低浓度消毒因子对铜绿假单胞菌生物膜的清除效果研究

Removal effect of disinfection factors in low concentration on Pseudomonas aeruginosa biofilm

本研究使用生物膜发生器法建立体外铜绿假单胞菌生物膜实验模型,评价2种流经杀菌树脂的水对铜绿假单胞菌生物膜清除效果,探讨低浓度消毒因子持续处理对口腔综合治疗台水路消毒的有效性。实验组选择消毒因子浓度为1~2 mg/L的聚碘树脂(iodinated resin,IR)滤过水、溴代聚苯乙烯海因树脂(简称:溴代海因树脂,bromined hydantoin resin,BHR)滤过水,对照组选择无菌蒸馏水,使用浸泡法持续处理生物膜片,处理时间为3、7、10、20、40 d。应用生物膜菌落总数计数法(total viable count,TVC)及激光共聚焦显微镜观察法(confocal laser scanning microscopy,CLSM)对生物膜的清除效果进行评价。结果显示,(1)TVC:IR组处理3、7、10、20 d后及BHR组处理3、7、10 d后,细菌清除率均低于99.9%,判定为消毒不合格;IR组处理40 d后及BHR组处理20、40 d后,细菌清除率分别达99.95%、99.99%、100.00%,消毒合格。(2)CLSM观察:处理前铜绿假单胞菌生物膜片呈片状、团块状分布,生物膜细菌覆盖率为19.24%±1.97%,活细菌比例为93.91%±1.39%,生物膜基质覆盖率为17.69%±1.11%。处理20 d后,IR组可见生物膜减少,但仍有基质残留,生物膜细菌覆盖率为6.77%±1.61%,活细菌比例为54.85%±5.65%,生物膜基质覆盖率为2.41%±0.85%,与处理前及对照组比较,差异均有统计学意义(F=359.996,P<0.001);BHR组未见生物膜样结构。处理40 d后,IR组仍存有少量生物膜基质残留,生物膜基质覆盖率为0.67%±0.47%,与处理前及对照组相比,差异有统计学意义(F=1 021.373,P<0.001),未见生物膜细菌覆盖;BHR组未见生物膜样结构。综上,BHR滤过水持续浸泡法在清除生物膜活、死细菌及生物膜基质方面更具优势,含相应低浓度消毒因子的诊疗用水在口腔综合治疗台水路系统生物膜控制领域可发挥重要作用。

Experimental model of Pseudomonas aeruginosa biofilm was established in vitro by using biofilm reactor.The aim of this study was evaluating the removal effect of two kinds of water flowing through bactericide resin on Pseudomonas aeruginosa biofilm,and exploring the effectiveness of continuous treatment with low concentration disinfection factor on dental unit waterlines.The experimental group selected 1-2 mg/L iodinated resin(IR)filtered water and bromined hydantoin resin(BHR)filtered water with the control group selecting the sterile distilled water.Biofilms were treated by using the immersion method for 3,7,10,20,and 40 days.Total viable count(TVC)and laser confocal microscopy method(CLSM)were selected to evaluate the biofilm removal effect.The result of TVC showed that in group IR,the bacterial clearance after the treatment of 3,7,10,and 20 days was lower than 99.9%and unqualified.The bacterial clearance after the treatment of 40 days was 99.9%,which is qualified.In group BHR,it was lower than 99.9%and unqualified after the treatment of 3,7,and 10 days.It was and 99.99%,100.00%after the treatment of 20,40 days,respectively.The result of CLSM showed that before treatment,Pseudomonas aeruginosa biofilm showed a sheet and mass distribution.The bacterial coverage was 19.24%±1.97%.The proportion of viable bacteria was 93.91%±1.39%,and the biofilm matrix coverage was 17.69%±1.11%.After 20 days of treatment,the biofilm was decreased in the IR group,with the biofilm bacterial coverage reducing to 6.77%±1.61%,the proportion of live bacteria reducing to 54.85%±5.65%,and the biofilm matrix coverage reducing to 2.41%±0.85%.There was significant difference from the pre-treatment and the control(F=359.996,P<0.001).No biofilm-like structure was found in the BHR group.After 40 days of treatment,there was still a small amount of biofilm matrix residue in the IR group,with no bacterial coverage observed.The biofilm matrix coverage was 0.67%±0.47%(F=1021.373,P<0.001).No biofilm-like structure was found in the BHR group.In conclusion,the continuous application of BHR filter water has more advantages in killing microorganisms in biofilms,removing live and dead bacteria and biofilm matrix in biofilms.Treatment water containing corresponding low concentration disinfection factors can play an important role in the field of biofilm control in dental unit waterlines.