碘伏对金黄色葡萄球菌生物膜的抗菌作用

Antibacterial effect of iodophor on Staphylococcus aureus biofilm

目的探讨碘伏对金黄色葡萄球菌生物膜(BBF)的抗菌作用。方法体外培养金黄色葡萄球菌,选取480枚钛合金板,分别在钛板表面建立7,14,21,28 d金黄色葡萄球菌生物膜体外模型,每个时相点120枚。再将每个时相点生物膜按随机数字表法分为无碘伏浸泡组(PBS组)、碘伏浸泡5 min组(5 min组)和碘伏浸泡10 min组(10 min组)。PBS组分别采用异硫氰酸荧光素标记的刀豆蛋白(FITC-ConA)与碘化丙啶(PI)、PI与SYT09染料将金黄色葡萄球菌染色,染色后使用激光共聚焦显微镜和扫描电镜观察细菌生物膜的形态结构,并采用活菌计数法(CFU计数)清点菌落数;5 min组、10 min组采用PI和SYT09染色,染色后使用激光共聚焦显微镜观察碘伏浸泡后的生物膜变化及细菌活力,采用活菌计数法评估碘伏的抗菌效果。结果PBS组采用FITC-ConA和PI染色后,通过激光共聚焦显微镜观察可见,随着培养时间的延长,细菌胞外多聚物逐渐增多,生物膜空间结构逐渐成熟,到21 d时生物膜空间结构变化明显,28 d时成熟;PI和SYT09染色后通过激光共聚焦显微镜观察可见细菌数量增多,外形呈山峰状。扫描电镜观察可见,随着培养时间的延长,细菌胞外多聚物逐渐增多,形成了结构化的微环境并逐渐成熟。5 min组、10 min组中培养7 d[0(0,0)CFU/ml]及14 d[0(0,0)CFU/ml]时细菌被全部杀灭,21 d时5 min组及10 min组抗菌作用均减弱,但10 min组[100(100,125)CFU/ml]较5 min组[300(275,425)CFU/ml]的抗菌效果好(P<0.05),28 d时碘伏浸泡5 min组[500(375,700)CFU/ml]和10 min组[250(175,400)CFU/ml]的抗菌效果差异无统计学意义(P>0.05)。结论生物膜的成熟是细菌和胞外多聚物的整体成熟并形成空间化的微环境,生物膜以21 d为界分成年轻生物膜和成熟生物膜,二者的主要区别在于胞外多聚物及微环境的成熟;对于培养时间≤21 d的细菌生物膜,碘伏浸泡10 min较5 min抗菌效果好,而对培养时间>21 d的细菌生物膜延长碘伏浸泡时间对于抗菌效果没有明显差别。

Objective To investigate the antibacterial effect of iodophor on Staphylococcus aureus biofilm(BBF).Methods Staphylococcus aureus were cultured in vitro and 480 pieces of titanium alloy plates were selected.On the surface of titanium plates,in vitro models of Staphylococcus aureus biofilms were established at days 7,14,21 and 28 respectively with 120 pieces of titanium plates at each time points.The biofilms at each time point were assigned to no iodophor immersion(PBS group),5 g/L iodophor immersion for 5 minutes(5 min group)and 5 g/L iodophor immersion for 10 minutes(10 min group),according to the random number table method.FITCConA,propidium iodide(PI)and SYT09 were used to dye Staphylococcus aureus in PBS group.After dyeing,confocal laser scanning microscopy and scanning electron microscopy were used to observe the morphological structure of bacterial biofilms,and the Colony forming unit(CFU)was counted by the viable count method.In the other two groups,PI and SYT09 were applied to dye Staphylococcus aureus,and then confocal laser scanning microscopy and scanning electron microscopy were used to observe the changes of biofilms and bacterial viability after iodophor immersion.The antibacterial effect of iodophor was evaluated by the viable count method.Results After dyeing Staphylococcus aureus with FITC-ConA and PI in PBS group,confocal laser scanning microscopy showed that the extracellular polymers of the bacteria increased gradually with the extension of culture time.The space structure of biofilm was gradually mature,changed significantly at day 21 and became mature at day 28.After staining Staphylococcus aureus with PI and SYT09 in PBS group,confocal laser scanning microscopy showed that the number of bacteria increased,and had a mountain-like shape.Scanning electron microscopy showed that the number of bacterial extracellular polymers increased gradually with the extension of culture time and a structured microenvironment was formed and gradually matured.In 5 min and 10 min groups,all bacteria were killed at days 7 and 14[0(0,0)CFU/ml],the antibacterial effect was weakened at 21 days,but the antibacterial effect of iodophor immersion in 10 min group[100(100,125)CFU/ml]was better than that in 5min group[300(275,425)CFU/ml](P<0.05).There was no significant difference in iodophor immersion in 5 min group[500(375,700)CFU/ml]and 10 min group[250(175,400)CFU/ml]at 28 days(P>0.05).Conclusions The maturation of biofilm is the overall maturation of bacteria and bacterial extracellular polymers and the formation of a spatialized microenvironment.Bounded by the 21st day,biofilms are divided into young biofilms and mature biofilms.The main difference between them lies in the maturation of extracellular polymers and microenvironment.For the bacterial biofilm with culture time less than 21 days,the antibacterial effect of the iodophor immersion for 10 min is better than that of 5 min.However,for the bacterial biofilm with culture time greater than 21 days,there is no significant difference in the antibacterial effect of the bacterial biofilm of prolonged iodophor immersion time.