Study On Drug Resistance Of Staphylococcus Aureus And The Antibacterial Effect Of Daphnetin On MRSA

Objective:1.To understand the distribution and drug resistance of Staphylococcus aureus(SA)in the burn patients in the First Affiliated Hospital of Nanchang university,this aim is to provide theoretical basis for effective control and rational antibacterial drugs usage.2.To investigate the antibacterial effect of Daphnetin on methicillin-resistant Staphylococcus aureus(MRSA),this aim is to provide new therapeutic ideas for effective prevention and control of MRSA infection and reduction of its drug resistance.Methods:1.A retrospective survey was conducted to explore the distribution characteristics of Staphylococcus aureus and the variability of SA drug resistance of patients in the burn unit of the First Affiliated Hospital of Nanchang University from January 1,2017 to December 31,2021.2.The K-B disk diffusion method was used to analyze MRSA drug-resistance to12 commonly antibiotics by adopting K-B method;the micro-broth dilution method was adopted to determine the Minimum Inhibitory Concentration(MIC)of Daphnetin and 8 antibiotics respectively,and to explore the anti-MRSA effect of Daphnetin and the degree of MRSA resistance.3.A checkerboard method was used to determine whether the combination of Daphnetin and five drug-resistant antibiotics had a synergistic antibacterial effect in vitro,and the inhibition effect of Daphnetin against MRSA in vitro was assessed by combining with the method of 24-hour growth curve;a method for constructing a infection model of Galleria mellonella was used to assess the antibacterial effect of Daphnetin on MRSA in vivo by the method of 72-hour survival curve after infection of Galleria mellonella.4.The effect of Daphnetin on the formation of MRSA biofilm was determined semi-quantitatively using crystalline violet staining;the effect of Daphnetin on MRSA biofilm was observed in three dimensions using confocal laser scanning microscopy;and the effect of Daphnetin on MRSA bacterial morphology and biofilm were observed directly using scanning electron microscopy.Results:1.A total of 3810 strains of pathogenic bacteria were isolated from the burn unit in the past 5 years,of which 437 strains were SA(11.5%),and the five-year average detection rates of MRSA were 68.4%,59.2%,55.0%,57.7% and 39.4% respectively.The top three sources of SA in the five years were traumatic secretions(80.7%),blood(6.8%)and sputum(5.4%).The detection rate of MRSA in the burn ICU was the highest(91.5%),followed by the general burn ward(65.5% in the third burn ward,and 42.7% in the fourth burn ward)and the chronic wound repair ward(42.7%),and lowest in the fifth burn ward(22.9%).The five-year average resistance rates of SA and MRSA to β-lactams were over 54.4% and 80.0%,respectively.The resistance rates of SA to Quinolones,Aminoglycosides,Macrolides and Tetracyclines antibiotic ranged from 44.1% to 60.0%,while that of MRSA to these antibiotics were more than70.0%.The resistance rates of SA and MRSA to Rifampicin were 35.1% and 61.4%,respectively.And the resistance rates to Nitrofurantoin,Quinupristin-dalfopristin and Trimethoprim/sulfamethoxazole were all less than 10.0%.SA and MRSA were sensitive to Tigecycline,Linezolid,Daptomycin and Vancomycin.2.The five strains of MRSA isolated from the burn unit were resistant to at least7 of the 12 common antibiotics,with a high level of resistance to several antibiotics such as β-lactams,Lincosamides,Quinolones,Aminoglycosides,Tetracyclines and Macrolides.The results of MIC value determined by the micro broth dilution method showed that the MIC values of Daphnetin to 6 strains were 128μg/ml,the MIC values of all six antibiotics were much higher than that of CLSI-22 drug sensitivity standard,among which the MIC values of Erythromycin were as high as 2048μg/ml,and the six MRSA strains were all sensitive to both Linezolid and Vancomycin.3.Daphnetin had a combined inhibitory effect with Levofloxacin,Gentamicin and Clindamycin,the measured FICI values were less than or equal to 1.The FICI value of Daphnetin and Levofloxacin against USA300 strain was 0.375,which has a synergistic antibacterial effect,while Daphnetin combined with Oxacillin and Erythromycin was showed antagonistic effect.The results of 24-hour growth curve showed that Daphnetin at MIC concentration completely inhibited the growth of USA300 strain,and the growth rate of 1/2MIC Daphnetin on USA300 strain was significantly slower than that of the positive control group.The other sub-inhibitory concentrations of Daphnetin had little effect on the growth activity of USA300 strain.in the infection model of Galleria mellonella,Daphnetin at MIC concentration decreased the virulence of MRSA and increased the 72-hour survival rate of the larvae.4.Daphnetin could slightly promoted the formation of MRSA biofilm in a rang of sub-inhibitory concentration.And the substrate distribution of MRSA biofilm treated with Daphnetin was dense under the confocal laser scanning microscope,the distribution of MRSA biofilm base after Daphnetin treatment was dense,the volume and thickness of biofilm increased.Under the scanning electron microscope,it was directly observed that the adhesion between cells was enhanced,the cell morphology was changed,and the cell wall was obviously wrinkled and damaged after Daphnetin treatment.Conclusion:1.The drug resistance rate of SA and MRSA in our burn unit from 2017 to 2021 showed a decreasing trend year by year,but the resistance rate was still significantly higher than the national average level,and the overall drug resistance situation in the burn department was more severe.2.Daphnetin can exert stable anti-MRSA activity in vitro and in vivo,at the same time,it can improve the sensitivity of MRSA to drug-resistant antibiotics and play a role in reducing the resistance of MRSA.3.Daphnetin can slightly promote the formation of MRSA biofilm and it can also play an anti-MRSA role by destroying the bacterial cell wall.