抗生素锁技术治疗中心静脉导管相关感染的实验室研究

发布时间：2018-05-29 04:25

本文选题：抗生素锁 + 导管相关感染　；参考：《长江大学》2015年硕士论文

【摘要】：中心静脉置管术以其操作简单、留置时间长等优点广泛应用于临床,成为急危重病救护的必要手段之一。但由置管引发的感染率也呈逐年增加的趋势,其中表皮葡萄球菌的分离概率高达60%-80%。导管相关感染与细菌容易形成生物膜有很大关系,生物膜的形成使全身用药疗效较差,患者被迫拔管的机率大大增加,使患者的治愈率降低,死亡率增加。抗生素锁技术(Antibiotic Lock Technique, ALT)是将高浓度的抗生素注入导管内,有效清除细菌生物膜,达到保留导管目的的一种技术。但是针对该技术治疗表皮葡萄球菌引起的导管相关感染的基础研究相对较少,其使用的有效性、使用时是否产生负作用以及合适的使用浓度和时间都有待于进一步探讨。为了揭示这些问题,我们将最易引起导管相关感染的表皮葡萄球菌产膜阳性菌株ATCC35984作为实验菌株,常用抗生素头孢他啶作为ALT抗菌溶液做相关性研究,希望能为今后临床开展此技术治疗导管相关感染提供科学的理论依据。目的：第一：通过动物实验比较ALT与全身用药治疗表皮葡萄球菌引起的导管相关感染的效果,同时观测使用ALT是否产生细菌耐药、对局部血管有无损伤,由此来确认该技术能否作为治疗导管相关感染的常规疗法。第二：在常规的药物浓度范围内和常用的锁定时间内,找到灭菌效果最好的药物溶液浓度和最有效的锁定时间,为以后临床使用该技术提供理论依据。方法：第一阶段：将形成表皮葡萄球菌生物膜的导管植入动物体内,将动物随机分为三组：全身用药组(A组)每天全身使用头孢他啶,剂量为100mg/kg×体重,导管内注入无菌生理盐水；导管注药组(B组)在导管内注入0.3m1头孢他啶-肝素混合液,剂量为7.2mg/ml;联合用药组(C组)既进行全身用药又进行导管注药。三组动物干预8天后停止干预,三天后拔管。这期间分别在置管后第2、4、6、8、11天采集导管血和外周血。采用稀释平板计数法进行细菌定量培养,银染法检测管腔生物膜情况,比较三种方法清除细菌生物膜的效果；应用酶联免疫吸附法测定外周血血管性假性血友病因子(von Wilebrand Factor, vWF)的含量比较三组方法对血管内皮损伤程度,同时做细菌定性培养判断动物是否发生全身感染；采用纸片扩散法对动物体内分离出的表皮葡萄球菌做药敏实验,判断细菌是否对抗生素产生耐药性。第二阶段：采用置片法将含有细菌生物膜的硅胶薄膜按药物干预的不同浓度随机分成300倍最低抑菌浓度(Minimum Inhibitory Concentration,MIC)、600倍MIC、900倍MIC三个浓度组,每组在干预至6h、12h、18h、24h四个时间点取出处理后,通过稀释平板计数法进行细菌计数定量培养,交叉比较出ALT最有效的使用时间和使用浓度。所有数据统计均采用SPSS17.0统计软件,细菌计数结果使用均数±标准差表示,统计学分析的数据应用LSD-t检验、独立样本t检验,P0.05为差异有统计学意义。结果：(1)导管血计数结果：在干预期间,B组和C组细菌计数结果无统计学差异,但与A组之间有显著性差异,p均0.000；停止干预时,A组计数仍居高不下,B组和C组共有9只动物计数结果为0(其中B组4只占66.67%,C组5只占83.3%)；停止干预后,B组和C组在停止干预时计数结果为0的9只动物在停止干预后仍为0。(2)外周血定性培养结果：未进行干预前,三组外周血定性培养均为阴性；干预第2天,A组3例阳性、B组和C组无阳性；干预第4天,A组4例阳性、B组1例阳性、C组无阳性；干预第6天,A组5例阳性、B组1例阳性、C组无阳性。干预停止时A组全部呈现阳性、B组2例阳性、C组无阳性；在停止干预后,B组仍然2例阳性、C组出现1例阳性。(3)导管处理结果：计数结果显示：B组和C组的共有9个标本的细菌计数结果为0(其中B组4个C组5个,总计75%),其余标本平均计数结果则达到51ogcfu/ml；银染结果显示：A组的留置针管腔内呈现出黑色相连的絮状物与菌膜分界不清；B组和C药组中未完全清除生物膜的导管内可见明显的斑块和少量聚集成团的絮状物；已完全清除生物膜的导管内没有明显的黑色团块和斑点。(4)vWF结果：置管后第2天,A组vWF含量显著高于B、C组,随着置管时间延长,三组vWF含量均逐渐升高,且A组vWF含量仍然显著高于B、C组,差异性有统计学意义,p0.001；停止干预后,A、B、C组vWF含量持续升高,三组之间vWF含量均有统计学差异,p0.001。(5)耐药性检测：三组都没有出现耐药。(6)不同浓度下最有效时间：300倍MIC干预下的硅胶薄膜细菌计数与对照组无显著差异；600倍MIC干预下的硅胶薄膜细菌在18h的计数结果出现显著性差异,P0.001；900倍MIC干预下的硅胶薄膜细菌在12h的计数结果出现显著性差异,P0.001。(7)不同时间下最有效浓度：在相同的锁定时间内,经300倍MIC干预后的硅胶薄膜细菌计数结果最高,其次是600倍MIC,900倍MIC干预后的硅胶薄膜细菌计数结果最低,三组之间均有显著性差异,P均0.001。结论：根据以上研究结果可得到如下结论：(1)仅全身用药不能有效清除细菌生物膜里的细菌,而导管注药和联合用药均能有效地清除生物膜细菌,即：使用ALT可有效清除生物膜。(2)使用ALT可降低全身感染的发生,但要合理控制注药的量以及注意注药的方法。(3)在ALT的临床使用过程中,配合全身用药可更加有效的防治全身感染。(4)若导管内细菌生物膜已完全被清除,在短期内不会引起再次感染,若导管内细菌生物膜未被完全清除,在停止干预后仍然有复发的可能。(5)在正确使用ALT的前提下,不会损伤血管内皮细胞,在短期内且不会引起细菌耐药。(6)300倍MIC作为ALT溶液不能有效清除细菌生物膜,而600倍和900倍MIC的溶液均具有良好的清除细菌生物膜的作用。且在相同的锁定时间内,浓度越高,其清除生物膜的能力越强,效果越好。(7)600倍MIC作为ALT溶液,锁定18小时效果最好；900倍MIC作为ALT溶液,锁定12小时效果最好。因此,在今后的临床应用中,若以600倍和900倍MIC作为ALT溶液,在治疗允许的情况下建议分别锁定18小时和12小时,以最大程度的清除细菌生物膜。
[Abstract]:In order to reveal these problems , we can find out whether the technology can be used as a conventional therapy for treating catheter - related infections .
The catheter blood and peripheral blood were collected in three groups after 8 days of intervention and three days later . The results of bacterial quantitative culture , silver staining and silver staining were used to detect the biofilm in the lumen and the effect of three methods on bacterial biofilm was compared .
Using enzyme - linked immunosorbent assay ( ELISA ) to determine the vascular endothelial cell damage degree , the degree of vascular endothelial injury was determined by three groups of methods , while bacterial qualitative training was used to determine whether systemic infection occurred in animals .
The results were as follows : ( 1 ) The results were as follows : ( 1 ) The results showed that : ( 1 ) There was no statistical difference between group B and C group during the intervention period , but there was significant difference between group B and group C , p = 0.000 ;
When the intervention was stopped , the count of group A remained high , there were 9 animal counts in group B and C group ( 66.67 % in group B and 83.3 % in group C ) .
After stopping the intervention , 9 animals in group B and group C counted as 0 after stopping intervention were still 0 . ( 2 ) peripheral blood qualitative culture results after stopping intervention : before intervention , all three groups of peripheral blood were negative ;
in group A , 3 were positive , group B and C were not positive in group A ;
The 4th day of intervention , 4 positive in group A , 1 in group B and no positive in group C ;
At the 6th day of intervention , 5 cases were positive in group A , 1 in group B and no positive in group C . All groups were positive in group A and 2 in group B were positive in group B .
After stopping the intervention , there were 2 positive cases in group B and 1 positive in group C . ( 3 ) The results showed that the bacterial count of 9 specimens in group B and C was 0 ( 5 of 4 C groups in group B and 75 % in total ) , and the average count of remaining samples reached 51ogcfu / ml ;
The results of silver staining showed that the black - connected flocs in the indwelling needle lumen of group A were not clear .
In group B and group C , there were obvious plaques and small aggregates of flocs in the catheter without completely clearing the biofilm ;
There were no significant black lumps and spots in the catheter which had completely cleared the biofilm . ( 4 ) The results were as follows : ( 4 ) There was a significant difference in the levels of the levels of the levels in the group A and the group C after the treatment . The content in group A was significantly higher than that in group B and C , and the content of the A in group A was still significantly higher than that in group B and C . The difference was significant , p 0.001 ;
There was no significant difference between the two groups . ( 6 ) There was no significant difference between the bacterial count of silica gel membrane and the control group at the different concentration .
The results showed that there was a significant difference in the bacterial count of silica gel membrane bacteria at 600 times MIC intervention ( P < 0.01 ) .
The results showed that : ( 1 ) In the course of clinical use of ALT , the bacterial biofilm could not be effectively removed by using ALT .
As an ALT solution , 900 - fold MIC was best used to lock the 12 - hour effect . Therefore , in future clinical applications , if 600 - fold and 900 - fold MIC were used as ALT solutions , it was recommended to lock for 18 and 12 hours , respectively , in the case of treatment , to minimize bacterial biofilm .
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.3

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