产碳青霉烯酶肠杆菌科细菌的检测及耐药特点研究

发布时间：2018-05-23 19:07

本文选题：肠杆菌科细菌 + 碳青霉烯酶　；参考：《广州医科大学》2017年硕士论文

【摘要】：研究背景肠杆菌科细菌是社区获得性感染和医院获得性感染的重要条件致病菌,主要引起呼吸系统感染和泌尿系统感染,在免疫力低下的患者中可引起严重甚至致死性的感染。而碳青霉烯类抗菌药物是目前临床上作为治疗肠杆菌科细菌感染最强而有力的一类抗菌药物,包括亚胺培南、美罗培南、厄他培南等,其对极大多数由质粒或染色体介导的β-内酰胺酶都有较高的稳定性,且与青霉素结合蛋白(PBPs)亲和力强,能有效地渗透细菌外膜,并存在抗生素后效应,因其抗菌谱广、抗菌活性强、杀菌作用快,临床上碳青霉烯类抗菌药物广泛用于治疗产超广谱β-内酰胺酶(ESBLs)和头孢菌素酶(AmpC酶)细菌引起的感染。但是随着碳青霉烯类抗菌药物的大量使用,国内外开始报道对碳青霉烯类抗菌药物不敏感甚至耐药的肠杆菌科细菌,这极大地限制了碳青霉烯类抗菌药物的使用,产生这一现象的重要原因是菌株获得了碳青霉烯酶基因。其中由质粒携带的碳青霉烯酶基因由于其耐药基因环境存在介导转移的基因元件,使得其易于在不同细菌间转移,造成了碳青霉烯酶耐药性的广泛传播,成为近年临床微生物学领域备受关注的热点之一。本研究通过采用法国梅里埃Vitek2全自动微生物鉴定仪与药物敏感仪对收集的耐药菌株进行鉴定及药敏分析,对本院可疑的产碳青霉烯酶菌株进行多重PCR检测碳青霉烯酶基因,并对耐药基因阳性的菌株进行接合转移实验,以了解耐药基因的传播方式;通过高通量测序技术对其中一株接合转移成功的多重耐药菌株进行质粒全序列的测定,获得的全序列通过生物信息学分析,展开对肺炎克雷伯菌耐药基因环境及耐药机制的研究。研究目的了解本院临床分离的肠杆菌科菌株的耐药特点及产碳青霉烯酶情况,探讨产碳青霉烯酶肠杆菌科细菌耐药基因的传播方式及可能的耐药机制,为临床控制耐药基因的传播提供一定的实验依据。研究方法1.菌株收集和药敏实验收集广州医科大学附属第一医院检验科微生物室分离的来自不同科室的不同标本类型的耐碳青霉烯类抗菌药物的肠杆菌科菌株共18株。全部菌株的鉴定及药敏实验采用法国梅里埃Vitek2全自动微生物鉴定仪与药物敏感仪完成,药敏结果按美国临床实验室标准化协会(CLSI)M100-S22 2012版进行判读。2.菌株DNA模板的制备及多重PCR实验菌株DNA模板的制备采用煮沸法,利用多重PCR方法对所有的菌株进行11种碳青霉烯酶基因blaIMP、blaSPM、blaAIM、blaVIM、blaOXA、blaGIM、blaBIC、blaSIM、blaNDM、blaDIM、blaKPC的检测,阳性PCR产物送公司进行测序验证,所得序列于网上BLAST比对,最终确定其基因亚型。3.耐药基因传播方式研究对所有携带碳青霉烯酶基因的菌株进行质粒接合转移实验并采用法国梅里埃Vitek2全自动微生物鉴定仪与药物敏感仪对接合子进行鉴定和检测药敏,药敏结果按美国临床实验室标准化协会(CLSI)M100-S22 2012版进行判读。对携带碳青霉烯酶基因的肺炎克雷伯菌株进行MLST分型,以和国内外已报道的情况进行比较。4.将多重耐药菌株肺炎克雷伯菌(KP)DQ49与受体菌EC600进行接合实验,PCR验证接合成功后,利用细菌基因组DNA提取试剂盒提取接合子基因组DNA,并利用Illumina Miseq高通量测序平台对其进行序列测定,得到的数据用Edena软件拼接,并利用RAST网上注释工具对得到的质粒全序列进行注释,使用网上序列比对工具BLAST进行耐药基因环境分析,使用PlasmidFinder网上工具进行质粒不相容性分析,使用ResFinder网上工具进行耐药基因分析,使用MLST网上工具进行ST分型分析。研究结果1.药敏结果18株耐碳青霉烯类抗菌药物的肠杆菌科细菌中包括肺炎克雷伯菌12株、大肠埃希菌2株、阴沟肠杆菌2株、植生拉乌尔菌1株、弗氏柠檬酸杆菌1株;科室分布为重症监护病房13株、泌尿外科2株、心血管内科1株、肝胆科1株、胃肠外科1株;标本类型为痰7株、中段尿2株、胆汁2株、腹腔引流液2株、插管导管1株、导管头1株、血液1株、分泌物1株、其他1株。经检测,18株菌对碳青霉烯类、青霉素类、β-内酰胺类、单环内酰胺类抗菌药物表现出较强的耐药性,表现为多重耐药;但对氨基糖苷类的阿米卡星、庆大霉素,磺胺类的呋喃妥因、复方新诺明,甘氨酰环素类的替加环素,氟喹诺酮类的环丙沙星、左旋氧氟沙星的耐药性却有不同。2.多重PCR检测碳青霉烯酶基因结果用多重PCR对18株碳青霉烯类耐药菌进行碳青霉烯酶基因检测,发现这些菌株均携带碳青霉烯酶基因,全部送测序,其中8株为blaNDM-1型,8株为blaKPC-2型,1株为blaVIM-1型,1株为bla_(IMP-26)型。3.耐药基因水平传播方式及肺炎克雷伯菌MLST结果在接合实验中,有10株菌成功地将质粒传递给受体菌EC600,其中包括肺炎克雷伯菌7株,大肠埃希菌2株,阴沟肠杆菌1株;8株通过电转化实验成功将质粒转移至受体菌DH5α,其中包括肺炎克雷伯菌5株,阴沟肠杆菌1株,植生拉乌尔菌1株,弗氏柠檬酸杆菌1株。选取12株肺炎克雷伯菌进行MLST分析,其中ST11型有10株,占大部分;ST20型1株;1株为ST2460,为首次报道。4.通过接合实验得到携带耐药基因bla_(IMP-26)的质粒pIMP26_DQ49,对质粒进行全序列测定。测序结果表明,pIMP26_DQ49属于质粒不相容群(Inc)中的IncN群,是大小为55179bp的环状质粒,携带三个耐药基因,G+C含量为50.4%,预测编码52个功能基因。BLAST发现pIMP26_DQ49与已报道的p IMP_HZ1序列相似度高达99%,且携带的可移动基因元件也高度相似,其耐药基因环境包含可导致转座事件发生的IS903D、IS2、Tn2、Tn3、tnp、tnpA,以及能捕获和整合外源性基因的1类整合子基因intI1。结论耐碳青霉烯类抗菌药物的肠杆菌科菌株均携带碳青霉烯酶基因,这些基因均可以通过接合实验或电转化实验在同科细菌间水平转移;肺炎克雷伯菌的质粒p IMP26_DQ49携带超广谱β-内酰胺酶基因blaTEM-1、氟喹诺酮耐药基因qnrS1和金属型碳青霉烯酶基因bla_(IMP-26),其存在可能对耐药基因在肠杆菌科细菌中的传播发挥重要作用。
[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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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