Construction and optimization of a 'NG Morbidostat' - An automated continuous-culture device for studying the pathways towards antibiotic resistance in .

To obtain a detailed picture of the dynamics of antibiotic resistance development in , we built a morbidostat according to the protocol of Toprak ., adjusted to the specific characteristics required for the growth of . 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 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% CO 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 , with a 750 and 1000 fold increase in MIC for WHO-F and WHO-X, respectively.