Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria

Abstract

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell’s bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic. At sub-minimal inhibitory concentrations, phenothiazines have been shown to inhibit virulence as well as the formation of biofilms in a wide range of different bacterial pathogens. In this study, we analyzed the anti-bacterial effect of the FDA-approved drug, promethazine, on biofilm-grown Pseudomonas aeruginosa. We demonstrate that PMZ interferes with bacterial bioenergetics and sensitizes biofilm-grown P. aeruginosa cells to bactericidal activity of several different classes of antibiotics by several orders of magnitude. This effect was most pronounced when cells were grown under environmental conditions that induce low intracellular proton levels. Thus, it seems that a reduced proton efflux in cells that exhibit decreased respiratory activity due to their biofilm mode of growth might explain their general antimicrobial tolerance. The use of PMZ as an antibiotic sensitizer holds promise that targeting tolerance mechanisms of biofilm-grown bacteria could become a practicable way to change the way physicians treat biofilm-associated infections.