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Basic & Translational Science

Category: Mycoplasma genitalium

(P244) Phenotypic antibiotic resistance of Mycoplasma genitalium using culture: differential susceptibilities to azithromycin

Tuesday, July 25, 2023
16:00 – 18:00 CST
Background Mycoplasma genitalium (MG) is sexually transmitted and associated with urethritis in men. Over 60% of MG strains harbor macrolide resistance associated mutations (MRAM) in the 23S rRNA gene. Known resistance mutations do not always correlate with treatment outcome. We aimed to establish in vitro cultivation and phenotyping of wild type and macrolide resistant strains, in order to better understand the clinical relevance of antibiotic resistance associated mutations in MG strains.

Methods Urine samples positive for MG by transcription mediated amplification (TMA) were cultured in the presence of Vero cells. Laboratory-developed multiplex PCRs were used to determine the presence of MRAM . Isolated strains from culture were used to define a minimal inhibitory concentration (MIC) of azithromycin, levofloxacin, moxifloxacin, sitafloxacin, minocycline, doxycycline, spectinomycin and lefamulin. MICs were defined as antibiotic concentrations giving > 99% inhibition of MG-DNA production at 3 weeks in comparison to control cultures without antibiotics. In vitro growth of MG was quantified using MgPa targeted qPCR. MICs were determined in triplo

Results On average, it took 2-3 weeks to detect growth of MG in the presence of Vero cells. We were able to grow MG from 7/19 (37%) MG-TMA positive urine samples. Phenotypic resistance analysis was successful in 6 of these strains. The MIC results ranged as follows: azithromycin (< 0.016- >16), levofloxacin (1- >4), moxifloxacin (< 0.25-1), sitafloxacin (< 0.032-0.25), minocycline (0.25-≥1) doxycycline (0.125-2), spectinomycin (2.5- >25), lefamulin (0.004-0.064). One WT strain had a MIC for azithromycin of < 0.016 whereas 5 strains with a MRAM showed a MIC ≥8. One strain containing the A2058G mutation showed no growth inhibition at 16 mg/L azithromycin, while samples carrying the A2058G (n=2) or A2059T mutation (n=2) showed high levels of growth inhibition at 8 mg/L azithromycin (60-90% and 97-99% respectively).

Conclusion. We successfully cultured MG in vitro and defined MICs in the presence and absence of MRAM. Different mutations could cause different levels of resistance against azithromycin. Alternatively, other genes outside the 23SrRNA could also be involved. With the emergence of antibiotic resistance in MG, this study emphasizes the need to culture more strains to compare genotype with phenotype.