data_proof_of_concept

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.