Background: Bacterial infections persist throughout life for individuals with cystic fibrosis, with significant morbidity and mortality. The most common bacteria are Staphylococcus aureus (Sa) and Pseudomonas aeruginosa (Pa). Over time, these bacteria become increasingly resistant to antibiotics, and some particularly challenging pathogens can also arise, including Burkholderia cepacia (Bcc) and Mycobacterium abscessus (Mab). Treatment of these more resistant bacteria is very difficult and often unsuccessful despite multiple antibiotics and prolonged therapy. Better therapeutics are clearly needed. There have been several recent clinical trials using inhaled nitric oxide (NO) for treatment of patients with cystic fibrosis. NO has known antimicrobial and immunomodulatory properties, making inhaled NO a promising adjunctive therapy for pulmonary infections. However, little is known about optimal dosing or whether any specific combination with antibiotics may improve outcomes.
Methods: We compared the effects of NO, administered in gaseous form or via a chemical donor, on Sa, Pa, Bcc, and Mab. Bacterial ATCC strains were cultured in flasks for 24-72 hours and growth quantified by OD or colony forming 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 PBS and minimal medium (M63) and exposed Pa, Bcc, and Mab to 160 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 ppme) or 30-minute inhaled NO at 80 or 160 ppm (PAPA NONOate, 80 or 160 ppme). We tested antibiotic effect in the presence of NO by minimum inhibitory concentrations (MICs) using the broth microdilution method. Pa, Bcc, and Mab were treated with NONOate at the lowest ppme concentration resulting in growth inhibition concurrent with antibiotics in a 96-well plate.
Results: We found that Sa, Pa, Bcc, and Mab differ in their susceptibility to NO. Mab was most sensitive to gNO. Exposure of Pa, Bcc, and Mab to 160 ppm gNO resulted in growth inhibition of Mab only. Mab was also the most sensitive to NONOate, with inhibition of growth at all concentrations tested, followed by Bcc, inhibited by 80 and 160 ppme NONOate, then Pa, only inhibited by 160 ppme NONOate. Both methicillin sensitive and resistant Sa were resistant to NO. No concentration of NONOate inhibited growth. NONOate treatment also decreased the Mab MIC for several antibiotics, with the greatest effect on those with an intracellular mechanism of action. For Mab, the MIC (in mcg/mL) without and with NONOate (respectively) for azithromycin was 4 and 0.5, clofazimine 0.5 and 0.03, linezolid 16 and 2, and moxifloxacin 8 and 2. Amikacin, bedaquiline, ciprofloxacin, cefoxitin, imipenem, and tebipenem MICs did not change or only decreased 2-fold. MIC also decreased for Pa with amikacin (4 to 1 mcg/mL) and tebipenem (8 to 2 mcg/mL) and for Bcc with ciprofloxacin (0.5 to 0.06 mcg/mL).
Conclusions: Together, our data show that treatment of individuals with cystic fibrosis with inhaled NO may be particularly beneficial in the setting of Mab or Bcc infection and when combined with specific antibiotics. Further studies are ongoing to better characterize these effects.
Acknowledgements: This work was supported by a Clinical Fellowship Award from the Cystic Fibrosis Foundation.
