The Inhibition Effect Of DOXO On Antibacterial Activity And The Synergistic Anti-microbicidal Effect Of AUR And PEITC In Vitro And In Vivo

Objective1.Investigate the effect of doxofylline（DOXO）on the antibacterial activity of Gram-negative bacteria in vitro and in vivo.2.To determine the underlying mechanisms of action of DOXO on the activity of antibacterial drugs.3.To explore whether auranofin（AUR）and phenethyl isothiocyanate（PEITC）have a synergistic antimicrobial effect on S.aureus in vitro and in vivo and the underlying mechanisms.Materials and Methods1.Susceptibility testing,time-kill studies were used to evaluate the effect of DOXO on antimicrobial activity in vitro.2.A Klebsiella pneumoniae-induced murine lung infection model were used to evaluate the effect of DOXO on antimicrobial activity in vivo.3.By combining efflux pump inhibition tests,q RT-PCR assays,using the gene tol C knockout strain and reactive oxygen species（ROS）assays to study the mechanism of DOXO-induced protection.4.Susceptibility testing,checkerboard assays,time-kill studies and an S.aureus-induced subcutaneous abscess mouse model were used to evaluate antimicrobial activity in vitro and in vivo.5.By combining ROS assays and cellular morphology tests to study the mechanism of synergistic antimicrobial activity of AUR-PEITC.Results1.We report the discovery of DOXO-induced high minimum inhibitory concentrations of antibiotics,and then showed that DOXO blocked antimicrobial-mediated killing for Gram-negative pathogens in vitro and in vivo.2.Mechanistically,we found that DOXO positively regulated gene expression of the Acr AB-Tol C efflux pump and attenuated the effect of DOXO on antibacterial activities in Δtol C mutants.3.DOXO protects bacteria from rapid antimicrobial-mediated killing by reducing intracellular ROS levels.4.Through the time-killing kinetic test in vitro,the combinations of AUR and PEITC increased the antibacterial activity against sensitive and drug-resistant S.aureus.5.AUR and PEITC can significantly reduce the number of bacteria colonies under phase callus,accelerate cutaneous wound healing,and promote resistance to infection and healing efficacy compared to AUR monotherapy in the S.aureus skin infection models.6.Mechanistically,we found that the synergistic antimicrobial activity of AUR-PEITC also caused higher ROS generation and damaged bacterial cell structure.Conclusions1.The negative effect of DOXO on antibiotic activity in Gram-negative bacteria suggests DOXO should not be administered alongside antibiotics,as it would prolong the time of therapy,increase the necessary dose,and eventually accelerate the emergence of antibiotic resistance.2.Our study indicates that DOXO protects Gram-negative bacteria from antimicrobial lethality by regulating the Acr AB-Tol C efflux pump in a Tol C-dependent manner and suppressing antibiotic-induced ROS accumulation.3.AUR combined with PEITC showed good antibacterial activity against gram-negative bacteria,and provided a theoretical basis for further preparation of later clinical trials.4.By enhancing ROS production and aggravating cell structure,the AUR and PEITC combination had a synergistic antimicrobial impact on S.aureus pathogens both in vitro and in vivo.5.This work also suggests that AUR combined with PEITC may open a path for the treatment of infections caused by S.aureus strains,especially those that are drug resistant.