匹维溴铵与苯唑西林联用对表皮葡萄球菌的协同抗菌作用  

Synergistic antibacterial effects of pinaverium bromide and oxacillin against Staphylococcus epidermidis

作　　者：袁乐宏 佘鹏飞 YUAN Lehong;SHE Pengfei(Department of Emergency,Third Xiangya Hospital,Central South University,Changsha 410013;Department of Laboratory Medicine,Third Xiangya Hospital,Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅三医院急诊科,长沙410013 [2]中南大学湘雅三医院检验科,长沙410013

出　　处：《中南大学学报(医学版)》2024年第10期1601-1610,共10页Journal of Central South University :Medical Science

基　　金：湖南省自然科学基金(2021JJ40944)。

摘　　要：目的:表皮葡萄球菌能黏附于医疗器械表面形成高耐药的生物被膜,针对表皮葡萄球菌及其生物被膜的新型抗菌药物研发已成为研究热点。本研究通过药物重新利用,探讨L型钙离子通道阻断剂匹维溴铵(pinaverium bromide,PVB)与苯唑西林(oxacillin,OXA)联合使用对表皮葡萄球菌的抗菌作用。方法:表皮葡萄球菌临床菌株于2022年1至9月收集于中南大学湘雅三医院检验科。通过微量肉汤稀释实验检测PVB和OXA的最低抑菌浓度(minimal inhibitory concentration,MIC)。棋盘稀释实验和时间-杀菌曲线用于检测PVB和OXA联用的协同抑菌指数和协同杀菌效率。耐药筛选实验用于检测PVB抑制OXA耐药性形成的能力。生物被膜清除实验结合激光共聚焦显微镜观察和持留菌计数实验用于检测PVB与OXA联用对表皮葡萄球菌高耐药性生物被膜和持留菌的抗菌作用。通过透射电镜、活性氧(reactive oxygen species,ROS)定量实验和ATP定量实验探讨PVB的作用机制。结果:PVB和OXA对表皮葡萄球菌标准菌株RP62A的MIC均为8μg/mL。棋盘稀释实验显示PVB与OXA联用对表皮葡萄球菌标准菌株RP62A的协同抑菌指数为0.2500,对临床分离菌株的协同抑菌指数为0.1875~0.5000,提示均为协同作用。耐药筛选实验发现PVB不仅不会产生耐药,还能有效抑制OXA产生耐药。1×MIC的PVB和OXA联用时,可使生物被膜的吸光度(A_(570 nm))从(2.36±0.46)减少到(1.12±0.39)(t=3.504,P=0.02)。通过激光共聚焦显微镜观察可知,PVB和OXA联用还可明显破坏生物被膜的结构,使死亡细菌的比例增加。同时,1×MIC的PVB和OXA作用于持留菌4 h,可使持留菌的总量从lg(7.73±0.21)减少到lg(2.79±0.43)(t=4.143,P=0.014)。此外,PVB联用OXA的协同抗生物被膜效果在临床菌株中得到进一步验证。透射电镜观察发现PVB可使细菌结构明显破坏。OXA与PVB联用可显著诱导细菌产生ROS,使ROS相对荧光强度从(30000.00±2000.00)升Objective:Staphylococcus epidermidis(S.epidermidis)adheres to the surface of medical devices,forming highly drug-resistant biofilms,which has made the development of novel antibacterial agents against S.epidermidis and its biofilms a key research focus.By drug repurposing,this study aims to explore the combinational antimicrobial effects between pinaverium bromide(PVB),a L-type calcium channel blocker,and oxacillin(OXA)against S.epidermidis.Methods:Clinical isolates of S.epidermidis were collected from January to September 2022 at the Department of Clinical Laboratory of the Third Xiangya Hospital,Central South University.The minimal inhibitory concentrations(MICs)of PVB and OXA were determined using the broth microdilution method.Checkerboard assays and time-kill curves were performed to assess the fractional inhibitory concentration index and synergistic bactericidal efficiency of the drug combination.Resistance selection assays evaluated PVB’s ability to inhibit the development of OXA resistance.Biofilm eradication assays,combined with confocal laser scanning microscopy(CLSM)and the persister cell quantification,were conducted to evaluate the effect of PVB and OXA on drug-resistant biofilms and persister cells.The mechanisms of PVB action were further investigated using transmission electronic microscopy(TEM),reactive oxygen species(ROS)quantification,and ATP quantification.Results:The MICs of PVB and OXA against the standard strain S.epidermidis RP62A were both 8μg/mL.Checkerboard assays showed that the fractional inhibitory concentration index(FICI)for the combination was 0.2500 for RP62A and ranged from 0.1875 to 0.5000 for clinical isolates,indicating synergistic effects.Resistance selection assays demonstrated that PVB not only failed to induce resistance but also effectively inhibited the development of OXA resistance.The combination of 1×MIC of PVB and OXA reduced biofilm biomass(A_(570 nm))from(2.36±0.46)to(1.12±0.39)(t=3.504,P=0.02).CLSM revealed significant biofilm structural disruption and an

关 键 词：匹维溴铵 苯唑西林 抗生素 协同作用 生物被膜 

分 类 号：R965[医药卫生—药理学]