Chlorate And Antibiotic Treatment Dismantle Pseudomonas Aeruginosa Biofilm And Lead To Healing Of Chronic Wounds

Abstract

Background: Pseudomonas aeruginosa (PA) is an opportunistic pathogen commonly found in human chronic wound. When within biofilm, PA is tolerant or resistant to conventional antibiotic therapy by using metabolites/enzymes that significantly increase pathogenicity during chronic infections. To survive in hypoxic wound microenvironments and the interior of biofilm, PA uses nitrate reductase (nar), an enzyme that facilitates anaerobic respiration after oxygen is depleted from the microenvironment. In addition to reducing nitrates, nar can reduce internalized chlorate to hypochlorite. This reaction not only converts a non-toxic chemical into a toxic one, but also provides a targeted mechanism that kills anaerobically respiring bacteria which are difficult to eradicate with antibiotics. We hypothesize that treating PA-infected chronic wounds with chlorate and the antibiotic ciprofloxacin, will remove aerobic and anaerobic respiring bacterial populations, respectively, to facilitate wound healing. To test the efficacy of ciprofloxacin +chlorate treatment, we used a murine chronic wound model infected by with a nar activity-containing PA strain isolated from these mice chronic wounds. Briefly, wounds are treated with inhibitors of antioxidant enzymes at wounding to induce high oxidative stress and 24hr after injury, PA is inoculated into the wounds resulting in development of chronic wounds that contain strong biofilm and do not heal. We found that wounds treated daily with low doses of ciprofloxacin+chlorate increased survival of the mice with biofilm-containing chronic wounds compared to chlorate or ciprofloxacin alone. Treatment with ciprofloxacin+chlorate greatly reduced PA bioburden. The biofilm is dismantled, and the wounds go on to heal and close within 40 days. Wound swab samples taken 20 days after treatment began, show very few viable bacteria after treatment with ciprofloxacin+chlorate compared to ciprofloxacin or chlorate alone. Using MiPACT, a method to detect fluorescently label bacteria in wounds tissues, that have healed, show that PA infection is resolved and cleared from the tissue. These results indicate that in chronic wounds, chlorate kills PA in anaerobic areas of the wound, dismantling the biofilm and making the bacteria in aerobic regions planktonic and susceptible to ciprofloxacin. In conclusion, because our chronic wound model mimics biofilm-containing human chronic wounds, our results point to proof-of-concept studies in humans using chlorate and antibiotic to improve healing.