Mechanism Of New NorA Efflux Pump Inhibitors Combined With Ofloxacin Against MRSA

Objective Carbonyl cyanide m-chlorophenylhydrazone(CCCP),an efflux pump inhibitor,is often combined with antibiotics to improve susceptibility against Gram-negative bacteria.However,it is highly toxic and has poor antibacterial activity when used alone.We structurally modified and optimized CCCP in order to obtain CCCP derivatives with better antibacterial activity and lower toxicity.In addition,we explored its mechanism of against methicillin-resistant Staphylococcus aureus(MRSA)when used alone or in combination,and to elucidate the interaction between active efflux and biofilm and quorum sensing(QS).In order to provide new ideas for clinical resistance to MRSA infections.Methods 1.Screening for CCCPs those have the best activity and lowest toxicity:Seventeen CCCPs synthesized by chemical modification,determined their MIC against Gram-positive bacteria,Gram-negative bacteria and Candida albicans;determined their cytotoxicity against normal human liver cell line L02 by cytotoxicity experiments,and finally screened out the compound 2e with the best activity and lowest toxicity.2.To preliminarily determine the substrate of compound 2e:We preliminarily explored the substrate of 2e by combined antibacterial assay and molecular docking,detected the expression of norA gene by qRT-PCR and explored the related mechanism of action.3.Screening for stains those have bettest ability of biofilm formation:From 16 clinically isolated MRSA strains,the film-forming ability of MRSA strains was measured by the crystal violet assay.4.Determination of compound anti-MRSA biofilm activity in vitro:The inhibitory effect of compound 2e on biofilm was detected by minimum biofilm eradication concentration(MBEC),and the changes of bacteria were observed by electron microscopy.5.Inhibition rate of hemolysin activity:The inhibitory ability of 2e on MRSA virulence factor(a-hemolysin)was observed by hemolysis assay.6.Impacts of compound 2e alone or in combination on QS-associated genes:MRSA2858 was cultured with different concentrations of 2e and in combination for 24h,then the bacteria were collected.RT-PCR was used to detect the gene expression of agrA,icaA,sarA and hla.7.In vivo antibacterial effect of compound 2e:We established a mouse model of peritonitis caused by MRSA and injected with 2e,ofloxacin,and their combination subcutaneously 2h later,and the mice sacrificed 24h later for in vivo bacterial load and hematological tests to observe the pathological changes of the kidneys and evaluate the antibacterial effect of 2e in vivo.Results 1.Screening for CCCPs those have the best activity and lowest toxicity:Seventeen CCCP derivatives showed good narrow-spectrum antibacterial activity alone,and they only showed good inhibitory concentrations against Gram-positive bacteria.Among them,2e had the most significant anti-MRSA effect,with an MIC of 1.56 μmol/L.And 2e had the least toxic and had little effect on normal human liver cell line LO2.2.To preliminarily determine the substrate of compound 2e:Compound 2e had the best synergistic effect with ofloxacin,a known substrate of the NorA pump,with a FICI of 0.28;molecular docking showed that it bound to NorA protein better than CCCP,and it was preliminarily determined that 2e acted on NorA protein;2e could significantly inhibit the norA gene,and it was speculated that 2e was a NorA protein inhibitor.3.Screening for MRSA stains those have bettest ability of biofilm formation:The MRSA2858 strain was selected for its strongest biofilm-forming ability by crystal violet assay.4.Determination of compound anti-MRSA biofilm activity in vitro:2e could erase mature biofilm with MBEC of 0.78-1.56 μmol/L,which was superior to erythromycin(resistant to MRSA2858),ofloxacin and vancomycin.Electron microscopy showed that 2e inhibited and removed the biofilm,induced the formation of membrane pores in the outer membrane of MRSA,and altered outer membrane permeability caused bacterial death.5.Inhibition rate of hemolysin:The inhibition rate of a-hemolysin was 99.15%when 2e was combined with ofloxacin,which was superior to 93.75%when 2e was used alone.6.Compound 2e alone or in combination reduced the expression of QS-related genes agrA,icaA,sarA and toxin gene hla:RT-PCR results showed that 2e could significantly inhibit the expression of agrA,sarA,icaA and hla(P<0.01)alone or in combination.7.In vivo antibacterial effect of compound 2e:In vivo experiments showed that 2e combined with ofloxacin could significantly reduce the bacterial load in the blood and kidney of mice,control the changes of peripheral blood caused by infection,reduce the inflammatory infiltration of kidney,the histomorphology of kidney tended to be normal.We reflected in the animal experiments as mice in the combination group were significantly protected compared with the two separate treatment groups.Conclusions The new NorA protein inhibitor 2e combined with ofloxacin has a better antibacterial effect in vitro and in vivo,by inhibiting the expression of efflux genes,reducing the efflux of NorA pump,inhibiting the excretion of microbial metabolites,toxic substances and antibacterial drugs,thereby interfering with the population effect of bacteria,down-regulating the expression of related genes of QS system,and inhibiting the formation of biofilm and toxin.This study provides a theoretical basis for the combination of NorA efflux pump inhibitors and antibiotics,and provides a new strategy and means for clinical anti-MRSA infection treatment.Figure[15]table[7]reference[67]...