医用臭氧气浴干预对烧伤常见致病菌体外杀菌作用的实验研究

Experimental study of bactericidal in vitro of medical ozone gas bath intervention for burns pathogenic bacteria

目的应用医用臭氧气浴技术,行体外载体定量细菌杀菌实验,观察其杀菌效果,为临床治疗感染创面提供实验基础。方法受试菌株包括3株标准菌株和武汉市第三医院烧伤研究所烧伤患者创面分泌物中培养分离的耐药菌株27株。3株标准菌株为ATCC25923金黄色葡萄球菌,ATCC29592大肠杆菌,ATCC27853铜绿假单胞菌;烧伤患者创面分泌物中培养分离的耐药菌株:耐甲氧西林金黄色葡萄球菌10株、铜绿假单胞菌7株、大肠杆菌5株及白色念珠菌5株。制备各种菌株的菌悬液,比浊后浓度为0.5×108~1.5×108cfu/m L,均取0.1 m L涂布于无菌平皿中央,将涂有受试菌悬液的平皿按照干预方法的不同,随机分为两组,即臭氧气浴实验组和微波照射对照组。将臭氧气浴实验组染菌平皿放入密闭无菌玻璃罐内,分别依次放入罐内2个相同染菌平皿并揭开皿盖,连接臭氧发生器装置,并将通气管插入密闭无菌玻璃罐内,保持罐内臭氧浓度为50μg/m L,开启臭氧气机开始计时,分别干预10、30、60 min。同时,微波照射对照组分别依次将2个相同染菌平皿放入超净工作台上,采用低强度微波距染菌平皿3 cm垂直照射做对照,分别照射10、30、60 min。两组实验均重复3次。实验毕,两组每个时相点各取出2个平皿加入0.9 m L 0.9%氯化钠溶液,吹打稀释后,取1 m L倾注琼脂于平皿上,盖上皿盖待含菌培养基凝固后,置37℃恒温培养箱内培养24 h(白色念珠菌培养72 h),采用涂抹平板计数法作细菌菌落计数,计算并采用t检验比较两组平皿不同时相点的杀菌率。结果臭氧气浴实验组在通臭氧气浴10 min时,受试的3株标准菌株及临床烧伤患者创面分离的耐药菌株中的耐甲氧西林金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌的平均杀菌率为(68.51±10.48)%,与微波照射对照组微波照射10 min时同等菌株的平均杀菌率(50.65±13.28)%比较,差异有统计学意义(t=2.586,P<0.05)。臭氧气浴实验组30、60 min时细菌平均杀菌率均为100.00%,微波照射对照组微波照射30、60 min的平均杀菌率为(84.25±6.06)%、(90.50±4.55)%,两组比较差异有统计学意(t=6.364、5.362,P值均小于0.05)。对白色念珠菌,臭氧气浴实验组与微波照射对照组在作用10 min时细菌杀菌率均小于10.00%,作用30、60 min时两组杀菌率均为99.99%以上,两组比较差异无统计学意义(P>0.05)。结论医用臭氧气浴干预对烧伤常见致病菌显示了强大的体外杀菌作用,为控制烧伤创面感染提供一种新的治疗途径。

Objective Carrier quantitative bacteria sterilization experiments were performed in vitro by using the medical ozone air bath technology,to provide experimental basis for the clinical treatment of wound infection. Methods The tested strains included three standard strains,ATCC25923 Staphylococcus aureus,ATCC29592 Escherichia coli, ATCC27853 Pseudomonas aeruginosa, and twenty-seven strains isolated from burn wound secretions of inpatients in Institute of Burns,Wuhan Third Hospital,including10 strains of staphylococcus aureus,7 strains of pseudomonas aeruginosa,5 strains of escherichia coli and5 strains of candida albicans. The strains were divided into two groups according to the difference in sterilization methods : ozone air bath group and microwave irradiation control group. Both group were observed in 3 time point: 10,30,and 60 min after intervention. The prepared bacterial suspensions 0. 1 m L was taken,whose ratio turbidity concentration were 0. 5 × 108-1. 5 × 108 cfu / m L,and then coated it on the center of sterile petri dishs. The ozone generator device was connected and snorkel inserted into a airtight sterile glass jar. Two uncovered petri dishs with bacterial were put in the jar each time,then the ozone generator device was turned on with time start,and the ozone concentration in jars was kept 50 μg / m L for10 min,30 min,60 min,respectively. Meanwhile,two control petri dishs with bacterial were put onto clean bench and given low intensity microwave irradiation from a distance of 3 cm for 10 min,30 min,60 min,respectively. Then two dishes of every group in each time point were taken out and 0. 9 m L 0. 9% sodium chloride was added into dishes for pipetting dilution. After dilution 1 m L bacterium solution was poured on a agar plate. Covered the dishes,after bacterial culture medium solidified,put them into incubators for 24 h( candida albicans for 72 h). Bacterial colony were counted by using plate smearing counting method. The sterilizing rate of the two groups in different time and of different strains was compared. The experiment was repeated 3 times and calculated the average rate. Differrence between two groups were analyzed by LSD-t test. Results For three standard strains and strains isolated from burn wound including staphylococcus aureus,escherichia coli and pseudomonas aeruginosa,the average sterilizing rate of ozone air bath group after treatment for 10 min was( 68. 51 ± 10. 48) %. Compared with the sterilizing rate of microwave irradiation control group( 50. 65 ± 13. 28) %,the difference was significant( t = 2. 586,P < 0. 05). The average sterilizing rates of ozone air bath group to strains mentioned above after treatment for 30 and 60 min were both 100. 00%,while that of microwave irradiation control group after treatment for 30 and 60 min were( 84. 25 ± 6. 06) % and( 90. 50 ± 4. 55) %,respectively. The difference was both statistically significant( t = 6. 364,5. 362,both P values were less than 0. 05). For Candida albicans,the sterilizing rates of the two groups after treatment for 10 min were both less than 10. 00%,while it was over 99. 99% after treatment for 30 and 60 min in two groups. The difference was not significant( P > 0. 05). Concusion Medical ozone air bath intervention for burn pathogens shows strong bactericidal action in vitro,and provides a new treatment approach for burn wound infection.