Hijacking anaerobic metabolism to restore antibiotic efficacy in <i>Pseudomonas aeruginosa</i>

Bacterial pathogens regularly encounter oxygen (O₂) limitation at sites of infection in the human body. Many pathogens exhibit antibiotic recalcitrance under low O₂ conditions, spotlighting the need for new therapeutics that are effective in O₂-limited environments. We demonstrate that several classes of antibiotics display limited toxicity against hypoxic cultures of the opportunistic pathogen, <i>Pseudomonas aeruginosa</i>, where the pathogen exhibits antibiotic tolerance. In O₂-limited host environments, many pathogens employ nitrate respiration, marking this anaerobic bacterial metabolism as an excellent drug target. Chlorate is a nitrate analog that hijacks nitrate respiration to kill <i>P. aeruginosa</i>. Chlorate acts as a prodrug: nitrate reductase reduces chlorate, thereby forming the toxic oxidizing agent, chlorite. Chlorate treatment is most toxic to <i>P. aeruginosa</i> under anoxia and displays limited-to-no toxicity under hypoxic or oxic conditions. Thus, neither chlorate nor antibiotics alone efficiently kill O₂-limited <i>P. aeruginosa</i>. Excitingly, combined chlorate-antibiotic treatment showed that chlorate addition potentiates all tested classes of antibiotics, often eradicating hypoxic <i>P. aeruginosa</i> populations to below detection. Chlorate addition reduced the lethal dose of ceftazidime by >100-fold, further highlighting chlorate's capacity to potentiate antibiotic treatment. Unlike chlorate, we found that most antibiotics do not synergize with different classes of drugs, except for colistin. Given that combination therapy is a promising strategy for combating antibiotic treatment failure, future studies should continue exploring chlorate's therapeutic potential, including examining the mechanisms of chlorate-antibiotic synergy. Our work points to the critical relationship between bacterial physiology and drug efficacy and highlights anaerobic metabolism as a promising drug target.IMPORTANCEMany antibiotics are less effective at killing pathogens under oxygen (O₂)-limited conditions. Pathogens frequently encounter O₂ limitation within host environments, which helps explain why antibiotic therapies often fail to resolve chronic infections. We are investigating the relationship between O₂ availability and drug efficacy in the opportunistic pathogen, <i>Pseudomonas aeruginosa</i>. In agreement with prior work, we demonstrate that <i>P. aeruginosa</i> exhibits antibiotic recalcitrance under hypoxic conditions. We also explore the use of a novel therapeutic, chlorate, which kills <i>P. aeruginosa</i> under O₂-limited conditions when the pathogen utilizes anaerobic metabolism (nitrate respiration). Excitingly, we find that chlorate-antibiotic combinations are highly lethal to <i>P. aeruginosa</i> across a wide range of O₂ availabilities similar to those the pathogen encounters during infection. Our work demonstrates that we can leverage our understanding of pathogen physiology to propose novel drug combinations that hijack anaerobic metabolism to overcome antibiotic treatment failure in O₂-limited environments.