【摘要】：近年來(lái)多重耐藥菌株和泛耐藥菌株層出不窮且播散速度快,進(jìn)一步增加了臨床感染性疾病治療的困難。攜帶耐藥基因的質(zhì)粒所導(dǎo)致的水平基因傳播在細(xì)菌耐藥中起著重要的作用,耐藥基因一般通過(guò)插入序列、整合子和轉(zhuǎn)座子等移動(dòng)元件進(jìn)行水平轉(zhuǎn)移,質(zhì)粒可以介導(dǎo)耐藥基因在不同的細(xì)菌個(gè)體間轉(zhuǎn)移,而移動(dòng)元件則只能介導(dǎo)耐藥基因在細(xì)菌個(gè)體內(nèi)的DNA分子間轉(zhuǎn)移。IncHI質(zhì)粒是腸桿菌科細(xì)菌對(duì)抗生素耐藥的重要載體。IncHI型質(zhì)粒先前根據(jù)研究的次序分為IncHI1、IncHI2和IncHI3。本研究中,根據(jù)代表性質(zhì)粒的復(fù)制起始基因及骨架保守序列特征將IncHI質(zhì)粒進(jìn)一步分為五類(lèi)亞組,增加IncHI4和IncHI5兩個(gè)亞型。經(jīng)分析顯示每個(gè)亞型內(nèi)質(zhì)粒骨架區(qū)域序列均存在著很高的遺傳保守性,同時(shí),五類(lèi)IncHI質(zhì)粒均保留有接合轉(zhuǎn)移區(qū)tra1和tra2,還有碲耐藥基因簇ter;即便如此,五類(lèi)IncHI亞型質(zhì)粒之間還是存在著很大的差異。從中國(guó)三家教學(xué)醫(yī)院臨床病人分離出來(lái)的三株多重耐藥菌:陰溝腸桿菌T5282,弗氏檸檬酸桿菌112298和解鳥(niǎo)氨酸拉烏爾菌YNKP001,分別得到了 IncHI2型質(zhì)粒pT5282-mphA、p112298-catA 和 IncHI5 型質(zhì)粒 pYNKP001-dfrA;根據(jù)三個(gè)質(zhì)粒的序列從全球核酸數(shù)據(jù)庫(kù)中挑選出IncHI2型參考質(zhì)粒R478和IncHI5型參考質(zhì)粒pKOX_R1,并對(duì)該五個(gè)質(zhì)粒序列進(jìn)行精細(xì)的生物學(xué)信息分析及比較基因組學(xué)分析。序列分析顯示整合子,轉(zhuǎn)座子和以插入序列介導(dǎo)的移動(dòng)元件等一系列插入?yún)^(qū)域分布在質(zhì)粒骨架區(qū)的不同位置,而這些元件構(gòu)建起來(lái)的多重耐藥區(qū)里一般攜帶著三種及其三種以上不同的耐藥基因,其種類(lèi)包括氨基糖苷類(lèi)抗性基因、氯霉素抗性基因、四環(huán)素抗性基因、β內(nèi)酰胺類(lèi)抗性基因、大環(huán)內(nèi)酯類(lèi)抗性基因、磺胺類(lèi)抗性基因、喹諾酮抗性基因、利福平抗性基因、甲氧芐啶抗性基因、磷霉素抗性基因、季銨鹽類(lèi)抗性基因等。在了解多重耐藥質(zhì)粒結(jié)構(gòu)的同時(shí),發(fā)現(xiàn)并命名了一系列新的移動(dòng)元件,包括7個(gè)轉(zhuǎn)座子Tn6321、Tn6322、Tn6338-Tn6340、Tn6346 Tn6347;還有插入序列 ISEc16、ISCfr8-ISCfr11、ISKox1-ISKox3。自抗生素應(yīng)用于臨床感染性疾病的治療,臨床上不久便出現(xiàn)其相應(yīng)耐藥菌株,細(xì)菌在不同的抗生素選擇壓力及環(huán)境條件的變化下,使其快速獲得并保留不同的抗性基因,并因此加速耐藥菌株的持續(xù)擴(kuò)散。本課題把目前出現(xiàn)具有代表性IncHI型質(zhì)粒進(jìn)行整理,重新定義劃分標(biāo)準(zhǔn),添加至5個(gè)亞型;精細(xì)分析呈現(xiàn)了多藥耐藥IncHI型質(zhì)粒的特征性及多樣性,揭示IncHI型質(zhì)粒是臨床分離菌多藥耐藥特征的重要載體,為腸桿菌科細(xì)菌性感染疾病的治療提供分子依據(jù),為臨床上多藥耐藥腸桿菌科細(xì)菌的傳播提供研究依據(jù),以期預(yù)防或延緩細(xì)菌耐藥發(fā)展的趨勢(shì)。
[Abstract]:In recent years, multidrug resistant strains and pan-resistant strains have emerged in endlessly and spread rapidly, which has further increased the difficulties in the treatment of clinical infectious diseases. Horizontal gene transmission caused by plasmids carrying drug resistance genes plays an important role in bacterial drug resistance. Drug resistance genes are generally transferred horizontally by inserting sequences, integrons and transposons, etc. Plasmids can mediate the transfer of drug resistance genes between different bacterial individuals. However, the mobile element can only mediate the transfer of drug resistance genes between DNA molecules. IncHI plasmid is an important vector of Enterobacteriaceae for antibiotic resistance. Previously, it was classified into IncHI1, IncHI 2 and IncHI3 according to the order of research. In this study, IncHI plasmids were further divided into five subgroups according to the characteristics of replication initiation genes and cytoskeleton conserved sequences of representative plasmids, and two subtypes of IncHI4 and IncHI5 were added. Analysis showed that the sequence of plasmid skeleton region in each subtype was highly conserved, and that the five IncHI plasmids retained the conjugate transfer region tra1 and tra2, and the tellurium resistance gene cluster, ter. even so, There are still great differences among the five subtypes of IncHI plasmids. Three strains of multidrug resistant bacteria, Enterobacter cloacae T5282, Citrobacter flexneri 112298 and ornithine Laurus YNKP001, were isolated from clinical patients in three schools of education in China. The IncHI2 plasmid pT5282-mphAp112298-catA and the IncHI5 plasmid pYNKP001-dfrA were obtained respectively. According to the sequences of the three plasmids, the IncHI2 type reference plasmids R478 and IncHI5 type reference plasmids pKOXR1 were selected from the global nucleic acid database, and the five plasmids were analyzed for detailed biological information analysis and comparative genomics analysis. Sequence analysis showed that a series of insertion regions, such as integron, transposon and inserted sequence-mediated moving elements, were distributed in different positions in the plasmid skeleton region. The multidrug resistance region constructed by these elements generally carries three or more different resistance genes, including aminoglycoside resistance gene, chloramphenicol resistance gene, tetracycline resistance gene and 尾 -lactam resistance gene. Macrolides resistance genes, sulfonamides resistance genes, quinolone resistance genes, rifampicin resistance genes, trimethoprim resistance genes, fosfomycin resistance genes, quaternary ammonium salt resistance genes and so on. In order to understand the structure of multidrug resistant plasmids, a series of new mobile elements were identified and named, including seven transposons Tn6321, Tn6322, Tn6338-Tn6340, Tn6346 Tn6347, and the inserted sequence ISEc16, ISCfr8-ISCfr11ISKox1-ISKox3. Since antibiotics were used in the treatment of clinical infectious diseases, the corresponding drug-resistant strains appeared in clinic soon. Under the change of different antibiotic selection pressure and environmental conditions, bacteria could quickly obtain and retain different resistance genes. And thus accelerate the sustained spread of drug resistant strains. In this study, the representative IncHI type plasmids were sorted out, and the classification criteria were redefined and added to five subtypes. The fine analysis showed the characteristics and diversity of multidrug resistant IncHI type plasmids. It is revealed that IncHI plasmid is an important carrier of multidrug resistance in clinical isolates, which provides molecular basis for the treatment of bacterial infections in Enterobacteriaceae, and provides a basis for the study on the transmission of multidrug resistant Enterobacteriaceae in clinic. In order to prevent or delay the development of bacterial drug resistance.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R378

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本文編號(hào)：2199087