GENETIC PATHWAY TO HIGH LEVEL AZITHROMYCIN RESISTANCE IN NEISSERIA GONORRHOEAE

Background The in-vitro genetic pathways to high-level azithromycin (AZM) resistance have hitherto not been established in Neisseria gonorrhoeae (Ng). Methods A Ng morbidostat that dynamically increases AZM concentrations in response to Ng growth was built according to the protocol of Toprak et al. The reference strains Ng WHO-F and WHO-X were grown in 12 mL GC broth supplemented with IsoVitaleX™ (1%) and vancomycin, colistin, nystatin, trimethoprim selective supplement for 30 days in a 6% CO2 environment at 36°C. Depending on the turbidity and growth of the culture, 1 mL of fresh medium or AZM was added to the culture vials after each cycle of 21 minutes. The experiment started with a concentration of 20x minimal inhibitory concentration (MIC) of AZM in the drug reservoir which was increased up to 320x MIC for both strains by the end of the experiment. Samples of the cultures were taken 2–3 times a week and MICs of AZM were determined using E-tests. Whole genome sequencing will be performed using Illumina MiSeq. All experiments were run in triplicate. Results The initial MICs of WHO-F and WHO-X were 0,125 µg/mL and 0,25 µg/mL respectively. In the first week, the MICs of WHO-F and WHO-X increased approximately 24-fold for WHO-F and 48-fold for WHO-X. After 30 days, WHO-F and WHO-X had attained MICs of 96 µg/mL and ≥296 µg/mL, respectively. The genetic pathways to resistance will be analysed and presented. Conclusion We were able to induce high level AZM resistance in Ng within 30 days of AZM exposure using our Ng morbidostat. Disclosure No significant relationships.