Preliminary Study On In Vitro Antibacterial Properties And Mechanisms And Cytotoxicity Of A New Glycopeptide Compound LYWW01

As one of the last antibiotic against methicillin-resistant Staphylococcus aureus(MRSA),vancomycin has been used more and more frequently in clinics,and the subsequent emergence of vancomycin-resistant bacteria was followed.The concern of increasingly frequent vancomycin resistance has highlighted the need for the new aminoglycosides antibiotics.LYWW01 and LYSC16 are new structural aminoglycosides antibiotics that were developed in this laboratory independently.In this study,the in vitroefficacy and mechanism and toxicity of these three compounds was evaluated.The results will provide valuable date for their further development.In vitro antibacterial properties reaserch,the minimal inhibitory concentration(MIC)and the minimum bacterial concentration(MBC)of drugs were determined for each drug by broth microdilution;the effect of growth phase and stable phase of strains on the MIC and MBC of drugs;the vitro activities of different drugs in standard strains and clinical strains were compared.The results showed that the antibacterial activity of LYWW01 and LYSC16 were superior than vancomycin in vitro,especially in some vancomycin-resistant enterococci(VRE),the antibacterial activityof LYSC16 and LYWW01 were 64 times more than vancomycin.The different physiological states of bacterial had little influence on the MIC.In addition,We selected a bacterial named HCCB 20274 with the largest difference between vancomycin and other vancomycin analogs.The post-antibiotic effect(PAE)of this drugs on HCCB 20274 was determined by using a dilution-removal antibiotic and growth curve.The result showed that LYSC16 had longer PAE for both exponential and stationary bacterial and when in the same high concentration(512 ?g/ml),the PAE of LYSC16 was 40 h longer than vancomycin.In preliminary study of antimicrobialmechanism,we studied the effect of this drugs on death of HCCB 20274,a clinically vancomycin-resistant enterococci(VRE),by using flow cytometry.The result showed that LYSC16 can cause bacterial death in a shorter time,there was a significant bacterial mortality rate(43.5%)at 2 h.Thenwe studied the mechanism of drug-induced bacterial death byinvestigating the drug-induced production of reactive oxygen species(ROS) and the influence on zeta potential.The results of Zeta potential showed that the zeta potential on bacterial surface moved to positive direction in different degrees after stimulation with different concentrations of drugs,which mean that the surface of the bacteria electronegativity was weakened.LYSC16 has a stronger bacterial cell membrane binding capacity;the LYSC16 waekened the zeta potential value of the bacterial surface more than 10 m V,while other drugs only weakened 6?8 m V.The result of ROS showed that the ROS produced by bacterial increased with the time and the concentration of the drug;and at the same concentration of drugs(the fluorescence value is proportional to the amount of ROS produced),the fluorescence intensity of LYSC16 stimulated bacteria reached 6000 at17.5h,and the other three drugs was more than 10000 at this time.In Cytotoxicity studies,this article studied the effects of vancomycin and LYWW01 on proliferation of renal tubular epithelial cells(HK-2)by using CCK-8.and combined the cytotoxic between vancomycin and LYWW01 through flow cytometry and ROS.We founded that LYWW01 has more capacity for cell death and apoptosis,and it can lead cells to produce more ROS,which indicates that with the increases of antibacterial activity of drug,its toxicity to cells also increased.