Chlorate Specifically Targets Oxidant-Starved, Antibiotic-Tolerant Populations of Pseudomonas aeruginosa Biofilms.

Abstract

Nitrate respiration is a widespread mode of anaerobic energy generation used by many bacterial pathogens, and the respiratory nitrate reductase, Nar, has long been known to reduce chlorate to the toxic oxidizing agent chlorite. Here, we demonstrate the antibacterial activity of chlorate against Pseudomonas aeruginosa, a representative pathogen that can inhabit hypoxic or anoxic host microenvironments during infection. Aerobically grown P. aeruginosa cells are tobramycin sensitive but chlorate tolerant. In the absence of oxygen or an alternative electron acceptor, cells are tobramycin tolerant but chlorate sensitive via Nar-dependent reduction. The fact that chlorite, the product of chlorate reduction, is not detected in culture supernatants suggests that it may react rapidly and be retained intracellularly. Tobramycin and chlorate target distinct populations within metabolically stratified aggregate biofilms; tobramycin kills cells on the oxic periphery, whereas chlorate kills hypoxic and anoxic cells in the interior. In a matrix populated by multiple aggregates, tobramycin-mediated death of surface aggregates enables deeper oxygen penetration into the matrix, benefiting select aggregate populations by increasing survival and removing chlorate sensitivity. Finally, lasR mutants, which commonly arise in P. aeruginosa infections and are known to withstand conventional antibiotic treatment, are hypersensitive to chlorate. A lasR mutant shows a propensity to respire nitrate and reduce chlorate more rapidly than the wild type does, consistent with its heightened chlorate sensitivity. These findings illustrate chlorate's potential to selectively target oxidant-starved pathogens, including physiological states and genotypes of P. aeruginosa that represent antibiotic-tolerant populations during infections.IMPORTANCE The anaerobic growth and survival of bacteria are often correlated with physiological tolerance to conventional antibiotics, motivating the development of novel strategies targeting pathogens in anoxic environments. A key challenge is to identify drug targets that are specific to this metabolic state. Chlorate is a nontoxic compound that can be reduced to toxic chlorite by a widespread enzyme of anaerobic metabolism. We tested the antibacterial properties of chlorate against Pseudomonas aeruginosa, a pathogen that can inhabit hypoxic or anoxic microenvironments, including those that arise in human infection. Chlorate and the antibiotic tobramycin kill distinct metabolic populations in P. aeruginosa biofilms, where chlorate targets anaerobic cells that tolerate tobramycin. Chlorate is particularly effective against P. aeruginosalasR mutants, which are frequently isolated from human infections and more resistant to some antibiotics. This work suggests that chlorate may hold potential as an anaerobic prodrug.