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

2019; Faculty of 1000; Volume: 8; Linguagem: Inglês

10.12688/f1000research.18861.1

2046-1402

Els Verhoeven, Saïd Abdellati, Patrick Nys, Jolein Laumen, Irith De Baetselier, Tania Crucitti, Chris Kenyon,

Bacterial Identification and Susceptibility Testing

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% CO 2 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.