泛耐药鲍曼不动杆菌包膜蛋白对致病性的影响

发布时间：2018-05-07 07:06

本文选题：不动杆菌 + 多重耐药　；参考：《河北医科大学》2013年硕士论文

【摘要】：目的：探讨ICU患者下呼吸道检出不动杆菌属耐药情况、不同致病状态的检出菌包膜蛋白产生情况及其与致病状态的关系。我们共分离出10株定植菌株，32株感染菌株。通过分析其感染高危因素，探讨定植菌与感染菌宿主在临床方面差异。根据CPIS评分和定量培养的结果来判定细菌的定植与感染状态。CPIS评分包括：体温，外周血白细胞分类及计数，，气道脓性分泌物的量，氧合指数，胸部影像检查结果和呼吸道抽吸物革兰染色镜检及培养结果。当CPIS评分大于6分，同时定量培养结果大于菌落计数106CFU/ml诊断为呼吸机相关性肺炎，即患者为感染状态。并研究耐药的鲍曼不动杆菌在定植及感染状态下外膜毒力蛋白（OmpA/BauA）的表达与其致病性的关系。方法： 1菌株来源：收集2011-2012年来自河北省4家医院的42株鲍曼不动杆菌菌株。 2药敏试验：采用K-B法鉴定分离的鲍曼不动杆菌对头孢哌酮/舒巴坦、头孢哌酮、头孢噻肟、哌拉西林、头孢他啶、头孢吡肟、比阿培南、美罗培南、阿米卡星、环丙沙星、左氧氟沙星、庆大霉素、氯霉素、氨曲南、米诺环素、复方新诺明16种抗菌素的敏感性。并根据美国临床实验室标准化委员会（NCCLS）2008年版要求进行抗菌药物敏感性判断。 3分析患者临床高危因素：通过药敏试验从中筛选出多重耐药，泛耐药及全耐药的菌株，根据CPIS评分和患者分泌物定量培养的结果分离出定植菌株10株，感染菌株32株，并收集患者的临床资料分析其两组检出菌宿主之间感染高危因素差别。 4PCR扩增外膜蛋白Ompa及BauA基因：根据GenBank公布的序列，设计Ompa(AY485227)及BauA(AY571146)基因的特异性引物。Ompa基因上游引物OMP1-5’ATTACGACGGAAATTGTAAGTAATTT-3’定位于Ompa基因+1一+26bp位碱基，下游引物OMP25’-CGACTCGTTAGAGTCGCTTTTTTATG-3’，定位于Ompa基因+1291一+1317bp位碱基，BauA基因上游引物OMP15’-TGCAAGCCCAACCTCACT-3’定位于BauA基因+4060一+4078bp位碱基；下游引物OMP25’-TTCTCCAGACTTAGGGAT-3’，定位于BauA基因+6642一+6660bp位碱基进行PCR扩增，将产物在0.8%琼脂糖凝胶电泳。用凝胶成像系统摄像并保存。 5PCR产物序列分析。结果： 1药敏试验应用K-B法检测42株鲍曼不动杆菌均表现为多重耐药，其中对头孢哌酮/舒巴坦耐药率最低为30.95%，对头孢噻肟的耐药率95.23%。头孢哌酮为88.10%，氨曲南及米诺环素分别为97.6%和78.57%；头孢吡肟、头孢他啶均为76.20%，庆大霉素、阿米卡星均为85.72%；哌拉西林与左氧氟沙星均为83.34%；复方新诺明为73.81%，对亚胺培南及比阿培南耐药率分别为73.81%、92.86%，氯霉素的耐药性69.05%，环丙沙星为90.48%。 2分析患者临床高危因素：通过复习病历收集多重耐药鲍曼不动杆菌定植或感染患者的临床资料（年龄、性别、医学合并症、ICU住院时间、侵入性操作及其持续时间、预后等资料）、结合临床干预效果42株菌株分为定植组10例、感染组32例。最常见的暴露因素为：入住ICU，机械通气，其他侵入性操作。最常见的基础疾病为：脑血管病，其次为慢性阻塞性肺疾病。对于定植组与感染组临床收集的资料无明显统计学意义（P0.05）。感染组的死亡率大于定植组，但是两组的死亡率差异无统计学意义（15.66%vs10%，P0.05）。但是对于ICU患者的定植与感染组中，感染组患者ICU的住院时间明显大于定植组（P0.05）有统计学意义。 3PCR法细菌包膜蛋白测定：BauA定植组（阳性率70%，阴性率30%)，感染组（阳性率53.13%，阴性率46.87%）。通过卡方检验统计学方法分析其两者的构成比，P＞0.05，OmpA定植组（阳性率80%，阴性率20%)，感染组（阳性率87.5%，阴性率12.5%）。通过卡方检验统计学方法分析其两者的构成比，P＞0.05，说明两者之间包膜蛋白构成比差异无统计学意义，尚不能认为定植组与感染组之间细菌的致病力与这两种蛋白之间有关。结论： 1本实验通过药敏分析了河北省4家医院收集的鲍曼不动杆菌的耐药情况。对头孢哌酮\舒巴坦耐药率最低为30.95%，对氨曲南的耐药率最高，达到了97.60%，对碳青酶烯类耐药率也在73.81%到92.86%之间。 2定植组与感染组在临床高危因素方面没有明显区别，在死亡率上也没有区别。但ICU入住时间在定植与感染组中有统计学差异，说明入住ICU时间越长造成菌株侵袭性机会越高。 3通过PCR分子生物学技术分析包膜蛋白OmpA在两组之间无统计学差异，与铁相关的外膜转运蛋白BauA在定植组与感染组之间也无统计学差异。尚不能认为这两种蛋白产生水平与泛耐药鲍曼不动杆菌致病性相关。可能与其他毒力蛋白及患者免疫状态的关系可能更密切。
[Abstract]:Objective : To investigate the relationship between bacterial resistance and pathogenic state in patients with lower respiratory tract infection in ICU . 10 strains of colonization and 32 strains of infection were isolated .

Method :

1 . Source : Collection of 42 strains of acinetobacter baumanii from 4 hospitals in Hebei Province in 2011 - 2012 .

2 Drug sensitivity test : The susceptibility of isolated baumanii was identified by K - B method . The susceptibility to antibiotics was determined according to the 2008 edition of American Clinical Laboratory Standardization Committee ( NCCLS ) .

3 . The clinical and high risk factors of the patients were analyzed . The strains with multiple drug resistance , pan - resistance and total resistance were screened from the drug - sensitive test , 10 strains were isolated according to CPIS score and quantitative culture of the patient ' s secretions , 32 strains were infected , and the clinical data of the patients were collected to analyze the difference of the high risk factors between the two groups .

Ompa ( AY485227 ) and BauA ( AY571146 ) gene were amplified by 4PCR . Ompa ( AY485227 ) and BauA ( AY571146 ) gene specific primers were designed according to the published sequence . The upstream primer of Ompa gene was located at the base of Ompa gene + 1 + 26bp . The upstream primer OMP15 ' - TGCAAGCCCAACCTCACT - 3 ' was located at the base of BauA gene + 4060 + 4078bp .
The downstream primer OMP25 ' - TTCTCCAGACT TAGGGAT - 3 ' was subjected to PCR amplification at the base of the BauA gene + 6642 I + 6660bp , and the product was subjected to gel electrophoresis of 0.8 % agarose gel .

5PCR product sequence analysis .

Results :

The drug resistance rate was 30.95 % , the drug resistance rate was 95.23 % , 80.10 % , 97.6 % and 78.57 % respectively .
Cefepime and ceftazidine were 76.20 % and 85.72 % respectively .
Piperacillin and levofloxacin were 83.34 % ;
The drug resistance rates were 73.81 % , 73.81 % , 92.86 % , 69.05 % and 90.48 % respectively .

The clinical data ( age , sex , medical complications , ICU admission time , invasive operation , duration and prognosis ) of patients with multiple drug resistance were analyzed . The most common underlying diseases were cerebrovascular disease , followed by chronic obstructive pulmonary disease . The most common underlying diseases were : cerebrovascular disease , followed by chronic obstructive pulmonary disease . The mortality rate in the infection group was higher than that in the colonization group , but there was no significant difference between the two groups ( 15.66 % vs 10 % , P0.05 ) . However , in the patients with ICU and infection group , the hospitalization time of ICU in the infection group was significantly higher than that in the colonization group ( P0.05 ) .

Detection of bacterial envelope protein by 3PCR method : BauA colonization group ( positive rate 70 % , negative rate 30 % ) , infection group ( positive rate 53.13 % , negative rate 46.87 % ) . By means of Chi - square test , the composition ratio , P & gt ; 0.05 , OmpA colonization group ( positive rate 80 % , negative rate 20 % ) , infection group ( positive rate 87.5 % , negative rate 12.5 % ) were analyzed . By means of Chi - square test , the composition ratio of the two proteins was analyzed by means of Chi - square test , P > 0.05 . The difference between the two proteins was not considered statistically significant , and it was not considered that the pathogenicity of bacteria between the colonization group and the infection group was related to the two proteins .

Conclusion :

1 . The drug resistance of baumanii collected by 4 hospitals in Hebei Province was analyzed by drug sensitivity . The drug resistance rate was 30 . 95 % , and the drug resistance rate to aztreonam was the highest , reaching 97.60 % , and the drug resistance rate was 73.81 % to 92.86 % .

There was no significant difference between the 2 colonization group and the infection group in the clinical high risk factors , but there was no difference in mortality rate . However , the ICU stay time had statistical difference in the colonization and infection group , indicating that the longer the ICU time , the higher the chance of invasive ability of the strain .

3 . There was no statistical difference between the two groups by PCR molecular biology technique . There was no statistical difference between the two groups .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R516;R459.7

【参考文献】