發(fā)布時(shí)間：2018-05-23 19:07

  本文選題：腸桿菌科細(xì)菌 + 碳青霉烯酶��； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景腸桿菌科細(xì)菌是社區(qū)獲得性感染和醫(yī)院獲得性感染的重要條件致病菌,主要引起呼吸系統(tǒng)感染和泌尿系統(tǒng)感染,在免疫力低下的患者中可引起嚴(yán)重甚至致死性的感染。而碳青霉烯類抗菌藥物是目前臨床上作為治療腸桿菌科細(xì)菌感染最強(qiáng)而有力的一類抗菌藥物,包括亞胺培南、美羅培南、厄他培南等,其對極大多數(shù)由質(zhì)�；蛉旧w介導(dǎo)的β-內(nèi)酰胺酶都有較高的穩(wěn)定性,且與青霉素結(jié)合蛋白(PBPs)親和力強(qiáng),能有效地滲透細(xì)菌外膜,并存在抗生素后效應(yīng),因其抗菌譜廣、抗菌活性強(qiáng)、殺菌作用快,臨床上碳青霉烯類抗菌藥物廣泛用于治療產(chǎn)超廣譜β-內(nèi)酰胺酶(ESBLs)和頭孢菌素酶(AmpC酶)細(xì)菌引起的感染。但是隨著碳青霉烯類抗菌藥物的大量使用,國內(nèi)外開始報(bào)道對碳青霉烯類抗菌藥物不敏感甚至耐藥的腸桿菌科細(xì)菌,這極大地限制了碳青霉烯類抗菌藥物的使用,產(chǎn)生這一現(xiàn)象的重要原因是菌株獲得了碳青霉烯酶基因。其中由質(zhì)粒攜帶的碳青霉烯酶基因由于其耐藥基因環(huán)境存在介導(dǎo)轉(zhuǎn)移的基因元件,使得其易于在不同細(xì)菌間轉(zhuǎn)移,造成了碳青霉烯酶耐藥性的廣泛傳播,成為近年臨床微生物學(xué)領(lǐng)域備受關(guān)注的熱點(diǎn)之一。本研究通過采用法國梅里埃Vitek2全自動(dòng)微生物鑒定儀與藥物敏感儀對收集的耐藥菌株進(jìn)行鑒定及藥敏分析,對本院可疑的產(chǎn)碳青霉烯酶菌株進(jìn)行多重PCR檢測碳青霉烯酶基因,并對耐藥基因陽性的菌株進(jìn)行接合轉(zhuǎn)移實(shí)驗(yàn),以了解耐藥基因的傳播方式;通過高通量測序技術(shù)對其中一株接合轉(zhuǎn)移成功的多重耐藥菌株進(jìn)行質(zhì)粒全序列的測定,獲得的全序列通過生物信息學(xué)分析,展開對肺炎克雷伯菌耐藥基因環(huán)境及耐藥機(jī)制的研究。研究目的了解本院臨床分離的腸桿菌科菌株的耐藥特點(diǎn)及產(chǎn)碳青霉烯酶情況,探討產(chǎn)碳青霉烯酶腸桿菌科細(xì)菌耐藥基因的傳播方式及可能的耐藥機(jī)制,為臨床控制耐藥基因的傳播提供一定的實(shí)驗(yàn)依據(jù)。研究方法1.菌株收集和藥敏實(shí)驗(yàn)收集廣州醫(yī)科大學(xué)附屬第一醫(yī)院檢驗(yàn)科微生物室分離的來自不同科室的不同標(biāo)本類型的耐碳青霉烯類抗菌藥物的腸桿菌科菌株共18株。全部菌株的鑒定及藥敏實(shí)驗(yàn)采用法國梅里埃Vitek2全自動(dòng)微生物鑒定儀與藥物敏感儀完成,藥敏結(jié)果按美國臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化協(xié)會(huì)(CLSI)M100-S22 2012版進(jìn)行判讀。2.菌株DNA模板的制備及多重PCR實(shí)驗(yàn)菌株DNA模板的制備采用煮沸法,利用多重PCR方法對所有的菌株進(jìn)行11種碳青霉烯酶基因blaIMP、blaSPM、blaAIM、blaVIM、blaOXA、blaGIM、blaBIC、blaSIM、blaNDM、blaDIM、blaKPC的檢測,陽性PCR產(chǎn)物送公司進(jìn)行測序驗(yàn)證,所得序列于網(wǎng)上BLAST比對,最終確定其基因亞型。3.耐藥基因傳播方式研究對所有攜帶碳青霉烯酶基因的菌株進(jìn)行質(zhì)粒接合轉(zhuǎn)移實(shí)驗(yàn)并采用法國梅里埃Vitek2全自動(dòng)微生物鑒定儀與藥物敏感儀對接合子進(jìn)行鑒定和檢測藥敏,藥敏結(jié)果按美國臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化協(xié)會(huì)(CLSI)M100-S22 2012版進(jìn)行判讀。對攜帶碳青霉烯酶基因的肺炎克雷伯菌株進(jìn)行MLST分型,以和國內(nèi)外已報(bào)道的情況進(jìn)行比較。4.將多重耐藥菌株肺炎克雷伯菌(KP)DQ49與受體菌EC600進(jìn)行接合實(shí)驗(yàn),PCR驗(yàn)證接合成功后,利用細(xì)菌基因組DNA提取試劑盒提取接合子基因組DNA,并利用Illumina Miseq高通量測序平臺(tái)對其進(jìn)行序列測定,得到的數(shù)據(jù)用Edena軟件拼接,并利用RAST網(wǎng)上注釋工具對得到的質(zhì)粒全序列進(jìn)行注釋,使用網(wǎng)上序列比對工具BLAST進(jìn)行耐藥基因環(huán)境分析,使用PlasmidFinder網(wǎng)上工具進(jìn)行質(zhì)粒不相容性分析,使用ResFinder網(wǎng)上工具進(jìn)行耐藥基因分析,使用MLST網(wǎng)上工具進(jìn)行ST分型分析。研究結(jié)果1.藥敏結(jié)果18株耐碳青霉烯類抗菌藥物的腸桿菌科細(xì)菌中包括肺炎克雷伯菌12株、大腸埃希菌2株、陰溝腸桿菌2株、植生拉烏爾菌1株、弗氏檸檬酸桿菌1株;科室分布為重癥監(jiān)護(hù)病房13株、泌尿外科2株、心血管內(nèi)科1株、肝膽科1株、胃腸外科1株;標(biāo)本類型為痰7株、中段尿2株、膽汁2株、腹腔引流液2株、插管導(dǎo)管1株、導(dǎo)管頭1株、血液1株、分泌物1株、其他1株。經(jīng)檢測,18株菌對碳青霉烯類、青霉素類、β-內(nèi)酰胺類、單環(huán)內(nèi)酰胺類抗菌藥物表現(xiàn)出較強(qiáng)的耐藥性,表現(xiàn)為多重耐藥;但對氨基糖苷類的阿米卡星、慶大霉素,磺胺類的呋喃妥因、復(fù)方新諾明,甘氨酰環(huán)素類的替加環(huán)素,氟喹諾酮類的環(huán)丙沙星、左旋氧氟沙星的耐藥性卻有不同。2.多重PCR檢測碳青霉烯酶基因結(jié)果用多重PCR對18株碳青霉烯類耐藥菌進(jìn)行碳青霉烯酶基因檢測,發(fā)現(xiàn)這些菌株均攜帶碳青霉烯酶基因,全部送測序,其中8株為blaNDM-1型,8株為blaKPC-2型,1株為blaVIM-1型,1株為bla_(IMP-26)型。3.耐藥基因水平傳播方式及肺炎克雷伯菌MLST結(jié)果在接合實(shí)驗(yàn)中,有10株菌成功地將質(zhì)粒傳遞給受體菌EC600,其中包括肺炎克雷伯菌7株,大腸埃希菌2株,陰溝腸桿菌1株;8株通過電轉(zhuǎn)化實(shí)驗(yàn)成功將質(zhì)粒轉(zhuǎn)移至受體菌DH5α,其中包括肺炎克雷伯菌5株,陰溝腸桿菌1株,植生拉烏爾菌1株,弗氏檸檬酸桿菌1株。選取12株肺炎克雷伯菌進(jìn)行MLST分析,其中ST11型有10株,占大部分;ST20型1株;1株為ST2460,為首次報(bào)道。4.通過接合實(shí)驗(yàn)得到攜帶耐藥基因bla_(IMP-26)的質(zhì)粒pIMP26_DQ49,對質(zhì)粒進(jìn)行全序列測定。測序結(jié)果表明,pIMP26_DQ49屬于質(zhì)粒不相容群(Inc)中的IncN群,是大小為55179bp的環(huán)狀質(zhì)粒,攜帶三個(gè)耐藥基因,G+C含量為50.4%,預(yù)測編碼52個(gè)功能基因。BLAST發(fā)現(xiàn)pIMP26_DQ49與已報(bào)道的p IMP_HZ1序列相似度高達(dá)99%,且攜帶的可移動(dòng)基因元件也高度相似,其耐藥基因環(huán)境包含可導(dǎo)致轉(zhuǎn)座事件發(fā)生的IS903D、IS2、Tn2、Tn3、tnp、tnpA,以及能捕獲和整合外源性基因的1類整合子基因intI1。結(jié)論耐碳青霉烯類抗菌藥物的腸桿菌科菌株均攜帶碳青霉烯酶基因,這些基因均可以通過接合實(shí)驗(yàn)或電轉(zhuǎn)化實(shí)驗(yàn)在同科細(xì)菌間水平轉(zhuǎn)移;肺炎克雷伯菌的質(zhì)粒p IMP26_DQ49攜帶超廣譜β-內(nèi)酰胺酶基因blaTEM-1、氟喹諾酮耐藥基因qnrS1和金屬型碳青霉烯酶基因bla_(IMP-26),其存在可能對耐藥基因在腸桿菌科細(xì)菌中的傳播發(fā)揮重要作用。
[Abstract]:Background Enterobacteriaceae is an important opportunistic pathogen of community acquired infection and hospital acquired infection, which mainly causes respiratory infection and urinary tract infection. It can cause severe or fatal infection in patients with low immunity. The carbapenems are currently used as the treatment of Enterobacteriaceae. The most powerful and powerful antimicrobial agents, including imipenem, meropenem, and etamepenem, are highly stable to most of the plasmid or chromosome mediated beta lactamases, and have strong affinity with the penicillin binding protein (PBPs), and can effectively permeate the bacterial outer membrane, and have the post antibiotic effect, because of their resistance to the bacteria. The bacterial spectrum is wide, the antibacterial activity is strong, and the bactericidal effect is fast. In clinic, carbapenems are widely used to treat the infection caused by ESBLs and AmpC bacteria. However, with the extensive use of carbapenems, it is reported that the antibiotics are not sensitive to carbapenems. The resistance of Enterobacteriaceae bacteria, which greatly restricts the use of carbapenems, is an important cause of this phenomenon, which is caused by the strain acquired carbapenem gene. The plasmid carrying carbapenem gene has the gene element that mediates the transfer due to its resistant gene environment, making it easy to be in different bacteria. The drug resistance of carbapenem is widely spread, which has become one of the hot topics in the field of clinical microbiology in recent years. This study identified and analyzed the drug-resistant strains collected by the French Vitek2 automatic microorganism identification instrument and drug sensitive instrument, and the suspected carbapenem producing enzyme produced in our hospital. The strain performed multiple PCR detection of carbapenem gene, and the conjugative transfer experiment was carried out on the resistant gene positive strains to understand the transmission mode of the resistant gene, and the full sequence of the plasmid was measured by high throughput sequencing technology, and the whole sequence was obtained through the bioinformatics credits. Study on the resistance gene environment and drug resistance mechanism of Klebsiella pneumoniae. The purpose of this study is to understand the characteristics of drug resistance and the situation of carbapenem production in clinical isolates of Enterobacteriaceae in our hospital, and to explore the transmission mode and possible mechanism of resistance genes of carbapenem producing Enterobacteriaceae, and to control the drug resistance genes in clinical. Study method 1. strains collection and drug sensitivity experiment collected 18 strains of Enterobacteriaceae strains isolated from the laboratory of the first hospital of the first hospital of Guangzhou Medical University, which were isolated from different sections of the laboratory. All strains were identified and drug sensitivity experiments were adopted in France. The Vitek2 full-automatic microbial identification instrument and the drug sensitivity instrument were completed. The drug sensitivity results were prepared by the M100-S22 2012 version of the American clinical laboratory standardization association (CLSI) M100-S22 and the preparation of the DNA template of the.2. strain and the preparation of the DNA template for multiple PCR experimental strains were prepared by the boiling method, and 11 carbapenems were carried out to all the strains by the multiweight PCR method. The enzyme gene blaIMP, blaSPM, blaAIM, blaVIM, blaOXA, blaGIM, blaBIC, blaSIM, blaNDM, blaDIM, blaKPC were tested. The positive PCR products were sent to the company for sequencing verification. The experiment was carried out and the drug sensitivity was identified and detected by the joint zygote of the French Vitek2 automatic microorganism identification instrument and the drug sensitive instrument. The drug sensitivity results were read according to the M100-S22 2012 edition of the American clinical laboratory standardization association (CLSI). The Klebsiella pneumoniae strains carrying carbapenem gene were typed by MLST. The reports are compared with.4., which joins the multidrug-resistant strain Klebsiella pneumoniae (KP) DQ49 and the receptor bacteria EC600. After the PCR verification is successful, the genomic DNA extraction kit of the bacterial genome is used to extract the zygote genome DNA, and the Illumina Miseq high throughput sequencing platform is used to sequence it, and the data obtained are Edena. The whole sequence of the plasmid was annotated with the RAST online annotation tool, and the resistance gene environment was analyzed using the online sequence alignment tool BLAST. The PlasmidFinder online tool was used to analyze the incompatibility of the plasmid. The drug resistance gene was analyzed using the ResFinder online tool, and the MLST online tool was used to carry out the ST score. Results 1. drug sensitive results of 18 strains of carbapenem resistant Enterobacteriaceae included 12 strains of Klebsiella pneumoniae, 2 Escherichia coli, 2 Enterobacter cloacae, 1 strains of Raoul bacteria, 1 citrate bacilli, 13 in intensive care centers, 2 in Department of Urology, 1 in cardiovascular medicine, and hepatobiliary. 1 strains, 1 gastrointestinal surgery, 7 strains of sputum, 2 middle urine, 2 bile, 2 abdominal drainage, 1 catheters, 1 ducts, 1 blood, 1 secretions, 1 strains, and 1 strains of bacteria showed strong resistance to carbapenems, penicillins, beta lactam and mono lactam. Drugs for Amikacin, gentamicin, sulfonamides, sulfamethoxin, tetracycline, fluoroquinolone, levofloxacin, and levofloxacin have different.2. multiple PCR detection of carbapenem gene results with multiple PCR against 18 carbapenems resistant bacteria Carbapenenenase gene detection showed that all of these strains carried carbapenem gene and all were sequenced, of which 8 were blaNDM-1, 8 were blaKPC-2, 1 were blaVIM-1, 1 were bla_ (IMP-26).3. resistant gene and MLST knot of Klebsiella pneumoniae in joint experiment, and 10 strains successfully transferred the plasmid to the plasmid. EC600, including 7 strains of Klebsiella pneumoniae, 2 Escherichia coli and 1 strains of Enterobacter cloacae, 8 strains were successfully transferred to receptor bacteria DH5 alpha through electrical transformation experiments, including 5 Klebsiella pneumoniae, 1 Enterobacter cloacae, 1 plants of Raoul bacteria, 1 strains of citric acid bacilli, and 12 strains of Klebsiella pneumoniae for MLST In the analysis, there were 10 ST11 strains, most of them, 1 ST20 strains and 1 strains of ST2460. The plasmid pIMP26_DQ49 was first reported by.4. to carry the plasmid pIMP26_DQ49 carrying resistance gene bla_ (IMP-26). The sequencing results showed that pIMP26_DQ49 belonged to the IncN group in the plasmid incompatible group (Inc), and was a cyclic plasmid with the size of 55179bp. With three resistant genes, the G+C content was 50.4%, and the predicted encoding 52 functional genes.BLAST found that the similarity between the pIMP26_DQ49 and the reported P IMP_HZ1 sequences was up to 99%, and the portable gene elements were also highly similar, and the resistant gene environment contained IS903D, IS2, Tn2, Tn3, TNP, tnpA, which could lead to transposable events. Integration of 1 integron genes of exogenous genes intI1. conclusion that the Enterobacteriaceae strains of carbapenem antibiotics all carry carbapenem gene. These genes can be transferred to the same family by conjugation or electroconversion experiments, and the P IMP26_DQ49 of Klebsiella pneumoniae carries broad-spectrum beta lactamases The presence of blaTEM-1, fluoroquinolone resistance gene qnrS1 and metal carbapenem gene bla_ (IMP-26) may play an important role in the transmission of resistant genes in Enterobacteriaceae.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.5

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