Synergistic Inhibitory Effect Of Reyanning Mixture Combined With Linezolide On Methicillin-resistant Staphylococcus Aureus

ObjectiveTo observe the effect of Reyanning Mixture(RYN)combined with linezolid(LNZ)on methicillin-resistant Staphylococcus aureus(MRSA)and its drug-resistant biofilm(BF),and to explore its synergistic bacteriostatic mechanisms.MethodsThe minimal inhibitory concentration(MIC)of LNZ and RYN against MRSA was determined by microdilution method.The chessboard method was used to determine the fractional inhibition concentration index(FICI)of MRSA and the MIC ratio.The change of the number of living bacteria was determined with microplate method before and after the formation of MRSA BF.The effects of LNZ and RYN alone or in combination on the number of living and dead bacteria in MRSA BF were observed by confocal laser scanning microscope(CLSM).The morphological changes of MRSA BF after the treatment of LNZ and RYN alone and in combination were observed by scanning electron microscope(SEM).The effects of single and combined use of LNZ and RYN on the permeability of MRSA BF were observed by BF penetration experiments.RT-PCR was used to detect the effects of single and combined use of RYN and LNZ on the expressions of MRSA BF related genes sarA,agrA,agrB,agrC,agrD,atlA and RNAIII.The characteristics of endogenous metabolites of mature MRSA with BF treated with RYN and LNZ alone and in combination were analyzed by ultra-performance liquid chromatography with time of flight mass spectrometry(UPLC-TOF-MS).ResultsThe MIC of LNZ and RYN to MRSA were 4μg/ml and 1/2RYN stock solution respectively.The MIC ratio was 2μg/ml LNZ+1/32 RYN,FICI = 0.625,which showed additive effect.For MRSA before BF formation,the bacteriostatic effect of 2μg/ml LNZ alone on living bacteria was better than that of 1/16 RYN group,and the bacteriostatic effect of 2μg/ml LNZ+1/16 RYN was better than that of 2μg/ml LNZ or 1/16 RYN alone.For MRSA after BF formation,2μg/ml LNZ alone had no inhibition to living bacteria in BF,RYN(1/8RYN,1/16 RYN,1/32RYN)alone or in combination with 2μg/ml LNZ inhibited the living bacteria in BF,and 2μg/ml LNZ+1/16 RYN was the best bacteriostatic concentration ratio,as well the bacteriostatic effect of the combination was better than that of 2μg/ml LNZ or 1/16 RYN alone.For the MRSA after BF formation,there was no difference in fluorescence signal intensity of living and dead bacteria between 2μg/ml LNZ group and control group.The fluorescence signal intensity of living bacteria in 1/16 RYN group was lower than that in 2μg/ml LNZ group and control group,and the fluorescence signal intensity of dead bacteria was higher than both.The fluorescence intensity of live and dead bacteria in 2μg/ml LNZ+1/16 RYN group was lower than that in control group,2μg/ml LNZ group and 1/16 RYN group.The cell morphology of MRSA with BF treated with 2μg/ml LNZ was not different from that of the control group.The MRSA of 1/16 RYN group had no membrane structure coating and biofilm architecture was loose.In 2μg/ml LNZ+1/16 RYN group,MRSA had no membrane structure coating,and the arrangement between cells was loose and varied in size.For MRSA after BF formation,compared with control group,the expressions of seven BF related genes sarA,agrA,agrB,agrC,agrD,atlA and RNAIII in drug intervention group was inhibited,especially in 2μg/ml LNZ group and 2μg/ml LNZ+1/16 RYN group,while in 1/16 RYN group all were inhibited except for agrC.Compared with 2μg/ml LNZ group,1/16 RYN group had a higher agrD inhibitory effect.Compared with the combination group,the inhibitory effect of agrC,agrD,sarA and atlA in 2μg/ml LNZ+1/16 RYN group was higher than that in 2μg/ml LNZ group,and the inhibitory effect of agrC,sarA and atlA was higher than that in 1/16 RYN group.Metabonomic analysis showed that twenty seven metabolites such as 1,3,7-trimethyluric acid,2-methylbutanoyl-coenzyme A and 3-dehydrocarnitine were differential metabolites before and after the formation of BF,reflecting the metabolic spectrum characteristics of BF formation.Twenty eight metabolites such as 2-methylacetoacetyl-coenzyme A,2-succinylbenzoyl-coenzyme A and 3-dehydrocarnitine were differential metabolites of 2μg/ml LNZ before and after intervention of MRSA BF,reflecting the metabolic spectrum characteristics of the effect of 2μg/ml LNZ on MRSA BF.Twenty five metabolites such as 3-dehydrocarnitine,ADP-D-ribose,bis(3’,5’)-cyclic diguanylic acid and so on were differential metabolites of 1/16 RYN before and after intervention of MRSA BF,reflecting the metabolite spectrum characteristics of the effect of 1/16 RYN on MRSA BF.Twenty one metabolites such as 1-O-caffeoylglucose,3-(methylthio)propionic acid and 3-dehydrocarnitine and so on were differential metabolites before and after the intervention of MRSA BF with 1/32RYN+2μg/mL LNZ,which reflects the metabolic spectrum characteristics of the effect of 1/32RYN+2μg/mL LNZ on MRSA BF.Thirty two metabolites such as 1-O-caffeoylglucose,2-methylbutanoyl-coenzyme A and 3-dehydrocarnitine were differential metabolites before and after the intervention of MRSA BF with 16 RYN+ 2μg/mL LNZ,which reflected the metabolic spectrum characteristics of the effect of 16 RYN+ 2μg/mL LNZ on MRSA BF.Thirty metabolites such as 1-O-caffeoylglucose,2-methylacetoacetyl-coenzyme A and 3-(methylthio)propionic acid,are the differential metabolites before and after the intervention of MRSA BF with 1/8 RYN+2μg/mL LNZ,which reflects the metabolite spectrum characteristics of the effect of 1/8 RYN+2μg/mL LNZ on MRSA BF.Through comparison of the differential metabolites of RYN and LNZ alone and in combination groups,it was found that 10 metabolites such as 3-dehydrocarnitin,ADP-D-ribose and homocitrulline were common differential metabolites.Of those metablolites,3-dehydrocarnitine,homocitrulline,kynurenine,L-leucine and L-tyrosine were identified as metabolic markers.Through the comparison of differential metabolites in different RYN dose groups,it was found that 12 metabolites such as 1-O-caffeoylglucose,3-dehydrocarnitine and glycerol were common differential metabolites among which homocitrulline,sn-glycerol-3-phosphate and tyrosine methyl ester were dose-dependent metabolic markers.Conclusion1.RYN can increase the sensitivity of MRSA to LNZ,reduce the MIC of LNZ.RYN has a damaging inhibitory effect on MRSA BF,which could reduce the resistance protection of MRSA due to BF,help LNZ to break through BF and be exposed to dormant bacteria with deep metabolism,and promote the removal of MRSA by antibiotics.2.Enhancement of the inhibition of sarA and atlA,LNZ combined with RYN could inhibit the production of BF,limit the adhesion of MRSA,and reduce the release of extracellular DNA by reducing bacterial autolysis to achieve a synergistic bacteriostatic effect.3.3-dehydrocarnitine,homocitrulline,kynurenine,L-leucine and L-tyrosine were metabolic markers of LNZ combined with RYN taking synergistic inhibitory effect on MRSA and its BF.Homocitrulline,sn-glycerol-3-phosphate and tyrosine methyl ester were metabolic markers reflecting the efficacy of RYN in a dose-dependent manner.4.This study not only provides a theoretical basis for the clinical treatment of MRSA with RYN combined with LNZ but also lays a foundation for the treatment of traditional Chinese medicine combined with antibiotics against refractory drug-resistant bacterial infections.