OR01

Pulmonary non-tuberculous mycobacteria (NTM) diseases are primarily caused by M.abscessus and/or M.avium. Current treatment regiments are long and ineffective due to the lack of 1) clinical breakpoints for most antibiotics and 2) novel NTM specific antibiotics.

The current gold-standard for drug susceptibility and drug discovery testing relies on determining the minimal inhibitory concentration (MIC), a labour-intensive, slow and biased process. We have developed a novel high-content imaging based assay for the detection of bacterial growth in function of time for M.abscesssus and M.avium. Bacteria were stained with Syto9 and propidium iodide and treated with different concentrations of antibiotics in a 96-well. By using the Caps-It automation system, plates were transferred from the incubators to the high-content imagers and images were taken every 4 hours for rapid growers and every 12 hours for slow growers. Dose-response growth curves were generated after 32 hours for M.abscessus and after 3 days for M.avium subsequently IC90-values were computed. We compared a panel of 13 antibiotics using our novel method and the microbroth dilution assay for M.abscessus and found concordant results for 12 antibiotics. For M.avium we compared 5 antibiotics (rifabutin, clarithromycin, moxifloxacin, rifampicin and amikacin) and obtained concordant results as well. Our novel assay reduces assay duration significantly while yielding more precise and unbiased results by quantifying bacterial growth in function of time. We have validated our assay for M.abscessus and implemented it in a high-throughput screening setting for drug discovery. Simultaneously, we believe that we can adjust our assay towards drug susceptibility testing.