PROJECT SUMMARY Mycoplasma genitalium is a sexually transmitted bacterial pathogen that frequently causes genital tract syndromes including urethritis in men and cervicitis, pelvic inflammatory disease, and infertility in women. Sensitive diagnostic tests have been approved in the US since 2019, however, few treatment options for M. genitalium are available. M. genitalium lacks a cell wall and has only a single membrane so antibiotics targeting peptidoglycan synthesis, or the bacterial outer membrane, are completely inactive. Doxycycline is only 30-40% effective in eradicating M. genitalium infections. The efficacy of azithromycin, the preferred therapy, has decreased in recent years and now more than 50% of US strains are resistant. In high-risk populations in the US and worldwide azithromycin resistance reaches 100%. More than 10% of strains are resistant to moxifloxacin, the recommended second line therapy, and resistance to both macrolides and fluoroquinolones is increasingly reported. No effective treatment options are approved in the US to treat these dually resistant infections. In addition, moxifloxacin is not approved for certain patient groups (e.g., pregnant women, adolescents <18 years old), and the FDA discourages fluoroquinolone use unless no other options exist because of potentially severe and permanent side effects. There is an urgent and immediate need to identify additional therapies with activity against M. genitalium. We determined that M. genitalium is susceptible in vitro to nitroimidazoles, consistent with a recently published clinical trial demonstrating that metronidazole reduced M. genitalium infection in women with PID, and contrary to the accepted view that these drugs are inactive against mycoplasmas. Building on our preliminary data we now propose to define the activity of nitroimidazole and nitrofuran antibiotics against M. genitalium in detail. We will assess the in vitro susceptibility to more than a dozen nitro group-containing drugs for which clinical safety and efficacy data are known, including several that are approved for other indications in the US and elsewhere in the world. The in vitro activity of these drugs will be compared in terms of minimum inhibitory concentration, time kill kinetics, post antibiotic effect, activity against biofilms, and spontaneous resistance rates to inform future clinical trials. Differences in strain susceptibility will be determined by measuring the potency of top performing analogs against our existing collection of clinical isolates. We found that nitroimidazole resistance is associated with mutations affecting an oxidoreductase gene suggesting a mechanism for the bioreductive activation of the prodrug that is necessary for its bactericidal activity. We propose to expand and confirm these findings by isolating additional resistant mutants and defining drug-enzyme interactions with wild type and mutant alleles of the purified oxidoreductase. This study will have direct tra...