The Eliminated Effect Of N-acetylcysteine Combined With Vancomycin On MRSA And Its Biofilm

Objective:(1)Through cell proliferation-toxicity test,it is confirmed that acetylcysteine is safe to synovial tissue.(2)In vitro,the biofilm model of MRSA is established as the research object.(3)Acetylcysteine alone or combined with vancomycin at different concentrations is used to verify its ability to scavenge bacteria in biofilm by LB-agar culture plate counting method.(4)Crystal violet staining,scanning electron microscope and laser confocal are used to observe the changes of biofilm area and morphology in vitro after acetylcysteine alone and acetylcysteine with different concentrations combined with vancomycin.Methods:(1)Synovial cell HIG-82 was administered with different concentrations of N-acetylcysteine,and the safe concentration range of N-acetylcysteine was detected by CCK-8 method;(2)Methoxy-resistant Staphylococcus aureus(BAA-1556,ATCC),a standard strain in laboratory,was used as the research object,and the maximum biofilm formation time was determined by crystal violet staining.(3)The experimental groups were divided into control group,vancomycin group,acetylcysteine group,vancomycin + acetylcysteine low-dose group and vancomycin + acetylcysteine high-dose group.The control group was not given any drugs,and the bacteria grew naturally and formed biofilm as the control group.Vancomycin group was treated with 5mg/m L vancomycin 24 hours after bacterial growth.Acetylcysteine group was given 20mg/m L after 24 hours of bacterial growth.Vancomycin + acetylcysteine low dose group was given 5mg/m L vancomycin and5mg/m L acetylcysteine after 24 hours of bacterial growth.Vancomycin + acetylcysteine high dose group was given 5mg/m L vancomycin and 20mg/m L acetylcysteine 24 hours after bacterial growth.The number of residual bacteria in biofilm was measured by LB-agar culture plate counting method.(4)The morphological changes of MRSA bacterial biofilm in different groups were observed under scanning electron microscope and laser confocal microscope.Results:(1)By CCK-8 experiment,when the concentration of N-acetylcysteine was between 0.1m M and 0.8m M,the activity of HIG-82 cells did not change significantly.(2)The morphology of MRSA biofilm was observed by crystal violet staining,and the mature biofilm structure was formed in 48hours;(3)The number of bacteria in biofilm decreased most obviously in the high dose group of vancomycin + acetylcysteine,followed by the low dose group of vancomycin +acetylcysteine.After 72 hours of vancomycin alone,there were still many bacterial residues.(4)Under laser confocal microscope and electron microscope,there wer e a large number of living and dead bacteria in the control group.Although more live bacteria were distributed in acetylcysteine group,the adhesion of dead bacteria and biofilm area decreased significantly.In vancomycin group,a large number of dead bacteria adhered to the carrier surface and formed biofilm,coexisting with some living bacteria;The distribution of living and dead bacteria in vancomycin + acetylcysteine low dose group decreased significantly.In the high dose group of vancomycin +acetylcysteine,the biofilm area was less,and only a small number of live bacteria adhered to the carrier surface,which distributed in a single way.Conclusion:(1)When the concentration of N-acetylcysteine is between 0.1m M and 0.8m M,there is no obvious damage to HIG-82 cells.(2)MRSA bacteria can form mature biofilm structure in 48 hours.(3)The combined application of vancomycin and N-acetylcysteine can effectively remove bacterial biofilm and significantly increase the clearance rate of bacteria in biofilm.