The Properties of Linezolid, Rifampicin, and Vancomycin, as Well as the Mechanism of Action of Pentamidine, Determine Their Synergy against Gram-Negative Bacteria

Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB). However, which antibiotics pentamidine can and cannot synergize with and the reasons for the differences are unclear. This study aimed to identify the possible mechanisms for the differences in the synergy of pentamidine with rifampicin, linezolid, tetracycline, erythromycin, and vancomycin against GNB. Checkerboard assays were used to detect the synergy of pentamidine and the different antibiotics. To determine the mechanism of pentamidine, fluorescent labeling assays were used to measure membrane permeability, membrane potential, efflux pump activity, and reactive oxygen species (ROS); the LPS neutralization assay was used to evaluate the target site; and quantitative PCR was used to measure changes in efflux pump gene expression. Our results revealed that pentamidine strongly synergized with rifampicin, linezolid, and tetracycline and moderately synergized with erythromycin, but did not synergize with vancomycin against E. coli, K. pneumoniae, E. cloacae, and A. baumannii. Pentamidine increased the outer membrane permeability but did not demolish the outer and inner membranes, which exclusively permits the passage of hydrophobic, small-molecule antibiotics while hindering the entry of hydrophilic, large-molecule vancomycin. It dissipated the membrane proton motive force and inactivated the efflux pump, allowing the intracellular accumulation of antimicrobials that function as substrates of the efflux pump, such as linezolid. These processes resulted in metabolic perturbation and ROS production which ultimately was able to destroy the bacteria. These mechanisms of action of pentamidine on GNB indicate that it is prone to potentiating hydrophobic, small-molecule antibiotics, such as rifampicin, linezolid, and tetracycline, but not hydrophilic, large-molecule antibiotics like vancomycin against GNB. Collectively, our results highlight the importance of the physicochemical properties of antibiotics and the specific mechanisms of action of pentamidine for the synergy of pentamidine–antibiotic combinations. Pentamidine engages in various pathways in its interactions with GNB, but these mechanisms determine its specific synergistic effects with certain antibiotics against GNB. Pentamidine is a promising adjuvant, and we can optimize drug compatibility by considering its functional mechanisms.