Effects of CO-releasing molecules on planktonic and biofilm cells of Pseudomonas aeruginosa

The overuse of antibiotics to treat cystic fibrosis (CF) lung infections has led to high levels of antibiotic resistance. Novel antimicrobial agents that could replace or complement current therapies are needed to fight chronic infections in CF patients. The advent of carbon monoxide-releasing molecules (CORMs) has been useful in investigating the physiological effects of CO by allowing the controlled release of CO to specific targets in mammalian systems. Recent evidence shows that the bactericidal activity of CORMs against a number of species is more effective compared to CO gas. Here, we applied ruthenium- based CORMs (CORM-2, CORM-3) and manganese-based CORM (PhotoCORM [Mn(CO)3(tpa-κ3N)]+) to biofilms using a novel nebulizer technique in conjunction with a Modified Robbins Device, and compared these effects with laboratory planktonic cultures of the Gram-negative pathogen Pseudomonas aeruginosa. We demonstrate that both CORM-2 and CORM-3 rapidly kill planktonic cells in laboratory culture. Although CORM-3 inhibits surface-associated growth of P. aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm, cells growing in a biofilm are more resistant to CORM-3 than planktonic cells. The mechanisms behind the activity of CORM-3 resistance from P. aeruginosa biofilm cells are examined. Poor penetration and anaerobic microenvironment may contribute to this resistance, but not starvation. Aerosolized CORM-3 treatment has a similar bactericidal effect to aerosolized colistin, a commonly used antibiotic for P. aeruginosa lung infections. Although CORM-2 or colistin kills bacteria within the established biofilm, the combination effect of those two antimicrobial compounds was found to be only additive against P. aeruginosa planktonic cells and biofilm cells. Although activated PhotoCORM exhibits slightly inhibitory effect on P. aeruginosa planktonic cells, the compound significantly inhibits surface-associated growth of P. aeruginosa by both preventing biofilm formation and killing bacteria within the established biofilm. Anaerobic cultures of P. aeruginosa are less sensitive to CORM- 3 compared to aerobic cultures