycline-sitafloxacin (126 patients, including 13 failures). Results The parC G248T/S83I mutation was more common among patients who failed sequential doxycycline-moxifloxacin (present in 76.2% of failures vs 7.8% cures, p<0.001) and doxycycline-sitafloxacin (50% failures vs 16.8% cures, p = 0.014). Doxycycline-sitafloxacin was more efficacious than doxycycline-moxifloxacin against infections carrying the ParC S83I mutation. Infections with a ParC S83I were more likely to fail treatment with a concurrent gyrA mutation (M95I or D99N) (for doxycycline- moxifloxacin group p = 0.067, for doxycycline-sitafloxacin group p = 0.0093), suggesting an additive effect. Conclusions This study indicates that parC G248T/S83I mutations contribute to failure of moxifloxacin and sitafloxacin, and will inform the development of quinolone resistance assays needed to ensure optimal selection of antimicrobials for M. genitalium.

Background While single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs, and their contribution to fluoroquinolone failure. Methods Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019–February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n=194), the association between SNPs and microbiologic treatment outcome was analyzed. Results The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA co-occurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs, when compared to infections with single S83I SNP alone from analysis of (i) 194 cases in this study (81.2% vs 45.8%, p=0.047), and (ii) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; p=0.0027), indicating a strong additive effect. Conclusions Compared to parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. While AMR varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.

Prevalence, trends, and treatment outcome estimates were generated for parC variants in macrolide-resistant Mycoplasma genitalium . Among 539 cases, the most common amino acid change was S83I, which increased from 13% in 2012 to 2013, to 23% in 2019 to 2020 ( P trend  = 0.046).