| Objectives:With the increase of drug resistance of Staphylococcus aureus(S.aureus),the combination therapy may include strains that have developed drug resistance.At present,the research on linezolid combined with fosfomycin is mainly focused on the two drug sensitive strains,and the research on related drug-resistant strains is less.In this study,drug-resistant strains were induced in vitro,and the main purpose of this study is to assess the in vitro/vivo activities of linezolid plus fosfomycin against S.aureus isolates with varying susceptibility to the study drugs,and to provide some basic data for the rational use of drug combination.Methods:In this study,S.aureus ATCC29213 was used as the experimental strain.The increasing concentration stepwise method was used to induce S.aureus resistant strains.The homology between strains was analyzed by pulsed field gel electrophoresis(PFGE)and multilocus sequence typing(MLST).In vitro experiments,on the one hand,the time bactericidal curve was used to explore the bactericidal activity of drugs against strains within 24h,and to determine the type of interaction between drugs.On the other hand,PAE was used to study the effect of drug combination before and after drug resistance.S.aureus with middle resistance to linezolid or fosfomycin was selected as the representative.The transmission electron microscopy(TEM)was employed to observe the cell morphology of bacteria treated with drug,and the changes of cell wall thickness were recorded.The virulence of different drug-resistant strains and the antibacterial activity of linezolid combined with fosfomycin against different drug-resistant staphylococcus aureus were studied by establishing the Galleria mellonella infection model.Results:PFGE results showed that the similarity between the strains before and after induction was 90%-100%,MLST results showed that the strains before and after induction were ST5939,which indicated that the homology between before and after induction strains was good.The results of time kill curve and PAE showed that the drug combination showed good synergistic or additive activity against linezolid resistant strains and S.aureus ATCC29213,but showed indifferent effect for fosfomycin-resistant strain.Meanwhile,in the time kill curve,we also observed that the growth of the strain was obvious when the single drug of fosfomycin was applied to the resistant strains of linezolid.TEM results showed that the cell thickness of fosfomycin resistant strain was similar to that of the original strain,about35nm.The cell wall of linezolid resistant strain was significantly thicker than that of the original strain,about56nm.For linezolid resistant strains,fosfomycin alone or in combination can effectively reduce bacterial lysis and cell wall thickness.For fosfomycin resistant strains,linezolid monotherapy and combination had no significant effect on the morphology of fosfomycin resistant strains,and could increase the cell wall thickness of the strains.Different concentrations of bacteria were used to infect the larva of the Galleria mellonella.It was found that the concentration of bacteria causing 80%mortality of S.aureus ATCC29213 was 10~8CFU/ml,and the concentration of bacteria causing 80%mortality of drug-resistant strains was 10~7CFU/ml.In the Galleria mellonella infection model,the survival rate of the antibiotic combined was improved compared with that of the single drug.There was a good correlation between in vivo efficacy and in vitro susceptibility.Conclusion:The type of interaction expressed in the test combination was highly dependent on fosfomycin resistance.Therefore,if the bacterial strains are ignored,it may be wrong to directly define the interaction type of two drug combination. |
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