紫外线-核黄素光动力疗法对眼科常见致病细菌的体外杀菌作用

背景 金黄色葡萄球菌、表皮葡萄球菌和铜绿假单胞菌是眼科的常见致病菌,研究最佳杀菌方法对眼科的临床工作具有重要意义. 目的 观察紫外线-核黄素光动力疗法（PDT）对眼科常见致病细菌的体外杀菌作用. 方法 分别取金黄色葡萄球菌、表皮葡萄球菌和铜绿假单胞菌冻存菌各10株接种于血培养皿中,复活2次培养至对数生长期后刮取菌落,置于细菌稀释液中,制备0.5麦氏浓度的细菌悬液原液,原液稀释10倍用于PDT实验.分别用1、2、3 mW/cm2紫外线A（UVA）对不同质量分数核黄素作用后的细菌照射10、20、30 min,测量不同条件下抑菌圈直径的大小,确定最佳抑菌条件.将金黄色葡萄球菌、表皮葡萄球菌和铜绿假单胞菌分别分为正常对照组、单独核黄素作用组、单独紫外线照射组和PDT组,比较不同组间各种细菌菌落数的差异. 结果 当UVA强度为3 mW/cm2、作用30 min、核黄素质量分数为0.1％时,MH固体培养皿上抑菌圈直径接近6 mm,故上述参数为PDT法抑菌的最佳条件.正常对照组、单独核黄素作用组、单独紫外线照射组和PDT组的金黄色葡萄球菌、表皮葡萄球菌和铜绿假单胞菌的菌落数依次减少,差异均有统计学意义（F=110.990、190.841、118.197,均P=0.000）,单独紫外线照射组各种细菌的菌落数均明显少于正常对照组,差异均有统计学意义（t=14.640、7.550、5.320,P＜0.05）,PDT组各种细菌的菌落数亦明显少于正常对照组的菌落数,差异均有统计学意义（t=25.480、14.750、12.920,P＜0.05）.单独紫外线照射组细菌量较正常对照组降低46.4％～50.2％,PDT组的细菌量降低91.6％ ～96.7％,而单独核黄素作用组的细菌菌落数与正常对照组相比差异均无统计学意义（t=0.660、0.470、0.510,P＞0.05）.结论 在紫外线-核黄素PDT灭活微生物的最佳体外实验条件下,PDT法对金黄色葡萄球菌、表皮葡萄球菌和铜绿假单胞菌的杀菌效果最好.

In vitro antimicrobial efficacy of riboflavin-UVA for common bacteria in ophthalmology

Background Staphylococcus aureus,Staphylococcus epidermidis and Pseudomonas aeruginosa are common pathogenic bacteria in ophthalmology.To seek an optimal antimicrobial method is critical for the prevention of infection.Objective This study was to investigate the antimicrobial efficacy of riboflavin-ultraviolet A （UVA） on Staphylococcus aureus,Staphylococcus epidermidis and Pseudomonas aeruginosa.Methods Ten strains of frozen Staphylococcus aureus,Staphylococcus epidermidis and Pseudomonas aeruginosa were incubated to culture dishes and resuscitated for 2 times.The bacteria were put into bacterial dilution to prepare the suspension of 0.5 Mclntosh concentration and then the suspension was diluted 10-folds.UVA was used to irradiate the culture dishes added 0,0.1%,0.5% riboflavin for 10,20,30 minutes with the exposure intensities of 1,2,3 mW/cm2,and the optimal bacteriostatic condition was evaluated by measuring the diameters of inhibition zone.Staphylococcus aureus,Staphylococcus epidermidis and Pseudomonas aeruginosa were divided into normal control group,riboflavin group,UVA group and photodynamic therapy （PDT） group （riboflavin＋UVA）,and colony forming unit was compared among the groups.Results The optional experimental conditions of PDT inactivating microorganisms were determined as UVA wavelength 370 nm,UVA exposure rate 3 mW/cn2,UVA exposure time 30 minutes and riboflavin 0.1%.The colony forming units were decreased in turn in the normal control group,riboflavin group,UVA group and PDT group （F =110.990,190.841,118.197,all at P =0.000）.Compared with the normal control group,the colony forming units were significantly decreased in the UVA group （t =14.640,7.550,5.320,all at P〈0.05）,and the colony forming units were significantly decreased in the PDT group （t =25.480,14.750,12.920,all at P〈0.05）.The colony forming units decreased by 46.4%-50.2% in the UVA group and 91.6%-96.7% in the PDT group.There was no significant difference in colony forming units between the normal control group and the riboflavin group（t =0.660,0.470,0.510,all at P〉0.05）.Conclusions PDT can effectively inactivate common bacteria in ophthalmology.This result suggests that PDT has a potential in the treatment of microbial keratitis.