Combination of Antibiotic, Efflux Pump Inhibitor and Autolysis Inhibitor Protects Mice from S. aureus Peritonitis by Inhibiting α-hemolysin-induced MAPKs/NF-κB/NLRP3 Activation

Abstract

Background: The infection of staphylococcus aureus (S. aureus) is difficult to treat, our aim was to investigate the antibacterial abilities of a combination of antibiotic cloxacillin, efflux pump inhibitor thioridazine and autolysis inhibitor tetracycline and its anti-inflammation properties through inhibiting α-hemolysin induced MAPKs/NF-κB/NLRP3 activation in vitro and in vivo. Methods: The antibacterial susceptibility test and three-dimensional checkerboard method were utilized to investigate the synergistic antibacterial activity of the three-drug combination cloxacillin/thioridazine/tetracycline, α-hemolysin test and scanning electron microscope were used to assay the inhibition effects of the combination on the secretion of α-hemolysin and membrane-derived vesicles production from S. aureus, Western blot and pharmacological inhibition assays were performed to test α-hemolysin -induced MAPKs/NF-κB/NLRP3 activation in macrophage in vitro. Results: In vitro, the three-drug combination showed synergistic antistaphylococcal activity; the subinhibitory concentration of the combination significantly inhibited the secretion of α-hemolysin related to the number of vesicles produced by S. aureus and significantly inhibited the expression of MAPKs/NF-κB/NLRP3 proteins in the macrophages induced by S. aureus α-hemolysin. In vivo, the drug combination significantly reduced bacterial colony-forming unit counts of the viscera in mouse peritonitis model of S. aureus infection, the combination therapy reduced the primary inflammatory pathology and the release of bacterial-stimulated cytokines such as IL-1β and TNF-α, and inhibited the expression of MAPKs/NF-κB/NLRP3 proteins in peritoneal macrophage. Conclusions: Combination of antibiotic, efflux pump inhibitor and autolysis inhibitor owns good antistaphylococcal activity and significantly inhibit staphylococcal α-hemolysin and its inflammation, thus, the combination is a novel strategy of antibacterial and anti-inflammation.