Combinatory effects of chlorhexidine and azithromycin: Implications for therapeutic potential and mechanistic insights

The use of drug combinations to re-sensitize resistant strains is a promising strategy to overcome the stagnation in the drug discovery pipeline. Here, the results demonstrate that the combined application of the broadspectrum bisbiguanide antiseptic chlorhexidine (CHX) and the macrolide antibiotic azithromycin (AZM) significantly inhibits the growth of the Pseudomonas aeruginosa strain PAO1 (isolated from a wound) compared to the individual effects of each agent. Specifically, 1.5 mu g/mL CHX caused 11.4 +/- 4 % growth inhibition and 2 mu g/ mL AZM resulted in 14 +/- 4.5 % inhibition; however, the combination of 1.5 mu g/mL CHX and 2 mu g/mL AZM achieved 58 +/- 6 % inhibition, significantly exceeding the sum of their individual effects. Furthermore, the AZM and CHX combination reduced bacterial viability in biofilms. P. aeruginosa is a common pathogen in wounds, particularly chronic wounds, where it delays the healing process. An in vitro wound infection model further demonstrated that CHX and AZM combination reduced bacterial density and activity in a serum-supported collagen matrix. This combination was found to be effective not only against the Gram-negative P. aeruginosa but also against the Gram-positive Streptococcus mutans. To explain the observed combinatory inhibition effect mechanistically, Fourier Transform Infrared Spectroscopy (FTIR) was employed for the first time in the literature. The results reveal that CHX increases the cellular accumulation of AZM. Changes in the membrane lipid composition of the bacteria additionally suggest a mechanism for enhanced antibiotic accumulation in the presence of CHX. These findings suggest that the role of CHX as a potential partner in different syncretic combinations calls for comprehensive exploration in antibiotic resistant bacterial infections.