【摘要】：目的：觀察臭氧水對(duì)骨科感染性創(chuàng)面常見的四種耐藥菌，即耐甲氧西林金黃色葡萄球菌、產(chǎn)β-內(nèi)酰胺酶(ESBLS)大腸埃希菌、耐碳青霉烯銅綠假單胞菌、耐碳青霉烯鮑曼不動(dòng)桿菌的體外滅菌效果，為臭氧水治療臨床骨科創(chuàng)面相關(guān)耐藥菌感染提供初步的理論依據(jù)。 方法： 實(shí)驗(yàn)一：用臭氧發(fā)生器制取三種濃度為2.5ug/ml、5ug/ml和10ug/ml臭氧水（溫度20±1℃）各六次，立即用碘量法檢測(cè)其濃度；并檢測(cè)10ug/ml臭氧水分別在0、1、2、4、8、16、32、64和128min時(shí)濃度值； 實(shí)驗(yàn)二：用臭氧發(fā)生器制取濃度分別為2.5ug/ml、5ug/ml、10ug/ml臭氧水后，取4.5ml臭氧水六次對(duì)0.5ml耐甲氧西林金黃色葡萄球菌菌懸液進(jìn)行體外定量滅菌，分別作用0、0.5、1、2、4、8及16min時(shí)后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和劑中，振蕩5min后，取10ul中和后的混合液接種于普通營養(yǎng)瓊脂培養(yǎng)基上，在溫室培養(yǎng)箱中培養(yǎng)24h，進(jìn)行菌落計(jì)數(shù)，并計(jì)算滅菌率；用10ug/ml臭氧水對(duì)耐甲氧西林金黃色葡萄球菌體外滅菌分別作用0、2、4、8和16min后，加入中和劑，經(jīng)3200r/min離心15min，棄上清液，再加入磷酸鹽緩沖液1ml混懸5min，取等量混懸液和3%磷鎢酸染色2min后，滴于銅網(wǎng)上，待干燥后，在透射電子顯微鏡下觀察菌體超顯微結(jié)構(gòu)變化情況； 實(shí)驗(yàn)三：用臭氧發(fā)生器制取濃度分別為2.5ug/ml、5ug/ml和10ug/ml臭氧水，取4.5ml臭氧水分別六次對(duì)0.5ml產(chǎn)β-內(nèi)酰胺酶大腸埃希菌菌懸液進(jìn)行體外定量滅菌，分別作用0、0.5、1、2、4、8及16min時(shí)后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和劑中，振蕩5min后，取10ul中和后的混合液接種于普通營養(yǎng)瓊脂培養(yǎng)基上，在溫室培養(yǎng)箱中培養(yǎng)24h，進(jìn)行菌落計(jì)數(shù)，并計(jì)算滅菌率；用10ug/ml臭氧水對(duì)產(chǎn)β-ESBLS大腸埃希菌體外滅菌分別作用0、2、4、8和16min后，加入中和劑，經(jīng)3200r/min離心15min，棄上清液，再加入磷酸鹽緩沖液1ml混懸5min，取等量混懸液和3%磷鎢酸染色2min后，滴于銅網(wǎng)上，待干燥后，在透射電子顯微鏡下觀察菌體超顯微結(jié)構(gòu)變化情況； 實(shí)驗(yàn)四：用臭氧發(fā)生器制取濃度分別為2.5ug/ml、5ug/ml、10ug/ml臭氧水，取4.5ml臭氧水分別六次對(duì)0.5ml耐碳青霉烯銅綠假單胞菌菌懸液進(jìn)行體外定量滅菌，分別作用0、0.5、1、2、4、8及16min時(shí)后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和劑中，振蕩5min后，取10ul中和后的混合液接種于普通營養(yǎng)瓊脂培養(yǎng)基上，在溫室培養(yǎng)箱中培養(yǎng)24h，進(jìn)行菌落計(jì)數(shù)，并計(jì)算滅菌率；用10ug/ml臭氧水對(duì)耐碳青霉烯銅綠假單胞菌體外滅菌分別作用0、2、4、8和16min后，加入中和劑，經(jīng)3200r/min離心15min，棄上清液，再加入磷酸鹽緩沖液1ml混懸5min，取等量混懸液和3%磷鎢酸染色2min后，滴于銅網(wǎng)上，待干燥后，在透射電子顯微鏡下觀察菌體超顯微結(jié)構(gòu)變化情況； 實(shí)驗(yàn)五：用臭氧發(fā)生器制取濃度為2.5ug/ml、5ug/ml、10ug/ml臭氧水，取4.5ml臭氧水分別六次對(duì)0.5ml耐碳青霉烯鮑曼不動(dòng)桿菌進(jìn)行體外定量滅菌，分別作用0、0.5、1、2、4、8及16min時(shí)后，吸取0.5ml臭氧水作用的混合液，加入4.5ml中和劑中，振蕩5min后，取10ul中和后的混合液接種于普通營養(yǎng)瓊脂培養(yǎng)基上，在溫室培養(yǎng)箱中培養(yǎng)24h，進(jìn)行菌落計(jì)數(shù)，并計(jì)算滅菌率 用SPSS19.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，臭氧水濃度以及對(duì)四種耐藥菌的滅菌率均以均值±標(biāo)準(zhǔn)差（x±s）表示。臭氧水濃度檢測(cè)結(jié)果與設(shè)定值比較采用t檢驗(yàn)分析；不同時(shí)間臭氧水濃度比較以及相同時(shí)間不同濃度臭氧水和相同臭氧水濃度不同時(shí)間，臭氧水對(duì)四種耐藥菌的滅菌率的比較均采用方差分析，組內(nèi)兩兩比較采用LSD法。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 實(shí)驗(yàn)一：臭氧發(fā)生器制取三種設(shè)定濃度為2.5ug/ml、5ug/ml、10ug/ml臭氧水的測(cè)定濃度分別為(2.47±0.08)ug/ml、(5.04±0.07)ug/ml和(10.09±0.08)ug/ml,三種臭氧水濃度檢測(cè)結(jié)果與設(shè)定值均無統(tǒng)計(jì)學(xué)意義（P=0.38、0.10、0.06）。10ug/ml臭氧水在0、1、2、4、8、16、32、64和128min時(shí)檢測(cè)濃度分別為（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時(shí)分解率分別為：0.45%、0.93%、1.06%、1.40%、4.89%、18.32%、29.12%和49.87%。10ug/ml臭氧水在各時(shí)間點(diǎn)檢測(cè)濃度結(jié)果具有顯著性差異（P=0.00），但組內(nèi)經(jīng)LSD法比較，在0、1、2、4、8min時(shí)其濃度值無統(tǒng)計(jì)學(xué)差異（P=0.749）；在8、16、32、64、128min時(shí)其濃度有統(tǒng)計(jì)學(xué)意義（P=0.00）。 實(shí)驗(yàn)二：2.5ug/ml、5ug/ml、10ug/ml臭氧水0.5min時(shí)對(duì)耐甲氧西林金黃色葡萄球菌的平均滅菌率分別為14.9%、32.8%和42.1%；1min時(shí)分別為42.9%、70.4%和79.7%；2min時(shí)分別為54.8%、92.9%和96.8%；4min時(shí)分別為66.4%、98.8%和99.9%；8min分別為78.1%、100%和100%；16min分別為97.2%、100%和100%。相同濃度臭氧水不同時(shí)間以及不同濃度相同時(shí)間的滅菌率均有統(tǒng)計(jì)學(xué)意義(P=0.00)。電鏡觀察顯示未經(jīng)臭氧水作用的耐甲氧西林金黃色葡萄球菌菌體表面光滑，似葡萄狀聚集成串大小形狀相似；10ug/ml臭氧水作用2min時(shí)菌體變成橢圓、大小及著色不均；作用4min時(shí)部分菌體細(xì)胞壁明顯皺縮；作用8、16min時(shí)可見不規(guī)則菌體占多數(shù)且出現(xiàn)“空斑”現(xiàn)像。 實(shí)驗(yàn)三：2.5ug/ml、5ug/ml、10ug/ml臭氧水0.5min對(duì)產(chǎn)ESBLS大腸埃希菌的滅菌率時(shí)分別為17.7%、27.8%和43.7%；1min時(shí)分別為42.9%、72.4%和80.3%；2min時(shí)分別為53.3%、93.5%和96.5%；4min時(shí)分別為59.7%、98.8%和99.8%；8min分別為76.2%、100%和100%；16min分別為97.4%、100%和100%。相同濃度臭氧水不同時(shí)間以及不同濃度相同時(shí)間的滅菌率均有統(tǒng)計(jì)學(xué)意義(P=0.00)。電鏡觀察顯示未經(jīng)過臭氧水作用的產(chǎn)β-ESBLS大腸埃希菌菌體橢圓、扁平、邊緣整齊光滑、有鞭毛；10ug/ml臭氧水作用2min時(shí)菌體染色加深、鞭毛脫落斷裂；作用4min時(shí)細(xì)胞壁塌陷破壞；作用8、16min時(shí)菌體皺縮近似圓形、染色出現(xiàn)不均、失去固有扁平的形態(tài)。 實(shí)驗(yàn)四：2.5ug/ml、5ug/ml、10ug/ml臭氧水對(duì)耐碳青霉烯銅綠假單胞菌的平均滅菌率0.5min時(shí)分別為14.5%、23.3%和39.4%；1min時(shí)分別為41.1%、63.7%和77.8%；2min時(shí)分別為50.5%、93.0%和96.6%；4min時(shí)分別為56.6%、98.9%和99.9%；8min分別為76.1%、100%和100%；16min分別為97.5%、100%和100%。相同濃度臭氧水不同時(shí)間以及不同濃度相同時(shí)間的滅菌率均有統(tǒng)計(jì)學(xué)意義(P=0.00)。電鏡觀察顯示未經(jīng)過臭氧水作用的耐碳青霉烯銅綠假單胞菌菌體直或稍彎、有單端鞭毛；10ug/ml臭氧水作用2min多數(shù)細(xì)菌出現(xiàn)鞭毛脫落、大小不一；作用4min時(shí)仍可見鞭毛；作用8min菌體無鞭毛，菌體變短、變鈍；16min菌體出現(xiàn)“空斑”現(xiàn)象。 實(shí)驗(yàn)五：2.5ug/ml、5ug/ml、10ug/ml臭氧水對(duì)耐碳青霉烯鮑曼不動(dòng)桿菌的平均滅菌率0.5min時(shí)分別為15.1%、34.8%和41.0%；1min時(shí)分別為41.0%、70.1%和78.4%；2min時(shí)分別為50.5%、92.9%和97.1%；4min時(shí)分別為63.1%、98.8%和99.8%；8min分別為78.3%、100%和100%；16min分別為97.2%、100%和100%。相同濃度臭氧水不同時(shí)間以及不同濃度相同時(shí)間的滅菌率均有統(tǒng)計(jì)學(xué)意義(P=0.00)。 結(jié)論： 1.本研究所用臭氧發(fā)生器性能穩(wěn)定，能準(zhǔn)確制取實(shí)驗(yàn)所需濃度臭氧水，但易于分解，穩(wěn)定性較差，在實(shí)驗(yàn)過程臭氧水應(yīng)當(dāng)現(xiàn)場(chǎng)制取使用，保證實(shí)驗(yàn)的準(zhǔn)確性； 2.臭氧水對(duì)耐甲氧西林金黃色葡萄球菌、產(chǎn)β-ESBLS大腸埃希菌、耐碳青霉烯銅綠假單胞菌、耐碳青霉烯鮑曼不動(dòng)桿菌均有較好的殺滅效果，，同一種濃度臭氧水隨著作用時(shí)間延長滅菌率越高；相同時(shí)間內(nèi)臭氧水濃度越高對(duì)耐藥菌的滅菌率愈高。10ug/ml臭氧水是一種高效快速殺菌劑，可以消滅創(chuàng)面耐藥菌感染，用于骨科創(chuàng)面常見耐藥菌感染的局部治療。
[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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王豪;劉丁;陳萍;王政;成瑤;;創(chuàng)傷患者感染鮑曼不動(dòng)桿菌危險(xiǎn)因素及其死亡率的調(diào)查研究[J];重慶醫(yī)學(xué);2011年36期

2 喬登嫣;梁勤;程p

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