Efficacy of nitric oxide donors and EDTA against biofilms: Implications for antimicrobial therapy in chronic wounds.

Opportunistic pathogen plays a crucial role in chronic wound biofilms, increasing infection's morbidity and mortality. In recent years, the signalling molecule nitric oxide (NO) and chelating agent tetrasodium EDTA (T-EDTA) have been applied therapeutically owing to their multifactorial effects including bacterial killing, biofilm dispersal, and wound healing. However, previous studies assessing NO's antibiofilm efficacy have not considered the variable pH and temperature of the wound environment. Here, pH-dependent NO donors -diazeniumdiolates (NONOates), PAPA NONOate (PA-NO) and Spermine NONOate (SP-NO), and T-EDTA were applied in wound-relevant pH environments (pH 5.5-8.5) and temperatures (32 °C and 37 °C) to PAO1 biofilms grown for either 24 or 48 h. At 32 °C and pH 7.5, 250 μM PA-NO reduced 24-h biofilm biomass by 35 %. At 37 °C, 250 μM PA-NO and 4 % w/v T-EDTA caused 21 % and 57 % biomass reduction in 24-h biofilms, respectively. In 48-h biofilms, NONOates did not induce significant biomass reduction, while T-EDTA maintained its efficacy with a 64 % reduction. A subsequent experiment investigated the impact of NONOates and T-EDTA as pre-treatments before exposure to ciprofloxacin. Unexpectedly, NONOate pre-treatment decreased ciprofloxacin's effectiveness, resulting in approximately 1-log increase in viable planktonic and biofilm-residing cells compared to ciprofloxacin alone. It was hypothesized that this protective effect might stem from NO-induced decreased cellular respiration, which inhibits reactive oxygen species (ROS)-mediated bactericidal mechanisms. These findings highlight both the potential and complexities of developing effective antimicrobial strategies for chronic wound infections, emphasizing the need for further research to optimize treatment approaches.