476 RNase E: A newplayer in Pseudomonas aeruginosa nutritional virulence

Methods: We compared the effects of NO, administered in gaseous form or via a chemical donor, on S. aureus, P. aeruginosa, B. cepacia, and M. abscessus.Bacterial American Type Culture Collection strains were cultured in flasks for 24 to 72 hours, and growth was quantified by optical density or colonyforming units on agar.We established a delivery system for gaseous NO (gNO) to flasks using a sealed plexiglass box.Gaseous NO does not solubilize well in typical growth media, so we used phosphate-buffered saline and minimal medium (M63) and exposed P. aeruginosa, B. cepacia, and M. abscessus to 160 parts per million ( ppm) gNO three times daily.We also treated bacteria with chemical NO donors at concentrations estimated to mimic continuous inhaled NO of 20 ppm (DETA NONOate, 20 ppm e ) or 30-minute inhaled NO at 80 or 160 ppm (PAPA NONOate, 80 or 160 ppm e ).We tested antibiotic effect in the presence of NO by minimum inhibitory concentration (MIC) using the broth microdilution method.P. aeruginosa, B. cepacia, and M. abscessus were treated with NONOate at the lowest ppm e concentration resulting in growth inhibition concurrent with antibiotics in a 96-well plate.Results: We found that S. aureus, P. aeruginosa, B. cepacia, and M. abscessus differ in their susceptibility to NO. M. abscessus was most sensitive to gNO.Exposure of P. aeruginosa, B. cepacia, and M. abscessus to 160 ppm gNO resulted in growth inhibition of M. abscessus only.M. abscessus was also the most sensitive to NONOate, with inhibition of growth at all concentrations tested; followed by B. cepacia, inhibited by 80 and 160 ppm e NONOate; and then P. aeruginosa, only inhibited by 160 ppm e NONOate.Methicillinsensitive and -resistant S. aureus were resistant to NO.No concentration of NONOate inhibited growth.NONOate treatment also decreased M. abscessus MIC for several antibiotics, with the greatest effect on those with an intracellular mechanism of action.For M. abscessus, MIC without and with NONOate (respectively) for azithromycin was 4 µg/mL and 0.5 µg/ mL, clofazimine 0.5 µg/mL and 0.03 µg/mL, linezolid 16 µg/mL and 2 µg/ mL, and moxifloxacin 8 µg/mL and 2 µg/mL.Amikacin, bedaquiline, ciprofloxacin, cefoxitin, imipenem, and tebipenem MICs differed only by zero or log2 dilution.MIC also decreased for P. aeruginosa with amikacin (from 4 to 1 µg/mL) and tebipenem (from 8 to 2 µg/mL) and for B. cepacia with ciprofloxacin (from 0.5 to 0.06 µg/mL).Conclusions: Our data show that treatment of individuals with CF with inhaled NO may be particularly beneficial in the setting of M. abscessus or B. cepacia infection and when combined with specific antibiotics.Further studies are ongoing to better characterize these effects.