不同来源大肠杆菌中喹诺酮外排泵基因oqxAB和qepA的传播机制研究

发布时间：2018-03-04 09:02

本文选题：大肠杆菌　切入点：oqxAB　出处：《华南农业大学》2016年博士论文　论文类型：学位论文

【摘要】：氟喹诺酮类药物是临床和畜牧养殖上极其重要的抗菌药物之一,广泛应用于临床治疗和动物养殖生产实践。近年来,随着质粒介导的喹诺酮耐药基因(plasmid-mediated quinolone resistance,PMQR)的出现,喹诺酮耐药问题日趋严重。质粒介导的喹诺酮外排泵基因oqxAB和qepA在动物源或人源菌株中已有较多的报道,在不同来源(动物、食品和人)大肠杆菌中的传播机制和携带oqxAB和qepA的耐药质粒特征尚不明确。本研究以不同来源(动物源、食品源、宠物源以及医院临床)的大肠杆菌为研究对象,分析PMQR基因(主要是oqxAB和qepA)的流行情况;阐明不同来源大肠杆菌中oqxAB和qepA的传播规律和传播机制;分析携带oqxAB和qepA基因多重耐药质粒的特征;为喹诺酮类药物耐药性防控提供基础数据,为指导临床合理用药以及新药研发提供科学的数据支撑。采用琼脂稀释法测试1740株不同来源大肠杆菌对8类抗菌药的敏感性,结果显示,不同来源的大肠杆菌对磺胺甲VA唑/甲氧苄啶耐药率最高(80%);其次是对氨苄西林(73%)、四环素(73%)、链霉素(67%)、喹乙醇(64%);随后是头孢噻肟、头孢他啶、新霉素、庆大霉素、安普霉素、氯霉素、氟苯尼考和环丙沙星,耐药率在25%~53%之间;但头孢西丁(7%)、阿米卡星(4%)和粘菌素(11%)耐药率则较低。无论是动物源、食品源还是医院临床大肠杆菌对氨苄西林、四环素的耐药率均较严重,达55%以上。动物源大肠杆菌对链霉素、新霉素、安普霉素、氯霉素、氟苯尼考、黏菌素和喹乙醇的耐药率均高于食品源、宠物源和人源菌株;但对头孢西丁、头孢噻肟、头孢他啶的耐药率均低于人源菌株。值得注意的是猪、鸡源大肠杆菌对喹乙醇的耐药率达95%以上,远超于食品源(39%)和人源(38%)。食品源、人源和猪源大肠杆菌对环丙沙星的耐药率较低,分别为34%、29%、23%,但宠物源和鸡源大肠杆菌却达到50%以上。有趣的是,宠物源大肠杆菌对阿米卡星的耐药率(27%)最高,远高于动物源、食品源和人源。对随机挑取的1034株不同来源大肠杆菌进行PMQR检测,结果发现oqxAB基因在食品动物源和食品源大肠杆菌中的检出率(22.9%、22.9%)明显高于医院临床和宠物源大肠杆菌的检出率(12.3%、12.0%)。qnrS基因在食品动物源大肠杆菌中的检出率最高(30.3%),其次是在食品源(21.9%),显著高于医院临床和宠物源(7.86%、8.01%)。而qepA在医院临床(4.08%)和宠物源(6.0%)的检出率则要高于食品源(1.41%)和食品动物源(0.07%)。qnrB的检出率在不同来源的大肠杆菌中检出率均较低:宠物源2.0%、食品动物源1.76%、和食品源0.35%,人源大肠杆菌则未检出。所有菌株中均未检测到qnra、qnrc和qnrd。利用脉冲场凝胶电泳(pulsed-fieldelectrophoresispfge)对50株不同来源oqxab阳性大肠杆菌进行亲缘性分析,结果成功将45株oqxab阳性大肠杆菌分为32个型,大部分菌株不存在克隆关系,但在不同动物或人之间以及动物、食品和人源菌间均存在oqxab阳性大肠杆菌的克隆传播现象。通过电转化实验,共获得21株oqxab阳性转化子,且均表现出多重耐药,转化子snj43-1除外。s1-pfge和southern杂交结果显示,21株oqxab转化子中含有1~4个质粒,大小为28~500kb,除snx19-2无杂交信号,4个双质粒转化子有两个杂交信号外,其余16个转化子中oqxab均定位于单一质粒上。21株oqxab转化子中有13株利用复制子分型成功,其中1株为incn型质粒,3株为inchi2型质粒,9株为incf型质粒,包括5株多复制子融合质粒。质粒酶切结果显示,7株分离自猪、鸡或食品的oqxab阳性质粒属于同一rflp型(限制性片段长度多态性),另外2株分离自鸡和病人的oqxab阳性质粒属于同一谱型,表明相似质粒在不同来源大肠杆菌间的水平传播也是oqxab基因流行的原因之一。采用反向pcr和pcrmapping相结合的方式对oqxab阳性转化子进行oqxab基因侧翼序列分析,结果在19株转化子中oqxab基因侧翼均为2个同向插入序列is26,该结构与已报道的人医临床来源或动物源的大肠杆菌或沙门氏菌相应片段序列高度相似,提示插入序列is26对oqxab基因在不同型质粒间的整合和不同来源菌株间的传播扩散发挥了重要作用。利用接合实验成功获得2株不同来源且同时携带qepa和rmtb基因的接合子,均表现出多重耐药。2株接合子中qepa/rmtb均位于f2:a-:b-型质粒,而质粒酶切结果显示,与课题组2008年获得的宠物源接合子3a11-16j携带的f2:a-:b-型qepa/rmtb阳性质粒属于同一rflp型,提示f2:a-:b-型质粒的水平传播是qepa和rmtb在不同来源大肠杆菌间协同扩散的主要原因。通过pacbio测序分析比较oqxab阳性质粒phnzy32的多重耐药质粒结构特征,发现phnzy32属于多复制子融合质粒,包括incfii、incn和incx型复制子,转移区及其毗邻的先导区与f33:a-:b-型质粒高度相似,携带pemki、vagcd、hok-sok和srnbc四种质粒沉溺系统。phnzy32携带两个耐药区,分别含有耐药基因aph(3’)-iia、Δble、oqxab、blatem-1、rmtb、fosa3和blactx-m-55、,以及flor、teta、stra、strb和sul2。推断该质粒可能是以n1-f33:a-:b-质粒phnfp460-1的相似质粒为骨架,整合了包括oqxab基因和复制子在内的incx型质粒片段,以及包括耐药基因flor、teta、stra、str B和sul2在内的20 Kb片段,经过多步插入、重组作用进化形成的。质粒pHNZY32的IncX型复制子区域在来自动物和人的15个oqxAB转化子中检出,推测IncX型质粒可能与oqxAB基因在不同来源细菌间的传播有关。通过鸟枪随机打碎构建文库的方法进行qepA阳性质粒pHN3A11测序,经分析,发现pHN3A11质粒骨架和其他IncFII型F2:A-:B-质粒高度相似,如中国的pHK23a,pFOS-HK151325和pXZ质粒以及加拿大的pC15-1a质粒。质粒pHN3A11序列中存在能提高质粒在垂直传播过程中的稳定性能的编码基因,包括质粒沉溺系统(pemI/pemK,hok/mok/sok)和分配系统(parM、parB、和stbB)。pHN3A11质粒的多重耐药区包括rmtB,qepA,blaTEM-1和dfr基因,上下游插入着若干个完整或截断的插入序列和转座子,如ΔIS1,ΔTn2,ΔintI1,ISCR33,IS26和Tn21。采用PCR和测序的方法研究pHN3A11类似质粒的流行分布情况。发现pHN3A11类似质粒已广泛扩散于中国的宠物源、食品动物源以及养殖环境源大肠杆菌中。综上所述,oqxAB基因在我国不同来源(食品动物、宠物、动物性食品和医院临床)大肠杆菌中普遍流行。垂直传播以及多重耐药质粒和插入序列IS26等可移动元件介导的水平传播共同介导oqxAB基因在动物源、食品源和人源大肠杆菌间流行和传播。而qepA则常与rmtB协同传播,F2:A-:B-型pHN3A11相似质粒是qepA和rmtB在不同来源大肠杆菌间水平传播的主要载体。喹诺酮外排泵基因oqxAB和qepA在不同来源大肠杆菌中的传播扩散,不仅影响兽医临床的防治效果,也对人类健康和公共安全形成潜在威胁。
[Abstract]:Fluoroquinolones are extremely important in clinical and animal husbandry on one of the antibiotics, is widely used in clinical and animal production practice. In recent years, with the plasmid mediated quinolone resistance gene (plasmid-mediated quinolone resistance, PMQR) the emergence of quinolone resistance problem has become increasingly serious. The plasmid mediated quinolone efflux pump gene oqxAB and qepA in animal or human strains has been reported in different sources (animal, food and human) Escherichia coli propagation mechanism and carrying oqxAB and qepA plasmid characteristics is not clear. In this study, different sources (animal source food source, source and pet hospital clinical) E. coli as the research object, analysis of the PMQR gene (oqxAB and qepA) of the epidemic situation; clarify the spreading rules of oqxAB and qepA of different sources of Escherichia coli and spread mechanism; analysis with o The characteristics of gene multidrug resistant plasmid qxAB and qepA; to provide the basic data for the prevention and control of quinolone resistance, provide scientific data support for clinical rational drug use and drug sensitivity test of antibacterial drugs. And different sources of Escherichia coli to 8 types of 1740 strains using agar dilution method showed that different sources of Escherichia coli to sulfame VA triazole / trimethoprim resistance was the highest (80%); the second is to ampicillin (73%), tetracycline (73%), streptomycin (67%), (64%); olaquindox followed by cefotaxime, ceftazidime, neomycin, gentamicin, Apramycin, chloramphenicol, florfenicol and ciprofloxacin resistance rate 25%~53%; but the cefoxitin (7%), Amikacin (4%) and (11%) polymyxin resistance rate is low. Both animal source food source or hospital clinical Escherichia coli to ampicillin, tetracycline resistance rates were more serious, up to 55% Above. Neomycin of Escherichia coli isolated from animal to streptomycin, Apramycin, chloramphenicol, florfenicol, colistin and olaquindox resistance rate were higher than that of pet food sources, sources and human strains; but to cefoxitin, cefotaxime, ceftazidime resistance rates were lower than the human strain. It is worth noting pig, Chicken Escherichia coli resistant to olaquindox rate reached more than 95%, far more than in the food source (39%) and the source (38%). The food source, human and swine Escherichia coli resistance to ciprofloxacin was low, were 34%, 29%, 23%, but the source and pet Chicken Escherichia coli has reached more than 50%. Interestingly, the pet Escherichia coli resistant to Amikacin (27%) the highest rate is much higher than that of animal source food source and human source. On 1034 randomly selected different strains of Escherichia coli were detected by PMQR. The results showed that oqxAB gene in animal food and food source in Escherichia coli The detection rate (22.9%, 22.9%) the detection rate was significantly higher than that of pet hospital and Escherichia coli (12.3%, 12%) of.QnrS gene in food of animal origin in Escherichia coli had the highest detection rate (30.3%), followed by the food source (21.9%), significantly higher than that of hospital and pets (7.86%, 8.01%). QepA in hospital (4.08%) and (6%) pet source detection rate is higher than the food source (1.41%) and food of animal origin (0.07%) the detection rate of.QnrB in different sources of Escherichia coli infection rate was low: the pet animal food sources 2%, 1.76%, and 0.35% people food source, source Escherichia coli were not detected. All strains were not detected in qnra, qnrc and qnrd. by pulsed field gel electrophoresis (pulsed-fieldelectrophoresispfge) by phylogenetic analysis of 50 different strains of oqxab positive Escherichia coli, the success will be the 45 strains of oqxab E.coli is divided into 32 types, most There is no relationship between the cloned strains, but between different animal or human and animal cloning, propagation phenomena were oqxab positive Escherichia coli and human food source. Bacteria were obtained by electro transformation experiment, 21 strains of oqxab positive transformants, which showed multiple antibiotic resistant transformants, except snj43-1.S1-pfge and Southern hybridization showed 21, oqxab transformants containing 1~4 plasmids, the size of 28~500kb, except for snx19-2, no hybridization signal, 4 double plasmid transformants two hybridization signals, the remaining 16 oqxab transformants were located in a single plasmid.21 oqxab transformants in 13 strains of the replicon typing successfully. 1 of them were incn type plasmid, 3 strains of type inchi2 plasmid, 9 strains of type incf plasmid, including 5 strains of multi replicon fusion plasmid. The plasmid showed that 7 strains isolated from pigs, oqxab positive plasmid chicken or food belong to the same type RFLP (restriction fragment Length polymorphism), the other 2 strains isolated from chicken and oqxab in patients with positive plasmids belonging to the same pattern, one of the reasons that a similar level of communication in different sources of Escherichia coli plasmid between oqxab gene is also popular. Using reverse PCR and pcrmapping combination of oqxab positive transformants flanking the oqxab gene analysis results in the oqxab sequence, and 19 transformants in the flanking gene were 2 with insertion sequence is26, Escherichia coli or Salmonella corresponding fragment of human clinic or animal sources have been reported with the structure of the highly similar, played an important role that is inserted into the is26 sequence of oqxab gene in different media integration plasmid between and among the strains from different diffusion. By conjugation experiments successfully obtained 2 strains of different sources and simultaneously carrying qepa and rmtb gene of the zygote, showed multi drug resistant.2 strains of transconjugants QE Pa/rmtb are located in the f2:a-: b- plasmid and the plasmid was obtained and the results show that the research group in 2008 the pets TRANSCONJUGANT 3a11-16j carrying f2:a-: b- type qepa/rmtb positive plasmids belonging to the same type of RFLP, suggesting that f2:a-: horizontal transmission of b- type plasmid is a major cause of qepa and rmtb in Escherichia coli among different sources cooperative diffusion. Through pacbio sequencing analysis of multi drug resistant plasmid structures oqxab positive plasmid phnzy32, found that phnzy32 belongs to the multi replicon fusion plasmid including incfii, incn and INCX type replicon, transfer area and the pilot area and adjacent to the f33:a-: b- plasmid carrying pemki, highly similar to vagcd, hok-sok and srnbc four plasmid.Phnzy32 carrying two resistance indulge system, respectively containing resistance genes APH (3 ") -iia, ble, oqxab, blatem-1, rmtb, fosa3 and blactx-m-55, and flor, TetA, stra, STRB and sul2. concluded that the plasmid may Is n1-f33:a-: similar plasmid b- phnfp460-1 as the framework, the integration of the INCX plasmid including oqxab gene fragment and replicon, and expression of flor, TetA, stra, STR, B and sul2, the 20 Kb fragment through multi-step insertion, recombination evolution form. Detection of IncX type plasmid pHNZY32 replicon region was transformed in 15 oqxAB from animal and human child, speculated about the IncX plasmid and oqxAB gene may spread in different sources of bacteria. Through the method of random shotgun library were broken qepA positive plasmid pHN3A11 sequencing, after analysis, found that pHN3A11 plasmid backbone and other IncFII type: B- F2:A- the plasmid is highly similar, such as China pHK23a, pFOS-HK151325 and pXZ in Canada and pC15-1a plasmid plasmid. Plasmid pHN3A11 sequence can improve the existing stability of plasmid encoding gene in the process of vertical transmission , including plasmid addiction (pemI/pemK, hok/mok/sok) system and distribution system (parM, parB, and stbB).PHN3A11 multi drug resistant plasmid including rmtB, qepA, blaTEM-1 and DFR gene, the upstream and downstream insertion with a plurality of intact or truncated insertion sequences and transposable elements, such as delta IS1, Delta Tn2, Delta intI1, ISCR33 IS26 and Tn21., the popular distribution of PCR and sequencing method of pHN3A11 plasmid. PHN3A11 found similar similar plasmid has been widely diffused in the Chinese pet food source, animal source and culture environment of Escherichia coli. In summary, oqxAB gene in different sources in China (animal food, animal food and pet hospital clinical) prevalent in Escherichia coli. Vertical transmission and multi resistant plasmids and insertion sequence IS26 mobile element mediated horizontal transmission co mediated oqxAB gene in animal source food source and human Escherichia coli and popular Spread. While qepA is often combined with rmtB communication, F2:A-: B- pHN3A11 and rmtB qepA plasmid is similar in the main carrier level communication between different sources of Escherichia coli. The spread of quinolone efflux pump gene oqxAB and qepA in different sources of Escherichia coli spread, not only affect the control effect of veterinary clinic, also formed a potential threat on human health and public safety.

【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.612;R378.21

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