北京某医院鲍曼不动杆菌临床分离株耐药性及分子流行病学的研究

  本文关键词：北京某医院鲍曼不动杆菌临床分离株耐药性及分子流行病学的研究，由笔耕文化传播整理发布。

        目的：鲍曼不动杆菌（Acinetobacter baumannii）是一种革兰阴性非发酵菌，广泛存在于医院环境中，如医护人员手部、呼吸机设备、患者用具、透析机、消毒液，甚至水龙头等，是最常见的条件致病菌之一；该菌可以导致多种医院感染，如伤口感染、肺炎、败血症和脑膜炎等。近几年随着抗菌药物的广泛应用，鲍曼不动杆菌耐药株逐年增加，并从对单一抗菌药耐药向多重耐药、由低耐药向高耐药发展。尤其是多重耐药鲍曼不动杆菌的出现，为抗感染疾病的有效治疗提出了严峻的挑战，因此对鲍曼不动杆菌耐药机制及流行病学研究已成为国内外的热点。本次研究采用多位点序列分型（Multilocus sequence typing，MLST），多重PCR（multiplexpolymerase chain reaction，M-PCR）对菌株的部分耐药机制、群体遗传学结构及菌株变异情况进行研究，期待为鲍曼不动杆菌的流行病调查提供新的数据。方法：2002到2009年期间收集北京某三甲医院不同科室，不同时期患者的不同标本分离得到的多重耐药鲍曼不动杆菌341株。首先，对所有菌株采用苯酚-氯仿提取方法提取基因组DNA；然后，应用多重PCR方法鉴定导致鲍曼不动杆菌碳青霉烯类耐药的主要相关基因，OXA23型、OXA58型、OXA69型、OXA143型、OXA51型和OXA24型；最后，采用MLST对341株分离株的7个等位基因进行分析，获得各个分离株的等位基因谱(allelic profile)和序列型(sequence type，，ST)。随后以ST型和等位基因谱为基础，利用Bionumerics6.6、START2.0、RDP3.0、MEGA和eBURST等软件对鲍曼不动杆菌的群体遗传学结构进行深入分析。结果：341株测试菌株中，blaOXA-51-like基因携带率为97.95%，与初始鉴定不符率是2.05%。多重PCR发现了18种耐药基因组合，其中以blaOXA-51-like+blaOXA-23-like+blaOXA69+blaISF-R+blaISF-OXA23R组合为主（n=262，76.83%），且blaOXA-23-like、blaISAba1的检出率分别是89.44%，99.41%。但是本次实验未发现blaOXA-24-like、blaOXA-58-like、blaOXA-143-like阳性菌株。多重耐药鲍曼不动杆菌全基因组具有非常丰富的插入序列ISAba1，位于blaOXA-23-like、blaOXA-51-like等耐药基因的上游。文献报道插入片段位于blaOXA-23-like或blaOXA-51-like前端，但在本次研究中发现了blaISAba1F-OXA23及blaISAba1F-OXA51同时存在的情况。341株菌中MLST的成功扩增并测序率是97.95%，利用Bionumerics6.6对这334株菌株序列进行分析，确定了4个ST，其中2个为新ST型。多重耐药鲍曼不动杆菌主要流行克隆株的等位基因谱为ST2(2-2-2-2-2-2-2)，与世界上很多国家流行的携带blaOXA-23-like菌株的ST一致。本次研究中ST及其分布如下：ST-2(97.9%)、ST-113(0.9%)、 ST-185(0.9%)和ST-187(0.3%)。首次发现了等位基因rpoB-42，rpoB-43和新序列型ST-185，ST-187。利用eBURST软件分析得到ST2、ST185、ST187属于克隆群1(Clonal Complex1，CC1)，而ST113属于克隆群8；通过START2.0得到ISA的值为0.1436(P<0.0001)，提示连锁不平衡并存在重组事件；RDP3.0分析得出pyrG、cpn60、fusA、gltA、rplB、recA均有重组发生，共计10个重组事件。结论：341株多重耐药鲍曼不动杆菌blaOXA-51-like基因携带率达到97.95%，以blaOXA-51-like+blaOXA-23-like+blaOXA69+blaISF-R+blaISF-OXA23R组合为主（n=262，76.83%）。本次研究所调查医院的多重耐药鲍曼不动杆菌以ST2为主，该菌的种群内存在频繁的重组，但是等位基因型依旧保持连锁不平衡的特征，属于典型的Epidemic种群结构。

    Objective: Acinetobacter baumannii is a gram-negative non-fermentingbacteria, widely distributed in the hospital environment, including surfaces ofmedical staff, ventilator equipment, dialysis machines, and even water. A.baumannii has emerged as a leading cause of nosocomial infections, whichcan lead to a variety of hospital infections, for instance wound infection,pneumonia, septicemia and meningitis. In recent years, with the extensiveapplication of antibiotics, A. baumannii resistant strain emerged and spreadeddramatically, from single to multidrug resistance, and low to high resistance.Multidrug-resistant A. baumannii presents serious challenges for the effectivetreatment of infections diseases, so researches on the mechanism andepidemiology of Acinetobacter resistance has become a hot topic. The studyexpected to provide new data for epidemiological investigation of A.baumannii by using MLST (Multilocus sequence typing), multiplexpolymerase chain (M-PCR) reaction to study the mechanisms of drugresistance, and population structure of A. baumannii.Methods: During the period of2002-2009,341clinicalmultidrug-resistant isolates A. baumannii collected from different departments,different period patients and different specimens of a general hospital. First ofall, genomic DNA of all the strains was extracted by phenol-chloroformprotocol; secondly, potential carbapenems resistance genes were screened byM-PCR: OXA23, OXA58, OXA69, OXA143, OXA51and OXA24; finally,all341isolates were analyzed by a MLST schme including sevenhousekeeping genes to determine the ST (sequence type) of isolates. On thebasis of allelic profile and ST, we can further analyze the population structureof A. baumannii by using Bionumerics6.6, START2.0, RDP3.0, MEGA and eBURST softwares.Results:97.95%of the341isolates are positive for blaOXA-51-likegene. Ingeneral the discrepancy rate with preliminary indentification is2.05%.Meanwhile the positive rates of blaOXA-23-likeand blaISAba1were89.44%and99.41%, respectively. We failed to identify any positive strains of blaOXA-24-like,blaOXA-58-likeand blaOXA-143-like. Totally18types of resistance-associated genescombinations were revealed by the method of Multiplex PCR, and thedominating combination profile wasblaOXA-51-like+blaOXA-23-like+blaOXA69+blaISF-R+blaISF-OXA23R(n=262,76.83%) Itwas reported that ISAba1located upstream of blaOXA-23-likeor blaOXA-51-like, butin this study we found blaISAba1-OXA23and blaISAba1-OXA51existed at the sametime. The present study showed that the success rate of341strains is97.95%by MLST method.334strains were designated to4STs by Bionumerics6.6,including2novel STs. The predominating clone of multidrug resistant A.baumannii in this hospital is ST2(2-2-2-2-2-2-2), which is also the leadingepidemic clone of resistant A. baumannii worldwide. In this research, theMLST sequence types (STs)(and their percent distributions) were as follows:ST-2(97.95%), ST-113(0.9%), ST-185(0.9%), and ST-187(0.3%). ST-185,ST-187and the alleles rpoB-42, rpoB-43are described for the first time. ST2,ST185and ST187belonged to clone complex1（CC1）, and ST113belongedto clone complex8（CC8）by the analysis of eBURST software. START2.0showed ISA=0.1436(P<0.0001). It implies linkage disequilibrium andrecombination events. For a total of10recombination events were identifiedin pyrG, cpn60, fusA, gltA, rplB and recA by RDP3.0.Conclusion: The present study showed that the blaOXA-51-likegenecarrying rate was97.95%. And the major combination wasblaOXA-51-like+blaOXA-23-like+blaOXA69+blaISF-R+blaISF-OXA23R(n=262,76.83%).The spread of multidrug-resistant A. baumannii in the investigated hospitalwas closely related to ST2-CC1. A. baumannii had epidemic populationstructure. High levels of recombination happened in clonal structure, butallelic profile remained the characteristics of linkage disequilibrium.

北京某医院鲍曼不动杆菌临床分离株耐药性及分子流行病学的研究

摘要5-7ABSTRACT7-8引言10-12第一部分 应用多重PCR方法分析碳青霉烯类耐药鲍曼不动杆菌的部分耐药机制12-34    前言12    材料与方法12-15    结果15-17    附图17-19    附表19-32    讨论32-33    小结33-34第二部分 应用 MLST 技术鉴定北京某三甲医院多重耐药鲍曼不动杆菌的主要流行克隆谱系34-50    前言34    材料与方法34-36    结果36-39    附图39-45    附表45-48    讨论48-49    小结49-50参考文献50-54结论54-55综述 鲍曼不动杆菌耐药基因的研究55-63    参考文献59-63致谢63-64个人简历64

  本文关键词：北京某医院鲍曼不动杆菌临床分离株耐药性及分子流行病学的研究，由笔耕文化传播整理发布。