臭氧水对骨科感染性创面常见耐药菌灭菌作用的初步实验研究
发布时间：2018-08-21 11:53
【摘要】：目的：观察臭氧水对骨科感染性创面常见的四种耐药菌，即耐甲氧西林金黄色葡萄球菌、产β-内酰胺酶(ESBLS)大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌的体外灭菌效果，为臭氧水治疗临床骨科创面相关耐药菌感染提供初步的理论依据。 方法： 实验一：用臭氧发生器制取三种浓度为2.5ug/ml、5ug/ml和10ug/ml臭氧水（温度20±1℃）各六次，立即用碘量法检测其浓度；并检测10ug/ml臭氧水分别在0、1、2、4、8、16、32、64和128min时浓度值； 实验二：用臭氧发生器制取浓度分别为2.5ug/ml、5ug/ml、10ug/ml臭氧水后，取4.5ml臭氧水六次对0.5ml耐甲氧西林金黄色葡萄球菌菌悬液进行体外定量灭菌，分别作用0、0.5、1、2、4、8及16min时后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和剂中，振荡5min后，取10ul中和后的混合液接种于普通营养琼脂培养基上，在温室培养箱中培养24h，进行菌落计数，并计算灭菌率；用10ug/ml臭氧水对耐甲氧西林金黄色葡萄球菌体外灭菌分别作用0、2、4、8和16min后，加入中和剂，经3200r/min离心15min，弃上清液，再加入磷酸盐缓冲液1ml混悬5min，取等量混悬液和3%磷钨酸染色2min后，滴于铜网上，待干燥后，在透射电子显微镜下观察菌体超显微结构变化情况； 实验三：用臭氧发生器制取浓度分别为2.5ug/ml、5ug/ml和10ug/ml臭氧水，取4.5ml臭氧水分别六次对0.5ml产β-内酰胺酶大肠埃希菌菌悬液进行体外定量灭菌，分别作用0、0.5、1、2、4、8及16min时后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和剂中，振荡5min后，取10ul中和后的混合液接种于普通营养琼脂培养基上，在温室培养箱中培养24h，进行菌落计数，并计算灭菌率；用10ug/ml臭氧水对产β-ESBLS大肠埃希菌体外灭菌分别作用0、2、4、8和16min后，加入中和剂，经3200r/min离心15min，弃上清液，再加入磷酸盐缓冲液1ml混悬5min，取等量混悬液和3%磷钨酸染色2min后，滴于铜网上，待干燥后，在透射电子显微镜下观察菌体超显微结构变化情况； 实验四：用臭氧发生器制取浓度分别为2.5ug/ml、5ug/ml、10ug/ml臭氧水，取4.5ml臭氧水分别六次对0.5ml耐碳青霉烯铜绿假单胞菌菌悬液进行体外定量灭菌，分别作用0、0.5、1、2、4、8及16min时后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和剂中，振荡5min后，取10ul中和后的混合液接种于普通营养琼脂培养基上，在温室培养箱中培养24h，进行菌落计数，并计算灭菌率；用10ug/ml臭氧水对耐碳青霉烯铜绿假单胞菌体外灭菌分别作用0、2、4、8和16min后，加入中和剂，经3200r/min离心15min，弃上清液，再加入磷酸盐缓冲液1ml混悬5min，取等量混悬液和3%磷钨酸染色2min后，滴于铜网上，待干燥后，在透射电子显微镜下观察菌体超显微结构变化情况； 实验五：用臭氧发生器制取浓度为2.5ug/ml、5ug/ml、10ug/ml臭氧水，取4.5ml臭氧水分别六次对0.5ml耐碳青霉烯鲍曼不动杆菌进行体外定量灭菌，分别作用0、0.5、1、2、4、8及16min时后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和剂中，振荡5min后，取10ul中和后的混合液接种于普通营养琼脂培养基上，在温室培养箱中培养24h，进行菌落计数，并计算灭菌率 用SPSS19.0统计软件进行统计学处理，臭氧水浓度以及对四种耐药菌的灭菌率均以均值±标准差（x±s）表示。臭氧水浓度检测结果与设定值比较采用t检验分析；不同时间臭氧水浓度比较以及相同时间不同浓度臭氧水和相同臭氧水浓度不同时间，臭氧水对四种耐药菌的灭菌率的比较均采用方差分析，组内两两比较采用LSD法。P＜0.05为差异有统计学意义。 结果： 实验一：臭氧发生器制取三种设定浓度为2.5ug/ml、5ug/ml、10ug/ml臭氧水的测定浓度分别为(2.47±0.08)ug/ml、(5.04±0.07)ug/ml和(10.09±0.08)ug/ml,三种臭氧水浓度检测结果与设定值均无统计学意义（P=0.38、0.10、0.06）。10ug/ml臭氧水在0、1、2、4、8、16、32、64和128min时检测浓度分别为（10.09±0.08）、（10.05±0.22）、（10.00±0.11）、（9.98±0.10）、(9.95±0.21)、(9.60±0.51)、（8.24±0.26）、（7.15±0.23）和（5.06±0.11）ug/ml；在1、2、4、8、16、32、64和128min时分解率分别为：0.45%、0.93%、1.06%、1.40%、4.89%、18.32%、29.12%和49.87%。10ug/ml臭氧水在各时间点检测浓度结果具有显著性差异（P=0.00），但组内经LSD法比较，在0、1、2、4、8min时其浓度值无统计学差异（P=0.749）；在8、16、32、64、128min时其浓度有统计学意义（P=0.00）。 实验二：2.5ug/ml、5ug/ml、10ug/ml臭氧水0.5min时对耐甲氧西林金黄色葡萄球菌的平均灭菌率分别为14.9%、32.8%和42.1%；1min时分别为42.9%、70.4%和79.7%；2min时分别为54.8%、92.9%和96.8%；4min时分别为66.4%、98.8%和99.9%；8min分别为78.1%、100%和100%；16min分别为97.2%、100%和100%。相同浓度臭氧水不同时间以及不同浓度相同时间的灭菌率均有统计学意义(P=0.00)。电镜观察显示未经臭氧水作用的耐甲氧西林金黄色葡萄球菌菌体表面光滑，似葡萄状聚集成串大小形状相似；10ug/ml臭氧水作用2min时菌体变成椭圆、大小及着色不均；作用4min时部分菌体细胞壁明显皱缩；作用8、16min时可见不规则菌体占多数且出现“空斑”现像。 实验三：2.5ug/ml、5ug/ml、10ug/ml臭氧水0.5min对产ESBLS大肠埃希菌的灭菌率时分别为17.7%、27.8%和43.7%；1min时分别为42.9%、72.4%和80.3%；2min时分别为53.3%、93.5%和96.5%；4min时分别为59.7%、98.8%和99.8%；8min分别为76.2%、100%和100%；16min分别为97.4%、100%和100%。相同浓度臭氧水不同时间以及不同浓度相同时间的灭菌率均有统计学意义(P=0.00)。电镜观察显示未经过臭氧水作用的产β-ESBLS大肠埃希菌菌体椭圆、扁平、边缘整齐光滑、有鞭毛；10ug/ml臭氧水作用2min时菌体染色加深、鞭毛脱落断裂；作用4min时细胞壁塌陷破坏；作用8、16min时菌体皱缩近似圆形、染色出现不均、失去固有扁平的形态。 实验四：2.5ug/ml、5ug/ml、10ug/ml臭氧水对耐碳青霉烯铜绿假单胞菌的平均灭菌率0.5min时分别为14.5%、23.3%和39.4%；1min时分别为41.1%、63.7%和77.8%；2min时分别为50.5%、93.0%和96.6%；4min时分别为56.6%、98.9%和99.9%；8min分别为76.1%、100%和100%；16min分别为97.5%、100%和100%。相同浓度臭氧水不同时间以及不同浓度相同时间的灭菌率均有统计学意义(P=0.00)。电镜观察显示未经过臭氧水作用的耐碳青霉烯铜绿假单胞菌菌体直或稍弯、有单端鞭毛；10ug/ml臭氧水作用2min多数细菌出现鞭毛脱落、大小不一；作用4min时仍可见鞭毛；作用8min菌体无鞭毛，菌体变短、变钝；16min菌体出现“空斑”现象。 实验五：2.5ug/ml、5ug/ml、10ug/ml臭氧水对耐碳青霉烯鲍曼不动杆菌的平均灭菌率0.5min时分别为15.1%、34.8%和41.0%；1min时分别为41.0%、70.1%和78.4%；2min时分别为50.5%、92.9%和97.1%；4min时分别为63.1%、98.8%和99.8%；8min分别为78.3%、100%和100%；16min分别为97.2%、100%和100%。相同浓度臭氧水不同时间以及不同浓度相同时间的灭菌率均有统计学意义(P=0.00)。 结论： 1.本研究所用臭氧发生器性能稳定，能准确制取实验所需浓度臭氧水，但易于分解，稳定性较差，在实验过程臭氧水应当现场制取使用，保证实验的准确性； 2.臭氧水对耐甲氧西林金黄色葡萄球菌、产β-ESBLS大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌均有较好的杀灭效果，，同一种浓度臭氧水随着作用时间延长灭菌率越高；相同时间内臭氧水浓度越高对耐药菌的灭菌率愈高。10ug/ml臭氧水是一种高效快速杀菌剂，可以消灭创面耐药菌感染，用于骨科创面常见耐药菌感染的局部治疗。
[Abstract]:OBJECTIVE: To observe the in vitro sterilization effect of ozone water on four common antibiotic-resistant bacteria in orthopaedic infectious wounds, namely methicillin-resistant Staphylococcus aureus, Escherichia coli producing beta-lactamase (ESBLS), carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii, and to provide ozone water for the treatment of clinical orthopaedic wound-related antibiotic susceptibility. Dyeing provides a preliminary theoretical basis.
Method:
Experiment 1: Three concentrations of 2.5ug/ml, 5ug/ml and 10ug/ml ozone water (20
Experiment 2: After producing 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water with ozone generator, 0.5 ml ozone water was taken six times to sterilize 0.5 ml methicillin-resistant Staphylococcus aureus suspension in vitro. After 0,0.5,1,2,4,8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed and added into 4.5 ml neutralizer, and the suspension was shaken for 5 m. After in vitro sterilization of methicillin-resistant Staphylococcus aureus with 10 ug/ml ozone water for 0,2,4,8 and 16 minutes respectively, neutralizer was added and centrifuged for 15 minutes at 3200 r/min. After adding 1 ml phosphate buffer suspension for 5 minutes, taking the same amount of suspension and 3% phosphotungstic acid staining for 2 minutes, dropping on the copper mesh, and then drying, the ultrastructure of the bacteria was observed under transmission electron microscope.
Experiment 3: Ozone water with concentration of 2.5 ug/ml, 5 ug/ml and 10 ug/ml was produced by ozone generator. Ozone water with concentration of 4.5 ml was used to sterilize the suspension of E. coli producing beta-lactamase in vitro quantitatively for six times. After 0,0.5,1,2,4,8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed and added to 4.5 ml neutralizer, respectively. After 5 minutes, 10 UL neutralized mixture was inoculated on general nutrient agar medium, cultured in greenhouse incubator for 24 hours, colony counting and sterilization rate were calculated. After 0, 2, 4, 8 and 16 minutes of in vitro sterilization with 10 ug/ml ozone water, neutralizer was added, centrifuged for 15 minutes at 3200 r/min, supernatant was discarded, and then supernatant was added. Put 1 ml phosphate buffer into suspension for 5 minutes, take the same amount of suspension and 3% phosphotungstic acid staining for 2 minutes, then drop on the copper mesh. After drying, observe the ultrastructure of the bacteria under transmission electron microscope.
Experiment 4: Ozone water with concentration of 2.5 ug/ml, 5 ug/ml, 10 ug/ml was produced by ozone generator, and 4.5 ml ozone water was taken to sterilize 0.5 ml carbapenem-resistant Pseudomonas aeruginosa suspension in vitro for six times. After 0, 0.5, 1, 2, 4, 8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed, added to 4.5 ml neutralizer, and the suspension was shaken for 5 m. After in vitro sterilization of carbapenem-resistant Pseudomonas aeruginosa with 10ug/ml ozone water for 0,2,4,8 and 16 minutes respectively, neutralizer was added, centrifuged for 15 minutes by 3200r/min, and then supernatant was discarded. Add 1 ml phosphate buffer suspension for 5 minutes, take the same amount of suspension and 3% phosphotungstic acid staining for 2 minutes, then drop on the copper mesh. After drying, the ultrastructure of the bacteria was observed under transmission electron microscope.
Experiment 5: The concentration of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water was produced by ozone generator, and 4.5 ml ozone water was taken for six times to quantitatively sterilize 0.5 ml carbapenem-resistant Acinetobacter baumannii in vitro. After 0, 0.5, 1, 2, 4, 8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed and added to 4.5 ml neutralizer. After 5 minutes of oscillation, 10 UL was taken for 5 minutes. After neutralization, the mixture was inoculated on general nutrient agar medium and cultured in greenhouse incubator for 24 hours. Colony count and sterilization rate were calculated.
SPSS19.0 statistical software was used to analyze the concentration of ozone water and the sterilization rate of four kinds of drug-resistant bacteria. The results of ozone water concentration test were compared with the set values by t-test analysis. Meanwhile, analysis of variance was used to compare the sterilization rate of ozone water to four kinds of drug-resistant bacteria, and LSD method was used to compare the two groups. P<0.05 was statistically significant.
Result:
Experiment 1: Ozone generator produced three set concentrations of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water determination concentrations were (2.47.08) ug/ml, (5.04.07) ug/ml and (10.09.08) ug/ml, three ozone water concentration test results and set values were not statistically significant (P = 0.38, 0.10, 0.06). 10 ug/ml ozone water in 0, 1, 2, 4, 16, 32, 64 and 128 minutes. The detection concentrations were (10.09 + 0.08, (10.09 [(10.09 [(10.05 [(10.05 [(10.05 [0.22), (10.00 [0.22], (10.00 [0.11], (10.00 [(9.98 [0.11],, (9.95 [(9.95 [0.95 [0.21], (9.60 [.60 [.51], (8.60 [.60 [(8.24 [(8.24 [0.26), (7.15 [(7.15 [0.15 [0.23]] and (5.06 [(0.06 [(0.11 87%. 10ug/ml ozone water was detected at each time point. There was a significant difference in the concentration (P = 0.00), but there was no significant difference in the concentration (P = 0.749) at 0, 1, 2, 4 and 8 minutes by LSD, and there was a significant difference in the concentration (P = 0.00) at 8, 16, 32, 64 and 128 minutes.
The average sterilization rates of methicillin-resistant Staphylococcus aureus were 14.9%, 32.8% and 42.1% at 0.5 min in 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water, 42.9%, 70.4% and 79.7% at 1 min, 54.8%, 92.9% and 96.8% at 2 min, 66.4%, 98.8% and 99.9% at 4 min, 78.1%, 100% at 8 min and 100% at 16 min, respectively. The sterilization rates of methicillin-resistant Staphylococcus aureus were 97.2%, 100% and 100%. The sterilization rates of the same concentration of ozone water at different time and different concentration at the same time were statistically significant (P=0.00). The surface of methicillin-resistant Staphylococcus aureus without ozone water treatment was smooth and similar to that of grape-like aggregation. When the ozone water treatment lasted for 2 minutes, the sterilization rates of 10 ug/ml ozone water treatment were similar. The cells became oval, uneven in size and colouring, and the cell walls of some bacteria shrank obviously after 4 minutes of treatment, and irregular bacteria dominated and appeared "plaque" after 8 and 16 minutes of treatment.
The sterilization rates of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water at 0.5 min were 17.7%, 27.8% and 43.7% respectively, 42.9%, 72.4% and 80.3% at 1 min, 53.3%, 93.5% and 96.5% at 2 min, 59.7%, 98.8% and 99.8% at 4 min, 76.2%, 100% at 8 min, 97.4% and 100% at 16 min, respectively. Electron microscopic observation showed that E. coli producing beta-ESBLS without ozone water was oval, flat, with smooth margin and flagella, and the staining deepened and flagella exfoliated and fractured after ozone water treatment for 2 min. The cell wall collapsed and destroyed after 4 minutes of treatment, and the cells shrank approximately round after 8 and 16 minutes of treatment, showing uneven staining and losing the inherent flat shape.
The average sterilization rates of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water were 14.5%, 23.3% and 39.4% in 0.5 min, 41.1%, 63.7% and 77.8% in 1 min, 50.5%, 93.0% and 96.6% in 2 min, 56.6%, 98.9% and 99.9% in 4 min, 76.1%, 100% and 100% in 8 min, respectively, and 77.8% in 16 min, respectively. 97.5%, 100% and 100%. The sterilization rates of the same concentration of ozone water at different times and at different concentrations at the same time were statistically significant (P = 0.00). Electron microscopic observation showed that the carbapenem-resistant Pseudomonas aeruginosa strains were straight or slightly curved with one-terminal flagella, and most of the bacteria showed flagella exfoliation and large flagella after 2 minutes of ozone water treatment with 10 ug/ml ozone water. Flagella could still be seen after 4 minutes of treatment, but no flagella was found after 8 minutes of treatment, and the bacteria became shorter and blunt, and "plaque" appeared after 16 minutes of treatment.
The average sterilization rates of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water to Acinetobacter baumannii were 15.1%, 34.8% and 41.0% in 0.5 min, 41.0%, 70.1% and 78.4% in 1 min, 50.5%, 92.9% and 97.1% in 2 min, 63.1%, 98.8% and 99.8% in 4 min, 78.3%, 100% and 100.0% in 8 min, 16 min, respectively. 97.2%, 100% and 100%. The sterilization rates of the same concentration ozone water at different times and at the same time were statistically significant (P=0.00).
Conclusion:
1. The ozone generator used in this study has stable performance and can accurately produce the concentration of ozone water needed in the experiment, but it is easy to decompose and has poor stability.
2. Ozone water had good efficacy in killing methicillin-resistant Staphylococcus aureus, E.coli producing beta-ESBLS, Pseudomonas aeruginosa resistant to carbapenem, Acinetobacter baumannii resistant to carbapenem. The higher the sterilization rate of ozone water with the same concentration, the higher the concentration of ozone water at the same time. The higher. 10ug/ml ozone water is a highly effective and rapid bactericide, which can eliminate drug-resistant bacterial infections in wounds and be used for local treatment of common drug-resistant bacterial infections in orthopedic wounds.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
[Abstract]:OBJECTIVE: To observe the in vitro sterilization effect of ozone water on four common antibiotic-resistant bacteria in orthopaedic infectious wounds, namely methicillin-resistant Staphylococcus aureus, Escherichia coli producing beta-lactamase (ESBLS), carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii, and to provide ozone water for the treatment of clinical orthopaedic wound-related antibiotic susceptibility. Dyeing provides a preliminary theoretical basis.
Method:
Experiment 1: Three concentrations of 2.5ug/ml, 5ug/ml and 10ug/ml ozone water (20
Experiment 2: After producing 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water with ozone generator, 0.5 ml ozone water was taken six times to sterilize 0.5 ml methicillin-resistant Staphylococcus aureus suspension in vitro. After 0,0.5,1,2,4,8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed and added into 4.5 ml neutralizer, and the suspension was shaken for 5 m. After in vitro sterilization of methicillin-resistant Staphylococcus aureus with 10 ug/ml ozone water for 0,2,4,8 and 16 minutes respectively, neutralizer was added and centrifuged for 15 minutes at 3200 r/min. After adding 1 ml phosphate buffer suspension for 5 minutes, taking the same amount of suspension and 3% phosphotungstic acid staining for 2 minutes, dropping on the copper mesh, and then drying, the ultrastructure of the bacteria was observed under transmission electron microscope.
Experiment 3: Ozone water with concentration of 2.5 ug/ml, 5 ug/ml and 10 ug/ml was produced by ozone generator. Ozone water with concentration of 4.5 ml was used to sterilize the suspension of E. coli producing beta-lactamase in vitro quantitatively for six times. After 0,0.5,1,2,4,8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed and added to 4.5 ml neutralizer, respectively. After 5 minutes, 10 UL neutralized mixture was inoculated on general nutrient agar medium, cultured in greenhouse incubator for 24 hours, colony counting and sterilization rate were calculated. After 0, 2, 4, 8 and 16 minutes of in vitro sterilization with 10 ug/ml ozone water, neutralizer was added, centrifuged for 15 minutes at 3200 r/min, supernatant was discarded, and then supernatant was added. Put 1 ml phosphate buffer into suspension for 5 minutes, take the same amount of suspension and 3% phosphotungstic acid staining for 2 minutes, then drop on the copper mesh. After drying, observe the ultrastructure of the bacteria under transmission electron microscope.
Experiment 4: Ozone water with concentration of 2.5 ug/ml, 5 ug/ml, 10 ug/ml was produced by ozone generator, and 4.5 ml ozone water was taken to sterilize 0.5 ml carbapenem-resistant Pseudomonas aeruginosa suspension in vitro for six times. After 0, 0.5, 1, 2, 4, 8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed, added to 4.5 ml neutralizer, and the suspension was shaken for 5 m. After in vitro sterilization of carbapenem-resistant Pseudomonas aeruginosa with 10ug/ml ozone water for 0,2,4,8 and 16 minutes respectively, neutralizer was added, centrifuged for 15 minutes by 3200r/min, and then supernatant was discarded. Add 1 ml phosphate buffer suspension for 5 minutes, take the same amount of suspension and 3% phosphotungstic acid staining for 2 minutes, then drop on the copper mesh. After drying, the ultrastructure of the bacteria was observed under transmission electron microscope.
Experiment 5: The concentration of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water was produced by ozone generator, and 4.5 ml ozone water was taken for six times to quantitatively sterilize 0.5 ml carbapenem-resistant Acinetobacter baumannii in vitro. After 0, 0.5, 1, 2, 4, 8 and 16 minutes, the mixture of 0.5 ml ozone water was absorbed and added to 4.5 ml neutralizer. After 5 minutes of oscillation, 10 UL was taken for 5 minutes. After neutralization, the mixture was inoculated on general nutrient agar medium and cultured in greenhouse incubator for 24 hours. Colony count and sterilization rate were calculated.
SPSS19.0 statistical software was used to analyze the concentration of ozone water and the sterilization rate of four kinds of drug-resistant bacteria. The results of ozone water concentration test were compared with the set values by t-test analysis. Meanwhile, analysis of variance was used to compare the sterilization rate of ozone water to four kinds of drug-resistant bacteria, and LSD method was used to compare the two groups. P<0.05 was statistically significant.
Result:
Experiment 1: Ozone generator produced three set concentrations of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water determination concentrations were (2.47.08) ug/ml, (5.04.07) ug/ml and (10.09.08) ug/ml, three ozone water concentration test results and set values were not statistically significant (P = 0.38, 0.10, 0.06). 10 ug/ml ozone water in 0, 1, 2, 4, 16, 32, 64 and 128 minutes. The detection concentrations were (10.09 + 0.08, (10.09 [(10.09 [(10.05 [(10.05 [(10.05 [0.22), (10.00 [0.22], (10.00 [0.11], (10.00 [(9.98 [0.11],, (9.95 [(9.95 [0.95 [0.21], (9.60 [.60 [.51], (8.60 [.60 [(8.24 [(8.24 [0.26), (7.15 [(7.15 [0.15 [0.23]] and (5.06 [(0.06 [(0.11 87%. 10ug/ml ozone water was detected at each time point. There was a significant difference in the concentration (P = 0.00), but there was no significant difference in the concentration (P = 0.749) at 0, 1, 2, 4 and 8 minutes by LSD, and there was a significant difference in the concentration (P = 0.00) at 8, 16, 32, 64 and 128 minutes.
The average sterilization rates of methicillin-resistant Staphylococcus aureus were 14.9%, 32.8% and 42.1% at 0.5 min in 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water, 42.9%, 70.4% and 79.7% at 1 min, 54.8%, 92.9% and 96.8% at 2 min, 66.4%, 98.8% and 99.9% at 4 min, 78.1%, 100% at 8 min and 100% at 16 min, respectively. The sterilization rates of methicillin-resistant Staphylococcus aureus were 97.2%, 100% and 100%. The sterilization rates of the same concentration of ozone water at different time and different concentration at the same time were statistically significant (P=0.00). The surface of methicillin-resistant Staphylococcus aureus without ozone water treatment was smooth and similar to that of grape-like aggregation. When the ozone water treatment lasted for 2 minutes, the sterilization rates of 10 ug/ml ozone water treatment were similar. The cells became oval, uneven in size and colouring, and the cell walls of some bacteria shrank obviously after 4 minutes of treatment, and irregular bacteria dominated and appeared "plaque" after 8 and 16 minutes of treatment.
The sterilization rates of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water at 0.5 min were 17.7%, 27.8% and 43.7% respectively, 42.9%, 72.4% and 80.3% at 1 min, 53.3%, 93.5% and 96.5% at 2 min, 59.7%, 98.8% and 99.8% at 4 min, 76.2%, 100% at 8 min, 97.4% and 100% at 16 min, respectively. Electron microscopic observation showed that E. coli producing beta-ESBLS without ozone water was oval, flat, with smooth margin and flagella, and the staining deepened and flagella exfoliated and fractured after ozone water treatment for 2 min. The cell wall collapsed and destroyed after 4 minutes of treatment, and the cells shrank approximately round after 8 and 16 minutes of treatment, showing uneven staining and losing the inherent flat shape.
The average sterilization rates of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water were 14.5%, 23.3% and 39.4% in 0.5 min, 41.1%, 63.7% and 77.8% in 1 min, 50.5%, 93.0% and 96.6% in 2 min, 56.6%, 98.9% and 99.9% in 4 min, 76.1%, 100% and 100% in 8 min, respectively, and 77.8% in 16 min, respectively. 97.5%, 100% and 100%. The sterilization rates of the same concentration of ozone water at different times and at different concentrations at the same time were statistically significant (P = 0.00). Electron microscopic observation showed that the carbapenem-resistant Pseudomonas aeruginosa strains were straight or slightly curved with one-terminal flagella, and most of the bacteria showed flagella exfoliation and large flagella after 2 minutes of ozone water treatment with 10 ug/ml ozone water. Flagella could still be seen after 4 minutes of treatment, but no flagella was found after 8 minutes of treatment, and the bacteria became shorter and blunt, and "plaque" appeared after 16 minutes of treatment.
The average sterilization rates of 2.5 ug/ml, 5 ug/ml, 10 ug/ml ozone water to Acinetobacter baumannii were 15.1%, 34.8% and 41.0% in 0.5 min, 41.0%, 70.1% and 78.4% in 1 min, 50.5%, 92.9% and 97.1% in 2 min, 63.1%, 98.8% and 99.8% in 4 min, 78.3%, 100% and 100.0% in 8 min, 16 min, respectively. 97.2%, 100% and 100%. The sterilization rates of the same concentration ozone water at different times and at the same time were statistically significant (P=0.00).
Conclusion:
1. The ozone generator used in this study has stable performance and can accurately produce the concentration of ozone water needed in the experiment, but it is easy to decompose and has poor stability.
2. Ozone water had good efficacy in killing methicillin-resistant Staphylococcus aureus, E.coli producing beta-ESBLS, Pseudomonas aeruginosa resistant to carbapenem, Acinetobacter baumannii resistant to carbapenem. The higher the sterilization rate of ozone water with the same concentration, the higher the concentration of ozone water at the same time. The higher. 10ug/ml ozone water is a highly effective and rapid bactericide, which can eliminate drug-resistant bacterial infections in wounds and be used for local treatment of common drug-resistant bacterial infections in orthopedic wounds.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
【参考文献】
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