医用臭氧的体外体液灭菌作用研究
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摘要
臭氧(O_3)是由三个氧原子组成的分子,臭氧气体是一种淡蓝色带有刺激性酸味的气体,在空气中极不稳定,在常温常压空气中易还原为氧气(半衰期40min)。医用臭氧(Medical ozone)为医用纯氧通过臭氧发生器产生的臭氧与医用纯氧的混合气体,臭氧浓度在0.05%～5%之间。与日常生活用和工业用臭氧不同,医用臭氧由医用纯氧而非空气生成,故不含氮氧化合物(NO_2)等有毒气体分子,且要求其浓度能够被精确调控,另外由于其极不稳定性故必须即时制备即时使用(为叙述方便,如无特殊说明下文中的“臭氧”均代指“医用臭氧”)。
臭氧具有极强的氧化性,其氧化性仅次于氟和过硫酸盐。已有大量研究表明,体外在生物耐受剂量浓度下(20～80μg/ml)的臭氧就能有效杀灭或灭活水体中多种微生物,包括G~+或G~-菌、HAV、HIV等。
我国有慢性肝炎病毒包括乙肝病毒(HBV)和丙肝病毒(HCV)感染者约1.5亿,主要传播途径为血液传播。由于检测技术的限制及病毒感染窗口期的存在,血液制品的生物安全性尚未得到有效解决。降低血液肝炎病毒载量对移植肝再感染也有利。对于慢性乙型肝炎病毒感染者进行肝移植,目前主要预防方法为口服核苷类似物联合静脉用乙肝免疫球蛋白(HBIG),费用高昂,且HBIG国内暂无上市;对于HCV感染者的移植肝再感染目前尚无有效的预防方法。自体输血作为目前最为安全有效的输血方式虽具有节约血源、减少不必要的输血反应和感染性疾病传播等诸多优势,但可能发生细菌污染,输用后将造成严重甚至致命的后果。以上问题的解决均依赖于安全、有效的血液净化方式。
腹水是肝硬化最常见的并发症之一。自发性腹膜炎(Spontaneous bacterialperitonitis,SBP)在肝硬化病例中的发生率为10%-25%,在腹水病例中的平均发生率约30%,是肝病严重的标志,病死率很高。SBP主要发生机制为宿主免疫防御功能减弱、肠道细菌移位(BT),感染的病原菌半数是大肠埃希菌,其次是金黄色葡萄球菌、肠球菌等。如能对此类患者腹水进行有效灭菌处理,其腹水浓缩后亦可回输体内,避免蛋白丢失或可提高治疗效果。
本研究目的在于利用臭氧体外水介质中能有效杀灭多种微生物的特性,试验臭氧在体外对体液(血浆、腹水等)的杀菌效果,为利用臭氧在体外对体液进行生物净化的可行性进行评价并提供实验参数。
第一部分医用臭氧对血浆中细菌杀灭作用的研究
1.1研究方法
向5份10ml含等量(0.5麦氏浓度)细菌的生理盐水中分别通入浓度为5μg/ml、10μg/ml、15μg/ml、20μg/ml、90μg/ml的过滤臭氧作用5min。对照组为10ml含等量细菌的生理盐水,以同样方法通入等量过滤医用纯氧。取两组稀释液各1ml接种于细菌培养皿,倾注普通营养琼脂15～20ml并充分混匀,每组做3个培养皿重复,37℃培养24h后计数两组菌落数,计算杀菌率。杀菌率=(对照组平均菌落数-试验组平均菌落数)/对照组平均菌落数×100%。每个实验重复5次。验证臭氧对生理盐水中细菌的杀灭效果。
取等浓度的大肠埃希菌和金黄色葡萄球菌菌悬液0.5ml加入10ml血浆中。探索高浓度(90μg/ml)的过滤医用臭氧分别作用5min、10min和20min对其杀菌效果。
向无菌生理盐水中添加人新鲜冰冻血浆,按生理盐水与血浆体积比29:1、19:1、9:1、4:1、1:1、0:10混合,得到血浆终浓度分别为3.3%、5.0%、10.0%、20.0%、50.0%、100.0%的系列混合物。实验组于血浆浓度为分别为3.3%、5.0%、10.0%、20.0%、50.0%、100.0%生理盐水-血浆混合物各10ml中加入含菌量为0.5麦氏浓度的菌悬液0.5ml,通入80μg/ml过滤臭氧作用不同时间(5min、10min、20min);对照组为加入等量细菌的同稀释度稀释血浆,以同样流速通入等量过滤医用纯氧。探索血浆对臭氧杀菌的干扰作用。
1.2研究结果
20μg/ml以上臭氧作用5min对10ml生理盐水中大肠埃希菌、金黄色葡萄球菌杀灭率均可达100%,如减小臭氧浓度至5μg/ml对两种细菌的杀菌率分别降至46.72%和38.50%。臭氧对血浆中大肠埃希菌、金黄色葡萄球菌杀灭效果较生理盐水中差,90μg/ml臭氧作用5min对10ml血浆中大肠埃希菌、金黄色葡萄球菌的杀菌率分别只有18.14%和17.86%。延长作用时间可提高杀菌效果,臭氧作用20min对两种细菌的杀菌率可分别提高至50.35%和43.58%。向生理盐水中添加血浆,随血浆浓度的升高,不同血浆浓度(3.3%、5.0%、10.0%、20.0%、50.0%、100.0%)经浓度为80μg/ml的臭氧作用20min对大肠埃希菌及金黄色葡萄球菌的杀菌率从100%降低至50%以下。不同血浆浓度(3.3%、5.0%、10.0%、20.0%、50.0%、100.0%)组间对大肠埃希菌和金黄色葡萄球菌两种细菌的杀菌率均存在显著性差异(F=51420.241,P=0.000和F=75088.996,P=0.000),当血浆浓度在10.0%以上时以80μg/ml臭氧作用20min(相当于臭氧总量19.2mg)即可有细菌存活。不同作用时间组间对大肠埃希菌的杀菌率存在显著性差异(F=30885.690,P=0.000),对金黄色葡萄球菌亦然(F=27441.387,P=0.000),作用时间长者杀菌效果较好。
1.3研究结论
1、臭氧对生理盐水中大肠埃希菌和金黄色葡萄球菌有明显的杀菌效果,20μg/ml以上臭氧作用5min对10ml生理盐水中两种细菌杀灭率即可达100%。
2、臭氧对大肠埃希菌的杀菌效果优于金黄色葡萄球菌。
3、向生理盐水中添加血浆后,臭氧对其杀菌效率迅速降低。血浆浓度在10.0%以上时以80μg/ml臭氧作用20min(相当于臭氧总量19.2mg)即可有细菌存活。使用90μg/ml臭氧作用20min对血浆中的大肠埃希菌和金黄色葡萄球菌的杀灭率仅为50.35%和43.58%。说明血浆中存在抗氧化物质,能干扰臭氧的杀菌作用。进一步延长作用时间或可获得更高杀菌率。
第二部分医用臭氧对肝硬化所致腹水中细菌的杀灭作用研究
2.1研究方法
收集4例肝硬化顽固性腹水患者腹水标本共6份(其中一名患者于不同时间点取腹水3次)。4名患者的腹水按腹水TP含量由小到大的顺序编号1～4同一患者的不同腹水按腹水TP含量由小到大的顺序编号A～C(4号标本与C标本为同一份标本)。腹水采集前1周内未全身或局部使用抗生素。经检验腹水无菌,加入等量的大肠埃希菌和金黄色葡萄球菌,37℃培养24h后观察不同腹水中培养的菌落数。每份标本做7个培养皿重复,取其平均值。
取每份腹水标本各10ml中加入等浓度菌悬液0.5ml,均通入80μg/ml臭氧分别作用5min、10min、20min,含等量细菌的同来源腹水作为对照组以同样方法通入等量过滤医用纯氧。取两组的稀释液接种细菌培养皿,倾注普通营养琼脂后于37℃培养24h,进行活菌菌落计数,计算杀菌率,观察80μg/ml臭氧作用不同时间对4份不同来源腹水的杀菌效果以及同一病人不同腹水的杀菌效果。
实验当天同时检测腹水总蛋白(TP)等腹水常规及生化指标,观察臭氧对不同腹水的杀菌效果与该腹水TP含量的关系。
2.2研究结果
1～4号标本的腹水TP值分别为0.6、2.2、16.6、25.7 g/L。等量细菌加入4份腹水培养24小时后菌落数存在差异,1号标本形成菌落数最多,2号大于3号,4号菌落数最少。80μg/ml臭氧对不同来源腹水中大肠埃希菌的杀菌效果存在显著性差异(F=40499.603,P=0.000),对不同腹水中金黄色葡萄球菌杀菌率亦存在显著性差异(F=92525.40,P=0.000),其中80μg/ml臭氧对1号腹水标本(腹水TP含量为0.6g/L)和2号标本(腹水TP含量为2.0g/L)作用10 min(相当于臭氧总量9.6mg)后对两种细菌的杀菌率即可达100%,而对3号和4号标本(腹水TP含量分别为16.6g/L和25.7 g/L)作用10min后对两种细菌杀菌率仍均低于70%。臭氧不同作用时间(5min、10min、20min)间对大肠埃希菌的杀菌率存在显著性差异(F=28514.907,P=0.000),对金黄色葡萄球菌的杀菌率亦存在显著性差异(F=13066.314,P=0.000),作用时间长者杀菌效果较好,作用20min时对4份腹水中两种细菌的杀菌率均达到70%以上。不同腹水及不同臭氧作用时间对臭氧杀灭大肠埃希菌效率的影响存在交互效应(F=9775.527,P=0.000),对金黄色葡萄球菌的杀菌效果的影响亦存在交互作用(F=3242.915,P=0.00)(表2-3、表2-4)。臭氧作用5 min、10min、20min均显示80μg/ml的臭氧对4份腹水的杀菌效率1号优于2号,其次为3号,对4号标本杀菌效果最差(图2-3),即不同病人腹水其TP含量高者臭氧对其杀灭效果较差。
A～C号标本的腹水TP值分别为14.6、22.1、25.7 g/L。80μg/ml臭氧对同一病人3份不同腹水中大肠埃希菌的杀菌效果存在显著性差异(F=395.821,P=0.000),对不同腹水中金黄色葡萄球菌杀菌率亦存在显著性差异(F=520.915,P=0.000)。不同作用时间组间对大肠埃希菌的杀菌率存在显著性差异(F=38534.299,P=0.000),对金黄色葡萄球菌的杀菌率亦存在显著性差异(F=10800.111,P=0.000),作用时间长者杀菌效果较好。同一病人的不同腹水及不同臭氧作用时间对臭氧杀灭大肠埃希菌效率的影响存在交互效应(F=227.996,P=0.000),对金黄色葡萄球菌的杀菌效果的影响亦存在交互作用(F=140.123,P=0.00)(表2-6、表2-7)。臭氧作用5 min、10min、20min均显示80μg/ml的臭氧对3份腹水的杀菌效率A标本优于B标本,C标本最差(图2-5)。对同一病人的腹水来说,腹水中TP含量高者臭氧对其杀灭效果较差。
2.3研究结论
1、不同腹水环境对细菌生长有影响,腹水TP较高的标本细菌存活数量较少。
2、臭氧对腹水的杀菌效果优于血浆,但对不同腹水杀菌效果存在差异。验证了在蛋白含量较血浆低下的体液如腹水中生物安全剂量范围(不超过80μg/ml)内的臭氧即可发挥有效杀菌效果(即使对TP含量达16.6g/L的腹水以80μg/ml臭氧作用20min其杀菌率亦可达90%以上),腹水TP含量低者臭氧对其杀菌效率较高。
3、延长作用时间可提高臭氧杀菌效率。
Ozone is a chemical compound consisting of three oxygen atoms(O_3-triatomic oxygen),the pure gas has a soft sky-blue colour with pungent,acrid smell. Ozone is very instable in atmospheric,the life of the ozone molecule depends on the temperature,so that at 20℃the ozone concentration is halved within 40min.
Medical grade ozone(Medical ozone) must be made from pure medical oxygen,its concentration could be well controlled.Medical ozone is actually a mixture of pure oxygen and pure ozone in the ratio of 0.05%to 5%of O_3 and 95%to 99.95%of O_2.Due to the instability of the O_3 molecule medical ozone must be prepared immediately before use("ozone" was mentioned below instead of"medical ozone").
Among oxidant agents,ozone is the third strongest,after fluorine and persulfate.Because of its high oxidative power,it has sterilization effect on bacteria, HAV,and HIV with the ozone concentration of 20～80μg/ml in aqueous medium.
There are 150 million people with chronic hepatitis B virus(HBV) and hepatitis C virus(HCV) infection in our country.Blood transmission is the main hepatitis transmission route.Unfortunately,safety problems of blood products such as transfusion-transmitted virus and bacteria infections have not been solved yet. Because of the virus residue in blood,hepatitis B recurrence may still happen after liver transplantation to the patients whose end-stage liver disease was caused by chronic HBV infection.Currently,the most effective drugs used for preventing hepatitis B recurrence after liver transplantation are hepatitis B immunoglobulin (HBIG) combined with Lamivudine,but it costs a lot.There is still no effective preventive methods of hepatitis C recurrence after liver transplantation.
Among the major complications of cirrhosis,ascites seems to be the most frequent one.Patients with cirrhosis and ascites show a higher susceptibility to bacterial infections-mainly because of the inadequate defence mechanisms and bacterial translocation(BT).In these patients,the most frequent infectious complication that occurs(10%～25%of the cases),and at the same time the most severe one-is spontaneous bacterial peritonitis(SBP).SBP pathogenesis is considered to be the main consequence of bacterial translocation.More than 50% of the SBP episodes are produced by gram negative enteric bacilli-E.coli,followed by Staphylococcus aureus and Enterococcus faecalis.The efficiency of ascites concentration and reinfusion being enhanced after preconditioning ascites with disinfectant such as ozone is reasonable.
As ozone has such strong sterilization effect on kinds of microorganism,we test the bactericidal effect of ozone on body fluids(plasma,ascites),to get parameters of the sterilization effect of ozone for treating with body fluid such as plasma and ascites.
PartⅠStudy of the sterilization effect of ozone on plasma
1.Methods:
Escherichia coli(ATCC25922) and Staphylococcus aureus(ATCC25923) suspended in physiologic saline were treated by medical ozone with different concentration treating for 5 min.The killing rate was calculated according to the bactericidal assay of quantitative suspension.The bacteria killing rate were also tested in plasma with 90μg/ml ozone treating for different time.Then different ozone treating time was choosed to test the sterilization effect of ozone with the concentration of 80μg/ml on different saline-plasma mixture.
2.Results:
2.1 Ozone even with lower concentration has great sterilization effect on physiologic saline;
2.2 After treated with ozone of concentration of 90μg/ml for 20min,the killing rate to Escherichia coli and Staphylococcus aureus in whole plasma were only 50.35%and 43.58%respectively.
2.3 The bactericidal rate of ozone of the concentration 80μg/ml to Escherichia coli and Staphylococcus aureus decreased from 100%to less than 50%with plasma concentration increased.Significant difference exists in mass among groups of different ozone treating time(F=30885.690,P=0.000) and different plasma concentration(3.3%,5.0%,10.0%,20.0%,50.0%,100.0%)(F=51420.241,P=0.000 when treating with Escherichia coli,and the same condition occurs when treating with Staphylococcus aureus(F=27441.387,P=0.000 and F=75088.996,P=0.000 ).
3.Conclusions:
Ozone had better sterilization effect in physiologic saline than in plasma,even 10%plasma in bacterial suspensions inhibit oxidation and protect bacteria.Ozone had better sterilization effect on Escherichia coli than on Staphylococcus aureus.The sterilization effect depended on the ozone concentration,the treatment time and plasma concentration of samples.
PartⅡStudy of the sterilization effect of ozone on intractable ascites of cirrhosis
1.Methods:
4 samples of ascetic fluid was collected for bacterial culture.Numbered the samples from different patients in order of ascitic fluid TP as NO.1～4.Sample A～C from one patient were numbered on the same principle.NO.4 and Sample C were the same one.Antibiotics were not used for a week before ascites collection. Escherichia coli(ATCC25922) and Staphylococcus aureus(AYCC25923) was added to the ascitic fluid which had cultured no colony available,then cultured in 37℃.The number of colonies was counted 24h later.
Escherichia coli(ATCC25922) and Staphylococcus aureus(ATCC25923) was added to the 10 ml ascitic fluid and treated by medical ozone with the concentration of 80μg/ml treating for 5 min,10min and 20min respectively.The killing rate was calculated according to the bactericidal assay of quantitative suspension.
Routine tests(total amount of proteins,cultures in blood-culture bottles) of ascitic fluid were performed in the same day.
2.Results:
Total proteins(TP) of 4 ascitic fluid samples(No.1～No.4) were 0.6、2.2、16.6、25.7g/L respectively.There were differences between 4 samples in the cultured colony counts:No.l＞No.2＞No.3＞No.4.Ozone of the concentration 80μg/ml had better sterilization effect on ascites than on plasma.Significant difference exists in mass among groups of different ozone treating time(F=28514.907,P=0.000) and different ascitic fluid samples(F=40499.603,P=0.000) when treating with Escherichia coli,and the same condition occurs when treating with Staphylococcus aureus(F=13066.314,P=0.000 and F=92525.40,P=0.000).The bactericidal rate of ozone to Escherichia coli and Staphylococcus aureus increased with the ozone treating time increased,it all reached to more than 70%when ozone treating time was 20min.The same tendency of bactericidal efficiency was displayed among different time condition:No.1＞No.2＞No.3＞No.4,while the TP tendency seems to be opposite.
Total proteins(TP) of 3 ascitic fluid samples(Sample A～E) which collected from one patient were 14.6、22.1、25.7 g/L respectively.Significant difference exists in mass among groups of different ozone treating time(F=38534.299, P=0.000) and different ascitic fluid samples(F=395.821,P=0.000) when treating with Escherichia coli,and the same condition occurs when treating with Staphylococcus aureus(F=10800.111,P=0.000 and F=520.915,P=0.000).The bactericidal rate of ozone to Escherichia coli and Staphylococcus aureus increased with the ozone treating time increased.The same tendency of bactericidal efficiency was displayed among different time condition:Sample A＞B＞C,while the TP tendency seems to be opposite.
3.Conclusions:
3.1 The ascitic fluid samples with lower TP show a higher susceptibility to bacterial infections(more colony counts).
3.2 Ozone had better sterilization effect on ascites than on plasma.The sterilization effect differed in different ascitic fluid samples.The sterilization effect depended on ozone treating time and TP of ascitic fluid.The ascitic fluid with lower TP show a better sterilization effect.
臭氧具有极强的氧化性,其氧化性仅次于氟和过硫酸盐。已有大量研究表明,体外在生物耐受剂量浓度下(20～80μg/ml)的臭氧就能有效杀灭或灭活水体中多种微生物,包括G~+或G~-菌、HAV、HIV等。
我国有慢性肝炎病毒包括乙肝病毒(HBV)和丙肝病毒(HCV)感染者约1.5亿,主要传播途径为血液传播。由于检测技术的限制及病毒感染窗口期的存在,血液制品的生物安全性尚未得到有效解决。降低血液肝炎病毒载量对移植肝再感染也有利。对于慢性乙型肝炎病毒感染者进行肝移植,目前主要预防方法为口服核苷类似物联合静脉用乙肝免疫球蛋白(HBIG),费用高昂,且HBIG国内暂无上市;对于HCV感染者的移植肝再感染目前尚无有效的预防方法。自体输血作为目前最为安全有效的输血方式虽具有节约血源、减少不必要的输血反应和感染性疾病传播等诸多优势,但可能发生细菌污染,输用后将造成严重甚至致命的后果。以上问题的解决均依赖于安全、有效的血液净化方式。
腹水是肝硬化最常见的并发症之一。自发性腹膜炎(Spontaneous bacterialperitonitis,SBP)在肝硬化病例中的发生率为10%-25%,在腹水病例中的平均发生率约30%,是肝病严重的标志,病死率很高。SBP主要发生机制为宿主免疫防御功能减弱、肠道细菌移位(BT),感染的病原菌半数是大肠埃希菌,其次是金黄色葡萄球菌、肠球菌等。如能对此类患者腹水进行有效灭菌处理,其腹水浓缩后亦可回输体内,避免蛋白丢失或可提高治疗效果。
本研究目的在于利用臭氧体外水介质中能有效杀灭多种微生物的特性,试验臭氧在体外对体液(血浆、腹水等)的杀菌效果,为利用臭氧在体外对体液进行生物净化的可行性进行评价并提供实验参数。
第一部分医用臭氧对血浆中细菌杀灭作用的研究
1.1研究方法
向5份10ml含等量(0.5麦氏浓度)细菌的生理盐水中分别通入浓度为5μg/ml、10μg/ml、15μg/ml、20μg/ml、90μg/ml的过滤臭氧作用5min。对照组为10ml含等量细菌的生理盐水,以同样方法通入等量过滤医用纯氧。取两组稀释液各1ml接种于细菌培养皿,倾注普通营养琼脂15～20ml并充分混匀,每组做3个培养皿重复,37℃培养24h后计数两组菌落数,计算杀菌率。杀菌率=(对照组平均菌落数-试验组平均菌落数)/对照组平均菌落数×100%。每个实验重复5次。验证臭氧对生理盐水中细菌的杀灭效果。
取等浓度的大肠埃希菌和金黄色葡萄球菌菌悬液0.5ml加入10ml血浆中。探索高浓度(90μg/ml)的过滤医用臭氧分别作用5min、10min和20min对其杀菌效果。
向无菌生理盐水中添加人新鲜冰冻血浆,按生理盐水与血浆体积比29:1、19:1、9:1、4:1、1:1、0:10混合,得到血浆终浓度分别为3.3%、5.0%、10.0%、20.0%、50.0%、100.0%的系列混合物。实验组于血浆浓度为分别为3.3%、5.0%、10.0%、20.0%、50.0%、100.0%生理盐水-血浆混合物各10ml中加入含菌量为0.5麦氏浓度的菌悬液0.5ml,通入80μg/ml过滤臭氧作用不同时间(5min、10min、20min);对照组为加入等量细菌的同稀释度稀释血浆,以同样流速通入等量过滤医用纯氧。探索血浆对臭氧杀菌的干扰作用。
1.2研究结果
20μg/ml以上臭氧作用5min对10ml生理盐水中大肠埃希菌、金黄色葡萄球菌杀灭率均可达100%,如减小臭氧浓度至5μg/ml对两种细菌的杀菌率分别降至46.72%和38.50%。臭氧对血浆中大肠埃希菌、金黄色葡萄球菌杀灭效果较生理盐水中差,90μg/ml臭氧作用5min对10ml血浆中大肠埃希菌、金黄色葡萄球菌的杀菌率分别只有18.14%和17.86%。延长作用时间可提高杀菌效果,臭氧作用20min对两种细菌的杀菌率可分别提高至50.35%和43.58%。向生理盐水中添加血浆,随血浆浓度的升高,不同血浆浓度(3.3%、5.0%、10.0%、20.0%、50.0%、100.0%)经浓度为80μg/ml的臭氧作用20min对大肠埃希菌及金黄色葡萄球菌的杀菌率从100%降低至50%以下。不同血浆浓度(3.3%、5.0%、10.0%、20.0%、50.0%、100.0%)组间对大肠埃希菌和金黄色葡萄球菌两种细菌的杀菌率均存在显著性差异(F=51420.241,P=0.000和F=75088.996,P=0.000),当血浆浓度在10.0%以上时以80μg/ml臭氧作用20min(相当于臭氧总量19.2mg)即可有细菌存活。不同作用时间组间对大肠埃希菌的杀菌率存在显著性差异(F=30885.690,P=0.000),对金黄色葡萄球菌亦然(F=27441.387,P=0.000),作用时间长者杀菌效果较好。
1.3研究结论
1、臭氧对生理盐水中大肠埃希菌和金黄色葡萄球菌有明显的杀菌效果,20μg/ml以上臭氧作用5min对10ml生理盐水中两种细菌杀灭率即可达100%。
2、臭氧对大肠埃希菌的杀菌效果优于金黄色葡萄球菌。
3、向生理盐水中添加血浆后,臭氧对其杀菌效率迅速降低。血浆浓度在10.0%以上时以80μg/ml臭氧作用20min(相当于臭氧总量19.2mg)即可有细菌存活。使用90μg/ml臭氧作用20min对血浆中的大肠埃希菌和金黄色葡萄球菌的杀灭率仅为50.35%和43.58%。说明血浆中存在抗氧化物质,能干扰臭氧的杀菌作用。进一步延长作用时间或可获得更高杀菌率。
第二部分医用臭氧对肝硬化所致腹水中细菌的杀灭作用研究
2.1研究方法
收集4例肝硬化顽固性腹水患者腹水标本共6份(其中一名患者于不同时间点取腹水3次)。4名患者的腹水按腹水TP含量由小到大的顺序编号1～4同一患者的不同腹水按腹水TP含量由小到大的顺序编号A～C(4号标本与C标本为同一份标本)。腹水采集前1周内未全身或局部使用抗生素。经检验腹水无菌,加入等量的大肠埃希菌和金黄色葡萄球菌,37℃培养24h后观察不同腹水中培养的菌落数。每份标本做7个培养皿重复,取其平均值。
取每份腹水标本各10ml中加入等浓度菌悬液0.5ml,均通入80μg/ml臭氧分别作用5min、10min、20min,含等量细菌的同来源腹水作为对照组以同样方法通入等量过滤医用纯氧。取两组的稀释液接种细菌培养皿,倾注普通营养琼脂后于37℃培养24h,进行活菌菌落计数,计算杀菌率,观察80μg/ml臭氧作用不同时间对4份不同来源腹水的杀菌效果以及同一病人不同腹水的杀菌效果。
实验当天同时检测腹水总蛋白(TP)等腹水常规及生化指标,观察臭氧对不同腹水的杀菌效果与该腹水TP含量的关系。
2.2研究结果
1～4号标本的腹水TP值分别为0.6、2.2、16.6、25.7 g/L。等量细菌加入4份腹水培养24小时后菌落数存在差异,1号标本形成菌落数最多,2号大于3号,4号菌落数最少。80μg/ml臭氧对不同来源腹水中大肠埃希菌的杀菌效果存在显著性差异(F=40499.603,P=0.000),对不同腹水中金黄色葡萄球菌杀菌率亦存在显著性差异(F=92525.40,P=0.000),其中80μg/ml臭氧对1号腹水标本(腹水TP含量为0.6g/L)和2号标本(腹水TP含量为2.0g/L)作用10 min(相当于臭氧总量9.6mg)后对两种细菌的杀菌率即可达100%,而对3号和4号标本(腹水TP含量分别为16.6g/L和25.7 g/L)作用10min后对两种细菌杀菌率仍均低于70%。臭氧不同作用时间(5min、10min、20min)间对大肠埃希菌的杀菌率存在显著性差异(F=28514.907,P=0.000),对金黄色葡萄球菌的杀菌率亦存在显著性差异(F=13066.314,P=0.000),作用时间长者杀菌效果较好,作用20min时对4份腹水中两种细菌的杀菌率均达到70%以上。不同腹水及不同臭氧作用时间对臭氧杀灭大肠埃希菌效率的影响存在交互效应(F=9775.527,P=0.000),对金黄色葡萄球菌的杀菌效果的影响亦存在交互作用(F=3242.915,P=0.00)(表2-3、表2-4)。臭氧作用5 min、10min、20min均显示80μg/ml的臭氧对4份腹水的杀菌效率1号优于2号,其次为3号,对4号标本杀菌效果最差(图2-3),即不同病人腹水其TP含量高者臭氧对其杀灭效果较差。
A～C号标本的腹水TP值分别为14.6、22.1、25.7 g/L。80μg/ml臭氧对同一病人3份不同腹水中大肠埃希菌的杀菌效果存在显著性差异(F=395.821,P=0.000),对不同腹水中金黄色葡萄球菌杀菌率亦存在显著性差异(F=520.915,P=0.000)。不同作用时间组间对大肠埃希菌的杀菌率存在显著性差异(F=38534.299,P=0.000),对金黄色葡萄球菌的杀菌率亦存在显著性差异(F=10800.111,P=0.000),作用时间长者杀菌效果较好。同一病人的不同腹水及不同臭氧作用时间对臭氧杀灭大肠埃希菌效率的影响存在交互效应(F=227.996,P=0.000),对金黄色葡萄球菌的杀菌效果的影响亦存在交互作用(F=140.123,P=0.00)(表2-6、表2-7)。臭氧作用5 min、10min、20min均显示80μg/ml的臭氧对3份腹水的杀菌效率A标本优于B标本,C标本最差(图2-5)。对同一病人的腹水来说,腹水中TP含量高者臭氧对其杀灭效果较差。
2.3研究结论
1、不同腹水环境对细菌生长有影响,腹水TP较高的标本细菌存活数量较少。
2、臭氧对腹水的杀菌效果优于血浆,但对不同腹水杀菌效果存在差异。验证了在蛋白含量较血浆低下的体液如腹水中生物安全剂量范围(不超过80μg/ml)内的臭氧即可发挥有效杀菌效果(即使对TP含量达16.6g/L的腹水以80μg/ml臭氧作用20min其杀菌率亦可达90%以上),腹水TP含量低者臭氧对其杀菌效率较高。
3、延长作用时间可提高臭氧杀菌效率。
Ozone is a chemical compound consisting of three oxygen atoms(O_3-triatomic oxygen),the pure gas has a soft sky-blue colour with pungent,acrid smell. Ozone is very instable in atmospheric,the life of the ozone molecule depends on the temperature,so that at 20℃the ozone concentration is halved within 40min.
Medical grade ozone(Medical ozone) must be made from pure medical oxygen,its concentration could be well controlled.Medical ozone is actually a mixture of pure oxygen and pure ozone in the ratio of 0.05%to 5%of O_3 and 95%to 99.95%of O_2.Due to the instability of the O_3 molecule medical ozone must be prepared immediately before use("ozone" was mentioned below instead of"medical ozone").
Among oxidant agents,ozone is the third strongest,after fluorine and persulfate.Because of its high oxidative power,it has sterilization effect on bacteria, HAV,and HIV with the ozone concentration of 20～80μg/ml in aqueous medium.
There are 150 million people with chronic hepatitis B virus(HBV) and hepatitis C virus(HCV) infection in our country.Blood transmission is the main hepatitis transmission route.Unfortunately,safety problems of blood products such as transfusion-transmitted virus and bacteria infections have not been solved yet. Because of the virus residue in blood,hepatitis B recurrence may still happen after liver transplantation to the patients whose end-stage liver disease was caused by chronic HBV infection.Currently,the most effective drugs used for preventing hepatitis B recurrence after liver transplantation are hepatitis B immunoglobulin (HBIG) combined with Lamivudine,but it costs a lot.There is still no effective preventive methods of hepatitis C recurrence after liver transplantation.
Among the major complications of cirrhosis,ascites seems to be the most frequent one.Patients with cirrhosis and ascites show a higher susceptibility to bacterial infections-mainly because of the inadequate defence mechanisms and bacterial translocation(BT).In these patients,the most frequent infectious complication that occurs(10%～25%of the cases),and at the same time the most severe one-is spontaneous bacterial peritonitis(SBP).SBP pathogenesis is considered to be the main consequence of bacterial translocation.More than 50% of the SBP episodes are produced by gram negative enteric bacilli-E.coli,followed by Staphylococcus aureus and Enterococcus faecalis.The efficiency of ascites concentration and reinfusion being enhanced after preconditioning ascites with disinfectant such as ozone is reasonable.
As ozone has such strong sterilization effect on kinds of microorganism,we test the bactericidal effect of ozone on body fluids(plasma,ascites),to get parameters of the sterilization effect of ozone for treating with body fluid such as plasma and ascites.
PartⅠStudy of the sterilization effect of ozone on plasma
1.Methods:
Escherichia coli(ATCC25922) and Staphylococcus aureus(ATCC25923) suspended in physiologic saline were treated by medical ozone with different concentration treating for 5 min.The killing rate was calculated according to the bactericidal assay of quantitative suspension.The bacteria killing rate were also tested in plasma with 90μg/ml ozone treating for different time.Then different ozone treating time was choosed to test the sterilization effect of ozone with the concentration of 80μg/ml on different saline-plasma mixture.
2.Results:
2.1 Ozone even with lower concentration has great sterilization effect on physiologic saline;
2.2 After treated with ozone of concentration of 90μg/ml for 20min,the killing rate to Escherichia coli and Staphylococcus aureus in whole plasma were only 50.35%and 43.58%respectively.
2.3 The bactericidal rate of ozone of the concentration 80μg/ml to Escherichia coli and Staphylococcus aureus decreased from 100%to less than 50%with plasma concentration increased.Significant difference exists in mass among groups of different ozone treating time(F=30885.690,P=0.000) and different plasma concentration(3.3%,5.0%,10.0%,20.0%,50.0%,100.0%)(F=51420.241,P=0.000 when treating with Escherichia coli,and the same condition occurs when treating with Staphylococcus aureus(F=27441.387,P=0.000 and F=75088.996,P=0.000 ).
3.Conclusions:
Ozone had better sterilization effect in physiologic saline than in plasma,even 10%plasma in bacterial suspensions inhibit oxidation and protect bacteria.Ozone had better sterilization effect on Escherichia coli than on Staphylococcus aureus.The sterilization effect depended on the ozone concentration,the treatment time and plasma concentration of samples.
PartⅡStudy of the sterilization effect of ozone on intractable ascites of cirrhosis
1.Methods:
4 samples of ascetic fluid was collected for bacterial culture.Numbered the samples from different patients in order of ascitic fluid TP as NO.1～4.Sample A～C from one patient were numbered on the same principle.NO.4 and Sample C were the same one.Antibiotics were not used for a week before ascites collection. Escherichia coli(ATCC25922) and Staphylococcus aureus(AYCC25923) was added to the ascitic fluid which had cultured no colony available,then cultured in 37℃.The number of colonies was counted 24h later.
Escherichia coli(ATCC25922) and Staphylococcus aureus(ATCC25923) was added to the 10 ml ascitic fluid and treated by medical ozone with the concentration of 80μg/ml treating for 5 min,10min and 20min respectively.The killing rate was calculated according to the bactericidal assay of quantitative suspension.
Routine tests(total amount of proteins,cultures in blood-culture bottles) of ascitic fluid were performed in the same day.
2.Results:
Total proteins(TP) of 4 ascitic fluid samples(No.1～No.4) were 0.6、2.2、16.6、25.7g/L respectively.There were differences between 4 samples in the cultured colony counts:No.l＞No.2＞No.3＞No.4.Ozone of the concentration 80μg/ml had better sterilization effect on ascites than on plasma.Significant difference exists in mass among groups of different ozone treating time(F=28514.907,P=0.000) and different ascitic fluid samples(F=40499.603,P=0.000) when treating with Escherichia coli,and the same condition occurs when treating with Staphylococcus aureus(F=13066.314,P=0.000 and F=92525.40,P=0.000).The bactericidal rate of ozone to Escherichia coli and Staphylococcus aureus increased with the ozone treating time increased,it all reached to more than 70%when ozone treating time was 20min.The same tendency of bactericidal efficiency was displayed among different time condition:No.1＞No.2＞No.3＞No.4,while the TP tendency seems to be opposite.
Total proteins(TP) of 3 ascitic fluid samples(Sample A～E) which collected from one patient were 14.6、22.1、25.7 g/L respectively.Significant difference exists in mass among groups of different ozone treating time(F=38534.299, P=0.000) and different ascitic fluid samples(F=395.821,P=0.000) when treating with Escherichia coli,and the same condition occurs when treating with Staphylococcus aureus(F=10800.111,P=0.000 and F=520.915,P=0.000).The bactericidal rate of ozone to Escherichia coli and Staphylococcus aureus increased with the ozone treating time increased.The same tendency of bactericidal efficiency was displayed among different time condition:Sample A＞B＞C,while the TP tendency seems to be opposite.
3.Conclusions:
3.1 The ascitic fluid samples with lower TP show a higher susceptibility to bacterial infections(more colony counts).
3.2 Ozone had better sterilization effect on ascites than on plasma.The sterilization effect differed in different ascitic fluid samples.The sterilization effect depended on ozone treating time and TP of ascitic fluid.The ascitic fluid with lower TP show a better sterilization effect.
引文
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[22]叶英,余鑫之,徐元宏.严重肝病腹腔感染的致病菌变迁和耐药趋势研究[J].中华肝脏病杂志,2001,9(4):208
[23]刘丽敏,池艳春,朱丹,等.体外浓缩自体腹水回输治疗肝硬化顽固性腹水的临床观察[J].中华肝脏病杂志,2004,12(5):314
[24]杨晋辉,余曙光,尤丽英,等.自体腹水浓缩后腹腔回输治疗顽固性腹水[J].中华内科杂志,1996,35(7):476-477.
[25]李富欢,刘红生,黄六英,等.腹水浓缩回输腹腔治疗肝硬化难治性腹水的效果评价.实用肝脏病杂志,2005,8(3):157-159.
[26]Sharma M,Hudson JB.Ozone gas is an effective and practical antibacterial agent[J].Am J Infect Control,2008 Oct,36(8):559-63
[27]Thurston-Enriquez JA,Haas CN,Jacangelo J,et al.Inactivation of enteric adenovirus and feline calicivirus by ozone[J].Water Res,2005Sep,39(15):3650-6.
[28]Wells KH,Latino J,Gavalchin J,et al.Inactivation of human immunodeficiency virus type 1 by ozone in vitro[J].Blood,1991 Oct 1,78(7):1882-90.
[29]Mezzanotte V,Antonelli M,Citterio S,et al.Wastewater disinfection alternatives:chlorine,ozone,peracetic acid,and UV light[J].Water Environ Res,2007 Nov,79(12):2373-9.
[30]Coronel B,Duroselle P,Behr H,et al.In situ decontamination of medical wastes using oxidative agents:a 16-month study in a polyvalent intensive care unit[J].J Hosp Infect,2002 Mar,50(3):207-12.
[31]Roy D,Wong PKY,Engelbrecht RS,et al.Mechanism of enteroviral inactivation by ozone[J].Appl Environ Microbiol,1981,41(3):718.
[32]Mudd JB,Reavitt R,Ongun A,et al.Reaction of ozone with amino acids and their derivatives following exposure to ozone and ultraviolet radiations[J].Arch Biochem Biophys,1954,51:208.
[33]Kim CK,Gentile DM,Sproul OJ.Mechanism of Ozone Inactivation of Bacteriophage f2.[J].Appl Environ Microbiol,1980,39(1):210-218.
[34]Bocci,V.Oxidants in Biology[M].Netherlands:Springer Netherlands,2008.155-165
[35]谈智,吉钟山,徐燕.臭氧对水中微生物的杀灭效果及其影响因素[J].中国消毒学杂志,2001,16(3):154-156.
[36]王芳,刘育京,李汉忠,等.蛋白胨对臭氧水杀菌作用影响的试验研究[J].中国公共卫生,2002,18(4):421-422.
[37]Cross CE,Reznick AZ,Packer L,et al.Oxidative damage to human plasma proteins by ozone[J].Free Radic Res Commun,1992,15(6):347-52.
[38]Valacchi G.,Fortino V.,BocciV..The dual action of ozone on the skin[J].Br J Dermatol 2005,153(6):1096-1100
[39]中华医学会传染病和寄生虫病学分会、肝病学分会.病毒性肝炎防治方案[J].中华肝脏病杂志,2000,8(6):324-329.
[40]秦波,郭树华.自发性细菌性腹膜炎[J].中华肝脏病杂志,2003,11(7):439-440
[41]Moore KP,Wong F,Gines P,et al.The management of ascites in cirrhosis:report on the consensus conference of the International As-cites club[J]. Hepatology,2003,38(1):258-266.
[42]翁心华.自发性细菌性腹膜炎的一些共识与进展[J].中华肝脏病杂志,2003,11(7):389-390
[43]赵勇华,于建武,李树臣.腹水超滤浓缩-细菌过滤回输腹腔治疗肝炎肝硬化合并自发性细菌性腹膜炎的临床疗效分析[J].肝脏,2006,11(4):235-237
[44]饶敏,蒋音.肝硬化并发自发性细菌性腹膜炎患者腹水病原菌及其耐药分析[J].肝脏,2008,13(5):444-445
[45]郑临,杨益大,孔海深,等.肝硬化患者自发性腹膜炎细菌学及耐药性的变迁[J].中华传染病杂志,2001,19(3):181-182.
[46]Guarner C,Sol(?) R,Soriano G,et al.Risk of a first community-acquired spontaneous bacterial peritonitis in cirrhotics with low ascitic fluid protein levels[J].Gastroenterology.1999,117(2):414-9.
[47]Bette M,N(u|¨)sing RM,Mutters R,et al.Efficiency of tazobactam/piperacillin in lethal peritonitis is enhanced after preconditioning of rats with O_3/O2-pneumoperitoneum[J].Shock,2006,25(1):23-9.