Construction and optimization of a ‘NG Morbidostat’ - An automated continuous-culture device for studying the pathways towards antibiotic resistance in Neisseria gonorrhoeae

Abstract

To obtain a detailed picture of the dynamics of antibiotic resistance development in Neisseria gonorrhoeae, we built a morbidostat according to the protocol of Toprak et al., adjusted to the specific characteristics required for the growth of N. gonorrhoeae. In this article we describe the adaptations, specifications and the difficulties we encountered during the construction and optimization of the NG morbidostat. As a proof of concept, we conducted a morbidostat experiment by increasing concentrations of azithromycin in response to bacterial growth. We started the experiment with two N. gonorrhoeae reference strains WHO-F and WHO-X. These strains were grown in 12 mL GC Broth supplemented with IsoVitaleX™ (1%) and vancomycin, colistin, nystatin, trimethoprim (VCNT) selective supplement for 30 days in a 6% CO2 environment at 36°C. Samples of the cultures were taken 2-3 times a week and minimal inhibitory concentrations (MICs) of azithromycin were determined using E-test. The initial MICs of WHO-F and WHO-X were 0.125 µg/mL and 0.25 µg/mL, respectively. In less than 30 days, we were able to induce high level azithromycin resistance in N. gonorrhoeae, with a 750 and 1000 fold increase in MIC for WHO-F and WHO-X, respectively.