微生物靶向光动力疗法联合28 k Hz超声对MRSA生物被膜内细菌的杀伤作用

Combination of 28 k Hz Ultrasound Irradiation and Antimicrobial Photodynamic Therapy to Combat Methicillin-resistant Staphylococcus Aureus Biofilm

目的观察阳离子亚苄基环戊酮类光敏剂(P3)介导的微生物靶向光动力疗法(antimicrobial photodynamic therapy,a PDT)联合28 k Hz超声对耐甲氧西林的金黄色葡萄球菌(methicillin-resistant staphylococcus aureus,MRSA)生物被膜的杀伤作用,以及该联合方法作用对正常真核细胞的影响。方法(1)生物被膜内细菌杀伤实验:采用MRSA临床菌株。实验分为对照组和实验组两个大组。对照组再分别为空白对照组(C组)、单纯照光组(L组)、单纯光敏剂组(PS组)、单纯超声组(US组)四个亚组。实验组再分为超声加光敏剂(US+PS组)、光动力组(a PDT组)、光动力联合超声组(a PDT+US组)三个亚组。各组4个复孔,实验重复3次。激光照射波长532 nm、功率密度40 m W/cm2、时间10 min;超声辐照剂量为频率28 k Hz、功率密度500 m W/cm2、时间10 min。各组处理后采用XTT法检测OD值,以评估各组生物被膜内细菌活力。(2)细胞杀伤实验:实验采用人皮肤成纤维细胞WS-1,分组和处理方法同细菌杀伤实验,各组处理后继续培养24 h,采用CCK-8法检测OD值,以评估各组细胞活力情况。结果(1)光动力组和超声联合光动力组生物被膜内细菌活力分别为55.9%和15.1%,两组比较差异具有统计学意义(P<0.05),且超声联合光动力组的细菌活力抑制率(84.9%)高于单纯超声组与光动力组之和(6.7%+44.1%=50.8%),说明两种方法联合具有协同效应。(2)联合处理后,WS-1细胞活力仅出现轻度降低,保持在86.2%。结论微生物靶向光动力联合28k Hz低强度超声对MRSA生物被膜具有协同杀菌效应,并且该联合方法不会对正常真核细胞造成严重损伤。

Objective To observe the effect of 28 k Hz ultrasound irradiation combined with the antimicrobial photodynamic therapy(a PDT)mediated by a cationic benzylidene cyclopentanone photosensitizer(P3)against methicillin-resistant staphylococcus aureus biofilm and observe its influence on cell viability toward normal eukaryotic cells.Methods(1)Biofilm-killing experiment:A clinical strain of MRSA was used in this experiment.In the experiment,two large groups were established:the control group and experimental group.The control group was further divided into four subgroups:the blank control group(C group),sole light group(L group),sole photosensitizer group(PS group)and ultrasound group(US group).The experimental group was also divided into three subgroups:the ultrasound plus photosensitizer group(US+PS group),photodynamic therapy group(a PDT group)and ultrasound plus photodynamic therapy group(US+a PDT group).Each group had 4 complex wells and the experiment was repeated 3 times.For the laser irradiation,the wavelength was set at 532 nm,the power density at 40 m W/cm~2,and the time for 10 min.The ultrasonic irradiation dose was as follows:frequency 28 k Hz,power density 500 m W/cm~2 and time 10 min.After the treatment,the OD values of each group were detected with XTT method to evaluate the viability of bacteria in biofilm.(2)Cell killing experiment:Human skin fibroblasts WS-1 cells were used in the experiment.The grouping and treating methods used in the biofilm killing experiment were taken in the experiment.After the treatment,the cells were cultured continually for 24 h and the OD value of each group were detected with CCK-8 method to evaluate the cell viability.Result(1)The bacterial viability of biofilms in a PDT group and US+PDT was 55.9%and 15.1%respectively,and the difference was significant(P<0.05).The viability inhibition rate(84.9%)of the US+a PDT group was higher than the sum(6.7%+44.1%=50.8%)of the a PDT group and US group,suggesting the combination of a PDT and ultrasound irradiation had a synergistic effect.(2)WS-1 cell viability was marginally reduced after the combined treatment(86.2%).Conclusions The combination of a PDT and 28 k Hz low intensity ultrasound against MRSA biofilm will bring in a synergistic bactericidal effect,and will not cause serious damage to human dermal fibroblasts.