| Background:Staphylococcus aureus is a major human and veterinary pathogen,which is very common in various clinical infectious diseases.The most common sites of infection are skin and soft tissue,and serious infections include bacteremia,pneumonia,endocarditis,bone and joint infections,and toxic shock syndrome.The mortality of patients with S.aureus bacteremia in the preantibiotic era exceeded 80%.The introduction of the first β-lactam,penicillin G,dramatically improved prognosis.Subsequently,resistant strains that expressing β-lactamase were recognized.Theβ-lactamase-insensitive penicillin derivatives insensitive to-lactamase named methicillin were developed in response to the emergence and spread of penicillin resistance.Staphylococcus aureus strains acquired SCCmec cassettes by horizontal transfer which encoded a penicillin-binding protein(PBP2a)that could not be inactivated by β-lactams.These are called MRSA strains.Furthermore,the MRSA strains manifest as resistance to virtually all β-lactams with the exception of the latest generation of cephalosporin β-lactams(such as cefloroline).MRSA can also acquire resistance to other alternative antimicrobial agents,further complicating the treatment of infection.This resistance includes vancomycin,linezolid,and daptomycin,which were once considered as a last resort for the treatment of severe MRSA infections.Given the increase of these multiple-resistant MRSA strains and the slowdown of the antimicrobial drug development process,we urgently need new alternative treatments to overcome this fight against bacterial infections.The development cycle of new antibiotics is long and the cost is huge,which cannot keep up with the pace of bacterial drug resistance.Therefore,using approved drugs to restore the sensitivity of drug-resistant bacteria to antibiotics is the most rapid and economical strategy to restrain drug-resistant bacteria.Thousands of years of clinical application highlights the potential use of traditional Chinese medicine(TCM)or its effective components in treating antibiotic resistance associated infections.Many prescriptions of eliminate fever and detoxify have good antibacterial effect in clinical practice.The clinical combination with antibiotics can significantly shorten the course of treatment,reduce the abuse of antibiotics,and avoid the risk of drug-resistant.There are many guidelines and expert consensus on bacterial infectious diseases,with dozens of antibiotics recommended for each version.However,because of the different infected pathogens and the various antibiotics with different pharmacology,most physicians prescribe by experience.Antibacterial traditional Chinese medicine can only stay in the "adjuvant" position,and cannot give full use to its unique advantages,thus not improving the level of prevention and treatment.Objective:Based on previous work,the TRQ was selected as a research subject.Firstly,a synergistic antibacterial compatibility screen was performed.Secondly,efficacy validation was performed in the resistance phenotype,mammalian cells,and mouse skin infection model.And then,the best drugs combined with TRQ were determined.Additionally,we explore the applicable scope of these combinations and the synergistic mechanism combined with antibiotics.It provides a theoretical basis for guiding precise drug administration and new drug development.Methods:This study was conducted mainly from the following four levels.Screening:The checkerboard method with easy operation and high accuracy was used to extensively screen various of common clinical antibiotics for compatibility with TRQ.To determine the optimal compatibility of the TRQ and its active components with antibiotic combination was verified by drawing time-kill curves.Proving:(1)We designed a 30-day resistance induction regimen to explore whether the development of aminoglycoside resistance was affected under the pressure of TRQ(and its active components)alone or in combination with antibiotics.(2)We cultured biofilms,small colony variants(SCV)and persisters,to examine whether the compatibility of TRQ(and its active components)and aminoglycoside is suitable for the clinical environment.Mechanism:(1)Transcriptomic and proteomic studies of methicillin-resistant Staphylococcus aureus(MRSA)were conducted after CDCA treatment.(2)The membrane disturbance of CDCA was observed by three high-resolution microscopes,including of transmission electron microscopy(TEM),structured light illumination microscope(SIM),and Stimulated Emission Depletion microscope(STED).(3)Subsequently,we measured intracellular accumulation of aminoglycoside using fluorescence-labeled aminoglycosides after CDCA treatment and found that internalization of aminoglycoside in S.aureus was enhanced in a dose-dependent manner of CDCA.(4)We further evaluated membrane permeability using 1-N-phenylnapthylamine(NPN)as an indicator.To further examine whether CDCA had an effect on the Δψ of PMF,we monitored cytoplasmic membrane depolarization.We used carbonyl cyanide 3-chlorophenylhydrazone(CCCP)as a protonophore and uncoupling agent that causes PMF collapse and prevents aminoglycoside uptake.(5)The level of superoxide dismutase(SOD)was assessed by xanthine oxidase reaction.And the level of reactive oxygen species(ROS),was assessed with DCFH-DA as a luorescent probe.Potency:(1)We performed synergistic antimicrobial efficacy validation in 24 clinical multiresistant MRSA strains.(2)We observed that CDCA at 40 μg/ml or amikacin alone and the combination of CDCAA did not influence the growth of the cell line HEI-OC1.(3)The efficacy of CDCAA efficacy was then evaluated using a mouse model in vivo using multiple parameters,including histopathology and CFU density in situ.Results:In this study,the combination of antibiotic and non-antibiotic was screened to provide a viable strategy to solve this issue by broaden antimicrobial spectrum.We found that one of the cholic acid derivatives in traditional Chinese medicine(TCM)Tanreqing(TRQ),chenodeoxycholic acid(CDCA),synergizes with amikacin against Staphylococcus aureus in vitro,and this synergistic killing was effective against various MRSA variants,including the small colony variant(SCV),biofilms,and persisters.CDCA and amikacin combination protects a mouse model from S.aureus infections.Mechanistically,CDCA increases the uptake of aminoglycosides in a proton motive force-dependent manner by dissipating the chemical potential and potentiates ROS generation by inhibiting superoxide dismutase activity.This work highlights the potential use of TCM components in the treatment of S.aureus-associated infections and extends the use of aminoglycosides in the elimination of Gram-positive pathogens.Conclusion:Inhibition of MRSA by TRQ combined with aminoglycosides has synergistic antibacterial effects,and effective for the vast majority of Gram-positive bacteria;TRQ combined with carbapenems also has synergistic antibacterial effect against MRSA,however this synergy is specifically targeting MRSA strains.The material basis of these two synergy is UDCA and CDCA.The two synergistic mechanisms provided by bile acids ultimately stem from a perturbation to the bacterial envelope,and the disordered bacterial membrane function at low concentrations that cannot destroy the cell membrane can produce completely different synergistic effects with different classes of antibiotics.In short-term strategies,we offer more compatibility options;in the long-term strategy,membrane-targeting drugs may become a good research direction again.TRQ has several non-negligible advantages over other anti-MRSA drugs.First of all,TRQ is an approved drug,which has good clinical efficacy and can be omitted from the process of research and development.Second,the pharmacological properties of targeting the cell membrane make it more difficult for selective resistance to appear selective antibiotics resistance.Moreover,if bacteria want to avoid becoming a drug target by modifying the cell envelope,strategies to increase the number of division sites and the septum to combat the action of sputum heat can lead to their failure to proliferate properly.TRQ can be synergistic with antibiotics different from other targets(aminoglycosides and β-lactam),and can sensitize to the vast majority of Gram-positive bacteria.Additionally,can more inhibit the development of antibiotic resistance. |
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