In Vitro Susceptibility and Resistance of Mycoplasma genitalium to Nitroimidazoles

Abstract

Mycoplasma genitalium is a sexually transmitted reproductive tract pathogen of men and women. M. genitalium infections are increasingly difficult to treat due to poor efficacy of doxycycline and acquired resistance to azithromycin and moxifloxacin. A recent clinical trial suggested that metronidazole may improve cure rates for women with pelvic inflammatory disease and reduced the detection of M. genitalium when included with standard doxycycline plus ceftriaxone treatment. As data regarding susceptibility of mycoplasmas to nitroimidazoles are lacking in the scientific literature, we determined the in vitro susceptibility of 10 M. genitalium strains to metronidazole, secnidazole, and tinidazole. MICs ranged from 1.6 to 12.5 μg/mL for metronidazole, 3.1 to 12.5 μg/mL for secnidazole, and 0.8 to 6.3 μg/mL for tinidazole. None of these agents was synergistic with doxycycline in checkerboard broth microdilution assays. Tinidazole was superior to metronidazole and secnidazole in terms of MIC and time-kill kinetics and was bactericidal (>99.9% killing) at concentrations below reported serum concentrations. Mutations associated with nitroimidazole resistance were identified by whole-genome sequencing of spontaneous resistant mutants, suggesting a mechanism for reductive activation of the nitroimidazole prodrug by a predicted NAD(P)H-dependent flavin mononucleotide (FMN) oxidoreductase. The presence of oxygen did not affect MICs of wild-type M. genitalium, but a nitroimidazole-resistant mutant was defective for growth under anaerobic conditions, suggesting that resistant mutants may have a fitness disadvantage in anaerobic genital sites. Clinical studies are needed to determine if nitroimidazoles, especially tinidazole, are effective for eradicating M. genitalium infections in men and women.

Keywords: Mycoplasma genitalium; antibiotic resistance; metronidazole; nitroimidazole; secnidazole; tinidazole.

FIG 1

Growth inhibition dose response of M. genitalium strains G37, Sea-1, and Sea-2 to nitroimidazoles. Mtz, metronidazole; Sdz, secnidazole; Tdz, tinidazole. The y axis shows the mean number of genomes detected in triplicate qPCR measurements compared to untreated M. genitalium. Errors bars show standard deviation for duplicate drug-treated wells. The experiment was repeated three times with similar results.

FIG 2

Time-kill experiments. Killing curves for M. genitalium strain G37 treated with various concentrations (micrograms per milliliter) of metronidazole, secnidazole, tinidazole, or moxifloxacin over time. Curves show the average CFU per 10-μL aliquot spotted in triplicate on SP-4 agar plates. Error bars show standard deviation. DMSO, solvent control, is shown in the upper left panel. Results of a typical experiment repeated twice are shown.

FIG 3

Nitroimidazole resistance-associated mutations in the MG_342-MG_343 intergenic region. For clarity, the M. genitalium genome sequence is shown in reverse orientation (note genome coordinates). The large gray triangle indicates the deletion of 92 bp (boxed) in MtzR(A) that changes the last 6 amino acids of MG_343 to 21 missense amino acids (indicated by italics) and overlaps the MG342 start codon. Possible ATG start codons are underlined. The T-to-C base change in the MG_342 start codon of mutants MtzR(E) and TdzR(A) is indicated above the sequence (small arrow). The W93L mutation in MG_342 of MtzR(B) is not shown.

FIG 4

(A) Model of MG342. FMN binding (green) and active site (red) residues and Trp93 (blue) are shown. C score = 0.39; TM score = 0.77 ± 0.10; root mean square deviation (RMSD) = 4.2 ± 2.8 Å. (B) Model of MG383. Active site residues are shown in red, and Ala186 (involved in NAD binding) is shown in blue. C score = 0.43; TM score = 0.77 ± 0.10; RMSD = 4.2 ± 2.8 Å. Both proteins were modeled with I-TASSER. N and C termini are indicated in dark and light gray, respectively. Images were produced with UCSF ChimeraX (version 1.3).