【作者】 陈菁；

【导师】 严延生；

【作者基本信息】 福建医科大学 ， 病原微生物学， 2014， 博士

【摘要】 目的鲍曼不动杆菌是医院感染常见的病原菌之一,随着抗生素的广泛使用,已出现了多重耐药、广泛耐药,甚至全耐药的菌株。住院患者特别是重症患者一旦感染,将面临无药可救的局面。多重耐药鲍曼不动杆菌(MDRAB)引起的医院感染爆发流行已成为国内外学者关注的重要公共卫生问题。多项研究表明由于各地医院抗菌药物使用情况不同及耐药菌控制的差异,MDRAB在世界各地的流行也存在差异。本课题通过研究本地区MDRAB的流行特征及其耐药机制,为预防和控制MDRAB在本地区的爆发流行提供参考。方法1.回顾性调查分析2011年1月至2012年6月在某综合性医院住院且发生鲍曼不动杆菌感染的患者病历资料,以MDRAB感染者为病例组,同时期鲍曼不动杆菌敏感株感染者为对照组进行病例对照研究,调查结果采用t检验、χ2检验及多因素logistic回归进行数据分析。2.随机收集2009年~2012年某综合性医院各类临床标本分离出的MDRAB菌株286株,采用VITEK260细菌鉴定仪鉴定,K-B法进行药敏试验,采用脉冲场凝胶电泳(PFGE)进行同源性分析。3.细菌抗菌药物敏感性结果采用WHONET5.6软件进行统计分析,耐药率差别统计采用χ2检验。4.采用悬液定量杀菌试验测定MDRAB对常用消毒剂酒精、含氯消毒剂和碘伏的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并与鲍曼不动杆菌敏感株和铜绿假单胞菌标准株作平行比较。结合细菌药敏结果,分析消毒剂抗性与抗生素耐药性之间是否存在联系。采用聚合酶链反应(PCR)测定MDRAB中耐消毒剂基因qacE△1的携带率。5.采用PCR方法鉴定收集的MDRAB菌株,琼脂稀释法进行药物敏感性试验,PFGE进行菌株的同源性分析以及通过PCR方法筛查整合子的种类和基因盒,并对基因盒片段进行基因测序。结果1.某综合性医院鲍曼不动杆菌感染者以院内感染(58.76%)居多,常见感染部位为呼吸道(85.05%);mdrab感染的病人较敏感株感染病人延长的住院天数平均增加13.39天,治疗总费用平均增加95171.58元;mdrab感染患者的病死率及自动出院率总和较敏感菌株感染的患者增加了31.96%。多因素logistic回归分析表明,基础疾病个数(or=3.012,95%ci:1.153~7.868)、入住icu(or=2.935,95%ci:1.093~7.885)、病情的严重程度(or=6.481,95%ci:2.524~16.644)、碳青酶烯类药物使用(or=7.988,95%ci:2.961~21.547)和深静脉置管(or=2.875,95%ci:1.049~7.880)为mdrab感染的独立危险因素。敏感组和多重耐药病例组的耐药率统计,差异有意义(p<0.05);多重耐药组的耐药情况严重,14种测试抗菌药物中9种药物的耐药率达100%。2.某综合性医院286株多重耐药鲍曼不动杆菌按90%相似度可分为43种基因群,其中g23、g25为优势基因群,且2009年~2012年中均有检出,流行科室以外科片区为主,综合icu为高发科室,另环境中也检出同基因群菌株;结合药敏结果,各基因群除头孢哌酮/舒巴坦和米诺环素的耐药率低于40%外,其余耐药率均大于65%,且g23群的耐药比例最高,对碳青酶烯类的耐药率与g25群相比较,分布差异有统计学意义。3.某综合性医院mdrab主要克隆群的代表株对酒精、碘伏和含氯消毒剂进行抗性测试表明,与标准菌株及敏感株比较,酒精对mdrab的mbc和mic值较高,有一定程度的抗性,碘伏及含氯消毒剂的差别不大;但三种消毒剂的常规使用浓度均超过试验的mic值及mbc值。结合消毒剂抗性与菌株对抗菌药物的耐药情况,发现二者并无必然联系。mdrab的耐消毒剂基因qace△1检出率为57.97%。4.中国东南部地区及北京和烟台收集的425株mdrab菌株,同源性分析表明该地区主要有5种克隆群,流行于10个地市的13家医院;c25、c33为两个最主要的优势克隆群,其中c25群的分布除福建省外,杭州、温州及烟台也有检出,而c33群则为福建本地的流行群,分布于福州、南平及泉州。425株mdrab除米诺环素外,对其余药物的耐药率均大于70%,其中78%为耐碳青酶烯的鲍曼不动杆菌(crab)。其int?整合子检出率为69.6%,未检出intⅡ和intⅢ整合子。整合子阳性菌株的耐药率高于阴性菌株,也存在4种优势克隆群,且优势克隆群p4为本地流行株,其泛耐药株所占比例较高。研究中整合子所携带的基因盒为aacA4-catB8-aadA1和dfrⅫ-orfF-aadA2,属福建省首次报道。结论1.MDRAB的耐药情况严重,其感染者愈后和转归不佳,应加强对MDRAB感染者的监测和控制,特别是入住ICU病情严重合并基础疾病多的患者;强调抗菌药物的合理使用,特别是碳青酶烯类药物;应采取有效控制措施,避免其引起的爆发流行。2.控制MDRAB菌株的流行应加强对优势克隆群的监测,加强高发及流行科室的消毒灭菌工作及医务人员的手卫生,及时隔离感染病人,减少不必要的转科。3.临床工作中遇到可疑MDRAB爆发流行及污染的情况按常规浓度使用消毒剂能够有效地将其杀灭;根据qacE△1基因是主导对季胺盐类和双胍类消毒剂的外排泵基因,建议消毒MDRAB污染的环境应减少季胺盐类消毒剂的使用,对于皮肤的消毒也不建议单纯使用双胍类消毒剂,提倡使用含有酒精成份复配的消毒剂以保证消毒效果。4.中国东南部地区存在MRDAB的优势克隆群的流行,福建省内MDRAB的流行模式为本地克隆株和国内流行克隆株的流行共同存在。该地区MDRAB耐药形势严峻,CRAB的检出率高,甚至出现广泛耐药的菌株。5、中国东南部地区MDRAB检出的整合子均为int?;整合子可增加MDRAB的耐药性,其流行存在优势克隆群;福建省本地的优势克隆群中广泛耐药的比例高于中国东南部地区;所携带的基因盒既有全球广泛报道的种类,也有本地流行的种类;强调根据地区差别,合理使用抗菌药物,加强监控,防止流行株广泛蔓延。更多还原

【Abstract】 ObjectiveAcinetobacter baumannii is a common pathogen of nosocomial infection, which has been multidrug resistant, extensively drug-resistant,and even pan-resistant strains because of the antibiotic abuse. Once patients are infected especially those with severe infection, they wouldn’t be cured. The outbreak of nosocomial infection has become an important public health issue, which caused by multidrug-resistant Acinetobacter baumannii(MDRAB).Most studies have shown that the prevalence of MDRAB would be different due to the diversity of the antibiotic use and the measures about infection control.Therefore we studied the epidemic characteristics and resistance mechanisms of MDRAB in our region to effectively prevent and control the outbreaks by the bacteria.Methods1. By a case-control study, the data of cases infected by MDRAB from January2011 to June 2012 were analyzed in a general hospital.The cases infected by common Acinetobacter baumannii were selected randomly at the same time as control.Univariate analysis(t-test and chi-square test) and multivariate logistic regression were used for statistics analysis.2. A total of 286 strains of Acinetobacter baumannii were randomly collected from various clinical specimens from 2009 to 2012.The isolates were identified by VITEK260. The susceptibility tests were performed with the Kirby-Bauer agar diffusion method.The analysis of homology was performed with pulsed-field gel electrophoresis(PFGE).3. Results of susceptibility tests were analyzed by WHONET 5.6. The chi-square test was used for the statistical analysis of the diversity on drug-resistant rates.4. The susceptibility of MDRAB to three common disinfectants(alcohol,chlorine and iodophor) was evaluated by determination of minimum bactericidal concentration(MBC) and minimum inhibitory concentrati on(MIC),compared with the sensitive strains of Acinetobacter baumannii and Pseudomonas.And the correlation analysis was conducted between the susceptibility of MDRAB to the disinfectants and antibiotics. The gene of qac E △ 1 was analyzed using Polymerase Chain Reaction(PCR).5. MDRAB isolates were identified by PCR, agar dilution method for drug susceptibility testing, PFGE for homology analysis and screened about the type of integron and gene cassettes by PCR. The fragments of gene cassettes were sequenced.Results1. In a general hospital, 58.76% patients with MDRAB belonged to nosocomial infection, 85.05% found in the respiratory tract. Compared with patients infected by sensitive strains, those infected by MDRAB were higher in the length of hospital stay(the average growth of 13.39 days),the total cost of treatment(the average growth of￥ 95,171.58) and the sum rate of mortality and automatic discharge rate(the average growth of 31.96%).Multivariate logistic regression analysis revealed that the number of underlying disease(OR: 3.012,95% CI :1.153 to 7.868), ever staying at ICU(OR:2.935,95% CI: 1.093 to 7.885), disease severity(OR:6.481, 95% CI: 2.524 to 16.644),carbapenem drug use(OR:7.988,95% CI:2.961 to 21.547) and deep venous catheter(OR:2.875,95% CI:1.049 to 7.880) were significantly related to the infected risk.The difference of resistance rates between case group and control group had statistics significance.The resistance rates in case group were severe, which for 9 kinds of 14 drugs were 100%.2. A total of 43 gene groups were detected according to 90% identity for 286 strains of MDRAB. G23 and G25 were dominant groups and all detected from 2009 to2012, which were mainly prevalent in the surgical departments, especially general Intensive Care Unit(ICU).Moreover the same gene groups were detected from the ICU enviroment. The drug-resistant rate of Ab was higher than 65%, except Cefoperazone /sulbactam and minocycline(<40%). The drug-resistant rate of G23 group was highest.The statistical analysis was obviously significant about the Carbapenems-resistant rate between G23 and G25.3. The results of sensitive tests to three common disinfectant revealed that the value of MBC and MIC on MDRAB representative strains was higher than that of the sensitive strain and standard strain about iodophor, and there were little different between them about alcohol and chlorine disinfectant. But the routine concentration of them exceeded the MIC and MBC values. There was no correlation between antimicrobial drug resistance and disinfectant resistantance. The detection rate of the qac E△1 gene was 57.97%.4. The homology analysis revealed that 5 major clones of 425 strains of MDRAB were prevalent in 13 hospitals of 10 districts from southeastern China, Beijing and Yantai.Among them, C25 and C33 were the main clone,which C25 clone was epidemic in Hangzhou, Wenzhou and Yantai including Fujian Province and C33 clone was endemic in Fujian Province,including Fuzhou, Nanping and Quanzhou. The total drug resistance of 425 MDRAB was higher than 70%, except minocycline. The detective rate of carbapenem-resistant Acinetobacter baumannii(CRAB)was 78%.The detection of class ? integrase was 69.6%,wheras classⅡand Ⅲintegrase weren’t detected.The drug resistance of the positive strains about class ? integrase was higher than the negative.The homology analysis revealed that there were four major clones in the positive.The dominant clone P4 was endemic, where the extensively drug- resistant strains were in majority. The gene cassettes of class ? integron was aac A4-cat B8-aad A1 and dfr Ⅻ-orf F-aad A2, which was first reported in Fujian Province.Conclusions1. Because the drug-resistant status of MDRAB was severe and the prognosis of patients infected by MDRAB was poor, those patients should be supervised, especially those staying at ICU with severe diseases.To avoid the outbreak of MDRAB, and we should take effective measures and emphasize the rational use of antimicrobial agents,especially the carbapenems.2. The dominant clones should be monitored to control the epidemic strains of MDRAB. We should strengthen at disinfection and hand hygiene of the staff in the epidemic departments and timely isolate infected patients who should be reduced unnecessary transfer.3.When an outbreak of MDRAB happens,we can use the disinfectants to successfully deal with the contaminant according to the manual.Since the qac E△1 gene expresses the efflux pumps of the quaternary ammonium and biguanide disinfectant, we should reduce to use the quaternary ammonium disinfectant at disposing the contaminant of MDRAB and advocate using the compound disinfectants containing alcohol for hand hygiene instead of using the pure biguanide disinfectant.4. The dominant clones of MDRAB were epidemic in southeastern China.The popular model of MDRAB was the co-existence of the endemic strains and epidemic strains in Fujian Province.The drug-resistant status of MDRAB was servere in the region, which the detective rate of CRAB was high and even pan drug-resistant isolates were found.5. The kind of integron for MDRAB isolates of southeastern China was Class ?integron, which can increase the drug resistance of MDRAB. They had the dominant clones.The propotion of extensively drug-resistant strains about the dominant clones was higher in Fujian Province than those in southeastern China.The kinds of gene cassettes were both global and local type.So the measures for preventing the epidemic strains to widely spread should be taken of the rational antibiotics use and strengthening surveillance on epidemic strains according to regional differences.更多还原