本文關(guān)鍵詞：北京某醫(yī)院鮑曼不動(dòng)桿菌臨床分離株耐藥性及分子流行病學(xué)的研究，由筆耕文化傳播整理發(fā)布。

        目的：鮑曼不動(dòng)桿菌（Acinetobacter baumannii）是一種革蘭陰性非發(fā)酵菌，廣泛存在于醫(yī)院環(huán)境中，如醫(yī)護(hù)人員手部、呼吸機(jī)設(shè)備、患者用具、透析機(jī)、消毒液，甚至水龍頭等，是最常見(jiàn)的條件致病菌之一；該菌可以導(dǎo)致多種醫(yī)院感染，如傷口感染、肺炎、敗血癥和腦膜炎等。近幾年隨著抗菌藥物的廣泛應(yīng)用，鮑曼不動(dòng)桿菌耐藥株逐年增加，并從對(duì)單一抗菌藥耐藥向多重耐藥、由低耐藥向高耐藥發(fā)展。尤其是多重耐藥鮑曼不動(dòng)桿菌的出現(xiàn)，為抗感染疾病的有效治療提出了嚴(yán)峻的挑戰(zhàn)，因此對(duì)鮑曼不動(dòng)桿菌耐藥機(jī)制及流行病學(xué)研究已成為國(guó)內(nèi)外的熱點(diǎn)。本次研究采用多位點(diǎn)序列分型（Multilocus sequence typing，MLST），多重PCR（multiplexpolymerase chain reaction，M-PCR）對(duì)菌株的部分耐藥機(jī)制、群體遺傳學(xué)結(jié)構(gòu)及菌株變異情況進(jìn)行研究，期待為鮑曼不動(dòng)桿菌的流行病調(diào)查提供新的數(shù)據(jù)。方法：2002到2009年期間收集北京某三甲醫(yī)院不同科室，不同時(shí)期患者的不同標(biāo)本分離得到的多重耐藥鮑曼不動(dòng)桿菌341株。首先，對(duì)所有菌株采用苯酚-氯仿提取方法提取基因組DNA；然后，應(yīng)用多重PCR方法鑒定導(dǎo)致鮑曼不動(dòng)桿菌碳青霉烯類(lèi)耐藥的主要相關(guān)基因，OXA23型、OXA58型、OXA69型、OXA143型、OXA51型和OXA24型；最后，采用MLST對(duì)341株分離株的7個(gè)等位基因進(jìn)行分析，獲得各個(gè)分離株的等位基因譜(allelic profile)和序列型(sequence type，，ST)。隨后以ST型和等位基因譜為基礎(chǔ)，利用Bionumerics6.6、START2.0、RDP3.0、MEGA和eBURST等軟件對(duì)鮑曼不動(dòng)桿菌的群體遺傳學(xué)結(jié)構(gòu)進(jìn)行深入分析。結(jié)果：341株測(cè)試菌株中，blaOXA-51-like基因攜帶率為97.95%，與初始鑒定不符率是2.05%。多重PCR發(fā)現(xiàn)了18種耐藥基因組合，其中以blaOXA-51-like+blaOXA-23-like+blaOXA69+blaISF-R+blaISF-OXA23R組合為主（n=262，76.83%），且blaOXA-23-like、blaISAba1的檢出率分別是89.44%，99.41%。但是本次實(shí)驗(yàn)未發(fā)現(xiàn)blaOXA-24-like、blaOXA-58-like、blaOXA-143-like陽(yáng)性菌株。多重耐藥鮑曼不動(dòng)桿菌全基因組具有非常豐富的插入序列ISAba1，位于blaOXA-23-like、blaOXA-51-like等耐藥基因的上游。文獻(xiàn)報(bào)道插入片段位于blaOXA-23-like或blaOXA-51-like前端，但在本次研究中發(fā)現(xiàn)了blaISAba1F-OXA23及blaISAba1F-OXA51同時(shí)存在的情況。341株菌中MLST的成功擴(kuò)增并測(cè)序率是97.95%，利用Bionumerics6.6對(duì)這334株菌株序列進(jìn)行分析，確定了4個(gè)ST，其中2個(gè)為新ST型。多重耐藥鮑曼不動(dòng)桿菌主要流行克隆株的等位基因譜為ST2(2-2-2-2-2-2-2)，與世界上很多國(guó)家流行的攜帶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%)。首次發(fā)現(xiàn)了等位基因rpoB-42，rpoB-43和新序列型ST-185，ST-187。利用eBURST軟件分析得到ST2、ST185、ST187屬于克隆群1(Clonal Complex1，CC1)，而ST113屬于克隆群8；通過(guò)START2.0得到ISA的值為0.1436(P<0.0001)，提示連鎖不平衡并存在重組事件；RDP3.0分析得出pyrG、cpn60、fusA、gltA、rplB、recA均有重組發(fā)生，共計(jì)10個(gè)重組事件。結(jié)論：341株多重耐藥鮑曼不動(dòng)桿菌blaOXA-51-like基因攜帶率達(dá)到97.95%，以blaOXA-51-like+blaOXA-23-like+blaOXA69+blaISF-R+blaISF-OXA23R組合為主（n=262，76.83%）。本次研究所調(diào)查醫(yī)院的多重耐藥鮑曼不動(dòng)桿菌以ST2為主，該菌的種群內(nèi)存在頻繁的重組，但是等位基因型依舊保持連鎖不平衡的特征，屬于典型的Epidemic種群結(jié)構(gòu)。

    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.

北京某醫(yī)院鮑曼不動(dòng)桿菌臨床分離株耐藥性及分子流行病學(xué)的研究

摘要5-7ABSTRACT7-8引言10-12第一部分 應(yīng)用多重PCR方法分析碳青霉烯類(lèi)耐藥鮑曼不動(dòng)桿菌的部分耐藥機(jī)制12-34    前言12    材料與方法12-15    結(jié)果15-17    附圖17-19    附表19-32    討論32-33    小結(jié)33-34第二部分 應(yīng)用 MLST 技術(shù)鑒定北京某三甲醫(yī)院多重耐藥鮑曼不動(dòng)桿菌的主要流行克隆譜系34-50    前言34    材料與方法34-36    結(jié)果36-39    附圖39-45    附表45-48    討論48-49    小結(jié)49-50參考文獻(xiàn)50-54結(jié)論54-55綜述 鮑曼不動(dòng)桿菌耐藥基因的研究55-63    參考文獻(xiàn)59-63致謝63-64個(gè)人簡(jiǎn)歷64

  本文關(guān)鍵詞：北京某醫(yī)院鮑曼不動(dòng)桿菌臨床分離株耐藥性及分子流行病學(xué)的研究，由筆耕文化傳播整理發(fā)布。