Benzydamine Hydrochloride Enhances The Antibacterial Activity Of Tetracycline Antibiotics

The discovery and widespread application of antibiotics not only saved countless lives,but also played an important role in the prevention and control of bacterial diseases in livestock and poultry farming.However,with the widespread use and even abuse of antibiotics,the proportion of bacteria that are resistant to antibiotics is increasing,seriously weakening the clinical efficacy of existing antibiotics.The fact that few antibacterial drugs with new scaffolds or targets have been approved in the past two decades aggravates this crisis.Repurposing FDA-approved drugs as potential antibiotic adjuvants offers a cost-effective strategy to mitigate the development of resistance and tackle the increasing infections by multidrugresistant bacteria.Benzydamine hydrochloride,a non-steroidal anti-inflammatory drug that exerts anti-inflammatory and analgesic effects by inhibiting prostaglandin synthesis,has been reported to prevent mucositis and postoperative sore throat.Recently,benzydamine was also found to inhibit osteoclast differentiation and bone resorption.However,the adjuvant potential of benzydamine to existing antibiotics is still not fully understood.Therefore,this study explores whether benzydamine can be used as an antibiotic adjuvant to restore the sensitivity of drug-resistant pathogens to antibiotics and suppresses the development of antibiotic resistance.First,in order to evaluate the drug-drug interactions of benzydamine and antibiotics,this study conducted a checkerboard broth microdilution assay with eight classes of antibiotics and a multidrug-resistant strain of E.coli B2 as the research objects to determine the fraction inhibitory concentration index（FICI）of their combination of antibiotics and benzydamine.Out of these drugs,kanamycin,etc displayed an antagonistic effect with benzydamine,whereas colistin,ciprofloxacin,and tetracycline antibiotics（doxycycline,tigecycline,tetracycline,oxytetracycline,and minocycline）showed synergistic activity with benzydamine.Remarkably,the combination of benzydamine and tetracycline antibiotics possessed the highest synergistic effect（FICI=0.188）.Next,the research tested the activity of benzydamine in combination with tetracycline antibiotics against different types of resistant pathogens to determine the antibacterial spectrum of synergy.Results have shown that the combination has a synergistic effect on both drug-susceptible and drug-resistant bacteria.Importantly,the ues of benzydamine can significantly enhance the activity of doxycycline against a panel of clinically important pathogens,including MRSA,CRE,MCRPE and tet（X）-positive Gramnegative bacteria.Subsequently,two Gram-positive bacteria（S.aureus ATCC 29213,MRSAT144）and two Gram-negative bacteria（E.coli ATCC 25922,E.coli B2）as representative strains were selected for this surdy,the synergistic mechanism of benzydamine on tetracycline antibiotics was further confirmed by measuring proton motive force,intracellular antibiotic accumulation,time-killing curve and transcriptomic analysis.The results indicated that the synergistic activity of the combintion was mainly related to proton motive force（PMF）.Benzydamine can dissipate the membrane potential（ΔΨ）of bacteria,thereby upregulating the transmembrane proton gradient（ΔpH）and promoting the uptake of tetracyclines.Furthermore,benzydamine exacerbated the oxidative stress by triggering the overproduction of ROS and inhibiting GAD system-mediated oxidative defensive.This mode of action explains the efficient bactericidal activity of the doxycycline-benzydamine combination against different metabolic states of bacteria.Finally,the safety and efficacy in vivo of the combination were evaluated by hemolysis analysis,mice toxicity experiments and animal infection models.The study displayed that benzydamine plus doxycycline had no hemolytic activity on mammal’s RBCs and no toxic effects in mice.In addition,the combination therapy effectively improved the survival rate of Galleria mellonella and significantly reduced the bacterial load in the thigh muscle of mice,indicating that benzydamine effectively reversed the resistance of doxycycline in vivo and restore the clinical efficacy of doxycycline against drug-resistant bacteria.In conclusion,this study found that the use of the non-steroidal anti-inflammatory drug benzydamine can restore the sensitivity of drug-resistant pathogens to tetracycline antibiotics.It is a safe,efficacy,and novel tetracyclines adjuvant with a broad-spectrum,which provides a reference for the treatment of infectious by MDR bacteria.