产碳青霉烯酶革兰阴性杆菌的耐药及传播机制研究
发布时间：2018-07-16 15:44
【摘要】：研究背景随着各种抗生素的大量使用,多重耐药甚至泛耐药细菌不断增多,给临床诊治带来巨大的挑战,现已成为一个世界性难题。碳青霉烯类抗生素是治疗多重耐药革兰阴性杆菌严重感染的最后一道防线,耐碳青霉烯革兰阴性杆菌的快速传播越来越引起关注。产碳青霉烯酶是这类细菌对碳青霉烯类抗生素耐药的主要原因,碳青霉烯酶包括Ambler分子分类中的A、B、D三类酶,是指所有能明显水解亚胺培南或美罗培南等碳青霉烯类抗生素的一类β-内酰胺酶。大多数碳青霉烯酶基因位于可转移性基因元件上如质粒、整合子等,在同种属和不同种属的细菌可以通过接合、传导、转化、转座等方式获得耐药基因,导致更多细菌产碳青霉烯酶。耐碳青霉烯革兰阴性杆菌不仅对β-内酰胺类抗生素耐药,同时多数也对喹诺酮、氨基糖苷等其他类抗生素耐药,从而表现出多耐药甚至泛耐药的特性。因此,使用快速方法检测细菌是否产生碳青霉烯酶,对控制这类细菌的传播和感染具有重要意义。目前产碳青霉烯酶的检测方法主要有改良的Hodge实验(the modified Hodge test)这是一个表型实验,这是美国临床实验室标准化协会(CLSI)推荐的表型确证试验。另外还有亚胺培南-EDTA双纸片协同试验及Etest MBL商品化确认试条,专门针对B组碳青霉烯酶的检验[1]。但是,随着耐药菌株所含耐药基因的组合和种类越趋复杂,上述方法的特异性和敏感性受到挑战,产碳青霉烯酶的检测方法亟待革新。美国临床与实验室标准化协会(Clinical and Laboratory Standards Institute,CLSI)于2015年引入了Carba NP试验[2],作为肠杆菌科、铜绿假单胞菌和不动菌属细菌产生的碳青霉烯酶表型检测的确证试验,由法国南法医学院Patrice Nordmann教授研究团队于2012年首次报道[3],此后,陆续优化该方法用于检测产碳青霉烯酶假单胞菌及指示碳青霉烯酶类型的Carba NP试验Ⅱ已有报道[4],该试验能直接判定产碳青霉烯酶菌株的产酶类型,较改良的Hodge试验更简单、快速。随着分子生物学和生物信息分析学的快速发展,使得全基因组测序技术得到广泛的应用。全基因组测序是对物种未知基因组全序列进行测序。目前,全基因组测序技术应用比较多的主要是第二代测序技术(NGS)和第三代测序技术。第二代测序技术能够快速、低成本的进行全基因组测序,其设备供应商主要是454(罗氏公司),SOLi D(AB公司)和Solexa。第三代测序技术于2011年开始推广,其单分子实时测序技术(SMRT)与第二代测序完全不同,由Pacific Biosciences公司研发,它的序列读长高达3kb。高通量测序技术的出现使得人类获得基因组方式出现重大变革,特别是在基因组信息较小的微生物领域,已成为病原微生物的鉴定分型、致病机理研究等一项不可取代的工具。本研究采用CLSI推荐的Carba NP试验,对本院可疑产碳青霉烯酶多重耐药革兰阴性杆菌进行筛选,以多重PCR方法对筛选阳性的结果进行耐药基因的检测,以了解我院革兰阴性杆菌产碳青霉烯酶流行情况,并探讨Carba NP试验检测方法的优劣。对来源同一病人的4株产碳青霉烯酶肺炎克雷伯菌进行接合/转化试验,比较接合/转化菌和亲本菌株的药物敏感性差异,并分析4株菌的同源性。通过高通量测序技术对其中1株携带bla KPC-2的多重耐药菌株作全基因组测序,通过生物信息学分析,了解肺炎克雷伯菌的耐药机制和耐药基因环境等特点。研究目的1.了解广州医科大学附属第一医院临床分离产碳青霉烯酶多重耐药革兰阴性杆菌的流行及耐药分子机制。2.了解Carba NP试验检测革兰阴性杆菌产碳青霉烯酶的效果。3.来源同一病人4株肺炎克雷伯菌株耐药性及同源性分析。4.用高通量测序方法分析1株KPC阳性肺炎克雷伯菌株的基因环境。研究方法1.收集广州医科大学附属第一医院微生物室2010年1月-2013年10月临床分离的59株多重耐药的革兰阴性杆菌,所有菌株用VITEK2微生物全自动鉴定仪进行鉴定和药敏实验,筛选对三种以上抗菌药物耐药的革兰阴性杆菌菌株,且对至少一种碳青霉烯类抗生素耐药,即为可疑产碳青霉烯酶菌株,并对标本类型、碳青霉烯类药物敏感性等资料进行统计,分析其流行病学特点。2.采用Carba NP试验检测产碳青霉烯酶的菌株,并用多重PCR技术对碳青霉烯酶表型试验阳性菌株进行相关耐药基因检测,了解本院革兰阴性杆菌碳青霉烯酶的主要基因型,并探讨Carba NP试验检测革兰阴性杆菌碳青霉烯酶的效果。3.通过接合试验和电转化试验获得4株肺炎克雷伯菌接合子或转化子,受体菌携带耐药基因bla NDM-1或bla KPC-2,提取质粒DNA,通过酶切技术分析质粒类型。4.分别提取多重耐药菌株LJ1、LJ2、LJ3、LJ4的总DNA,ERIC-PCR方法分析同源性和质粒不相容群,通过PCR方法检测7个肺炎克雷伯菌管家基因,利用MLST网上工具进行ST分型,进一步分析这些菌株的同源性。5.提取转化子LJ4C总DNA,用Illumina Miseq和Pac Bio RS II平台进行高通量测序。由Celera Assembler软件进行组装。在其PBc R pipeline中,用Illumina Mi Seq Reads(二代测序)的数据进行纠错。RAST网上工具进行基因注释,Res Finder和NCBI BLAST网上工具进行耐药基因分析,NCBI BLAST网上工具进行耐药基因遗传环境分析,Plasmid Finder网上工具进行质粒序列分析。研究结果1.59株多重耐药的革兰阴性杆菌的流行病学和药敏结果分析。59株多重耐药革兰阴性杆菌对亚胺培南、美洛培南、厄他培南耐药率分别为62.71%,61.02%,64.41%。菌株主要来源于痰液35.6%(21/59)胆汁5.1%(3/59)、分泌物8.5%(5/59)、腹水5.1%((3/59)、尿液13.6%(8/59)、胸水3.4%(2/59)、血液23.7%(14/59)、引流液5.1%(3/59)。多重耐药革兰阴性杆菌中以携带NDM-1基因和携带KPC-2基因型菌株为主,而产生碳青霉烯酶的细菌以弗氏柠檬酸杆菌、铜绿假单胞菌和肺炎克雷伯菌等比较常见。2.Carba NP试验和多重PCR检测结果分析比较。Carba NP试验确定33株产碳青霉烯酶菌株,分别为A类酶12株、B类酶21株,PCR法检出多种碳青霉烯酶,包括KPC(12株)、IMP(7株)、NDM(12株)、VIM(3株)。与PCR法比较,Carba NP试验敏感性和特异性分别为97.06%和100%。3.3株肺炎克雷伯菌LJ1-LJ3携带耐药基因bla NDM-1,这些菌株可通过接合方式传递耐药基因,携带耐药基因bla KPC-2菌株LJ4接合不成功,需用电转化获得转化子。S1核酸内切酶分析4株菌有三种质粒类型。4.用ERIC-PCR方法分析LJ1、LJ2、LJ3、LJ4菌株同源性,LJ1和LJ2高度同源,与LJ3、LJ4菌有不同的带型。LJ3、LJ4不属于18个已发现质粒不相容群的任何一种,LJ1、LJ2属于不相容群Inc F II群。通过7个肺炎克雷伯菌管家基因检测,MLST网上工具进行分析,在LJ1和LJ2为新发现ST型,均为ST1416,LJ3为ST20,LJ4为ST11。5.PCR法证实多重耐药产碳青霉烯酶菌株LJ4携带bla KPC-2型碳青霉烯酶基因,对其进行高通量测序,获得来自LJ4菌株的质粒p CT-KPC,是一个151466bp的环状分子,包括120783bp的质粒骨架,有编码,复制起始,转移,维持和稳定功能的编码区以及包含两段耐药基因的编码区(MRR)。p CT-KPC质粒含有53.81%的GC含量,总共有233个已鉴定的开放阅读框ORFs,138个编码基因与已知有功能的蛋白质有高度相似性。质粒LJ4C是由p HN7A8、p KPC-LK30的部分质粒结构及一个额外的tra区域构成一个嵌合体,接合转移基因簇tra与细菌接合转移相关。6.对质粒结构研究发现,LJ4菌株的转化子同时携带bla CTX-M-65、blafos A3、blarmt B、bla SHV、bla TEM1b及bla KPC-2多个耐药基因。blafos A基因在家禽和宠物之间广泛传播,本研究结果显示在病人身上分离的病原菌株质粒跟宠物来源的菌株质粒结构高度同源,不排除在病患宠物及家禽与人类来源细菌发生同源重组的可能。结论1.我院产生碳青霉烯酶的细菌以弗氏柠檬酸杆菌、铜绿假单胞菌和肺炎克雷伯菌等比较常见,提示临床上如果检出了此类多重耐药菌株,应注意筛查其产生碳青霉烯酶的状况,以便合理应用抗菌药物。2.Carba NP试验用于多重耐药革兰阴性杆菌产碳青霉烯酶的筛查简单、易行、快速、准确,可在临床实验室中推广使用。3.已出现同时携带ESBLs、fos A、rmt B、KPC基因质粒的菌株,质粒携带的bla NDM-1和bla KPC-2基因可通过接合或转化的方式在同种细菌间传播,预示这些多重耐药基因在细菌间有水平播散的可能。4.高通量测序可快速、全面、准确地对耐药基因、基因环境和细菌分型等开展详细的分析,便于完成细菌耐药机制的研究。
[Abstract]:Background with the extensive use of various antibiotics, multidrug-resistant and even pan resistant bacteria are increasing, which poses a great challenge to clinical diagnosis and treatment. It has become a worldwide problem. Carbapenems are the last line of defense for the severe infection of multiresistant gram-negative bacilli, carbapenem resistant gram-negative bacilli Carbon penicenase is the main cause of resistance to carbapenems. Carbapenenems include A, B, and D three enzymes in the classification of Ambler molecules, which are a class of beta lactamases, which can obviously hydrolyze imipenem or meropenem and other carbapenems. Most carbon green Mycophenase genes are located in transferable gene elements such as plasmids and integrons. Bacteria in the same genus and different species can obtain resistance genes through conjugation, conduction, transformation, transposing and so on, causing more bacteria to produce carbapenem. The carbapenems resistant bacteria are resistant to beta lactam antibiotics and most of them are also resistant to beta lactam antibiotics. The resistance to quinolone, aminoglycoside and other antibiotics, thus showing the characteristics of multidrug-resistant and even pan resistance. Therefore, the rapid detection of carbapenems by bacteria is of great significance in controlling the spread and infection of these bacteria. The main methods for the detection of carbapenems are the modified Hodge test (the m). Odified Hodge test) this is a phenotypic experiment. This is the phenotypic confirmatory test recommended by the American clinical laboratory standardization association (CLSI). There are also the imipenem -EDTA double paper synergistic test and the Etest MBL commercialization test, specifically for the test of carbopenicolenes in the B Group [1]. but with a group of resistant genes containing the resistant strain. The more complex and more complex, the specificity and sensitivity of the methods are challenged, and the detection methods for carbapenems are urgently needed to be innovated. The Clinical and Laboratory Standards Institute (CLSI) introduced the Carba NP test [2] in 2015 as the Enterobacteriaceae, Pseudomonas aeruginosa and the genus acomonas. The confirmatory test of the phenotypic phenotypic detection of carbapenenenes produced by bacteria was first reported in 2012 by the research team of Professor Patrice Nordmann of the French Academy of Southern forensic medicine (Prof Patrice Nordmann). Since then, the Carba NP test for the detection of carbapenem producing Pseudomonas sp. and the Carba NP test indicating the type of carbapenenenes has been reported. The test can be directly judged. The type of enzyme producing enzyme producing carbapenenenase is more simple and faster than the modified Hodge test. With the rapid development of molecular biology and bioinformatics, the whole genome sequencing technology is widely used. Whole genome sequencing is the whole sequence of unknown species of species. With more than second generation sequencing technology (NGS) and third generation sequencing technology, second generation sequencing technology can be quickly and low cost for whole genome sequencing. Its equipment suppliers are mainly 454 (Roche), SOLi D (AB) and Solexa. third generation sequencing technology began to be popularized in 2011, and their single molecule real time sequencing technology (SMRT) Different from the second generation sequencing, it was developed by Pacific Biosciences, and its sequence read long up to 3kb. high throughput sequencing technology has made human genome form a major change, especially in the field of microorganism with small genome information, which has become an identification of pathogenic microorganism, a study of pathogenic mechanism and so on. In this study, the Carba NP test recommended by CLSI was used to screen the multidrug-resistant gram-negative bacilli of suspected carbapenem production in our hospital. The multiple PCR method was used to detect the resistance genes of the screening positive results, so as to understand the prevalence of carbapenem production in Gram-negative bacilli in our hospital and to explore the Carba NP test. 4 strains of Klebsiella pneumoniae producing carbon penicylenzyme from the same patient were conjugation / conversion tests, and the differences in drug sensitivity of conjugation / transformed bacteria and parent strains were compared and the homology of 4 strains was analyzed. 1 strains of multiple resistant strains carrying bla KPC-2 were sequenced by high throughput sequencing. Through bioinformatics analysis, the drug resistance mechanism and drug resistance gene environment of Klebsiella pneumoniae were investigated. Objective 1. to understand the prevalence and molecular mechanism of multi resistant gram-negative bacilli of carbapenem production in the First Affiliated Hospital of Guangzhou Medical University and to understand the carbon production of gram-negative bacilli by Carba NP test. The effect of penicilenes on the resistance and homology of 4 strains of klebber strains from the same patient.3..4. high flux sequencing method was used to analyze the genetic environment of 1 strains of KPC positive klebber strain. Method 1. collected 59 strains of multiple tolerance of clinical isolation in the first hospital of Guangzhou Medical University, the first hospital microorganism room, October January 2010. Gram-negative bacilli of the drug, all strains were identified by VITEK2 microorganism automatic identification instrument and drug sensitivity test, screening the gram-negative bacilli resistant to more than three kinds of antimicrobial agents, and resistant to at least one carbapenene antibiotic, that is, suspected carbapenems, and the type of carbapenems, and the sensitivity of carbapenems. The epidemiological characteristics of.2. were analyzed by Carba NP test for the detection of carbapenem producing strains, and multiple PCR techniques were used to detect the related resistance genes of the positive strains of carbapenem test, to understand the main genotypes of carbapenems of gram-negative bacilli in our hospital and to explore the test of Carba NP test. The effect of gram-negative bacilli carbapenem.3. obtained 4 strains of Klebsiella pneumoniae zygote or transformant through conjugation test and electrical transformation test. The receptor bacteria carried the resistance gene bla NDM-1 or bla KPC-2 to extract plasmid DNA. The multiple resistant strain LJ1, LJ2, LJ3, LJ4 DNA were extracted by the enzyme cutting technique and analyzed the plasmid type.4. respectively. Methods the homologous and plasmid incompatible groups were analyzed, and 7 Klebsiella pneumoniae housekeeping genes were detected by PCR method. The ST typing was carried out by MLST online tools. The homologous.5. extracted transformants LJ4C total DNA was further analyzed. Illumina Miseq and Pac Bio RS II platform were used for high flux sequencing. In its PBc R pipeline, using the data of Illumina Mi Seq Reads (two generation sequencing) to carry on the error correction.RAST online tools to carry on the gene annotation, Res Finder and NCBI BLAST network tools to carry on the resistance gene analysis. Results the epidemiological and drug susceptibility results of 1.59 strains of gram-negative bacilli were analyzed. The resistance rates of.59 strains to imipenem, meropenem, and eopenem were 62.71% and 61.02% respectively. The strains of 64.41%. were mainly from 35.6% (21/59) bile 5.1% (3/59), 8.5% (5/59), ascites 5.1% (3/59), and 1 in urine. 3.6% (8/59), hydrothorax 3.4% (2/59), blood 23.7% (14/59), and drainage 5.1% (3/59). Multidrug-resistant gram-negative bacilli were mainly carried with NDM-1 gene and KPC-2 genotype strains, and carbapenem producing bacteria with citrate, Pseudomonas aeruginosa and Klebsiella pneumoniae were more common in.2.Carba NP test and multiple PCR. Analysis and comparison of the results of.Carba NP test to determine 33 strains of carbapenenenase producing carbapenenenase, respectively a class a enzyme 12, 21 B enzymes, and PCR method to detect a variety of carbapenems, including KPC (12 strains), IMP (7 strains), NDM (12 strains), VIM (3). The sensitivity and specificity of Carba NP test are 97.06% and 100%.3.3 strains Klebsiella pneumoniae respectively. Carrying resistance gene bla NDM-1, these strains can transfer drug-resistant genes through conjugation, carrying drug resistant gene bla KPC-2 strain LJ4 unsuccessfully, and use electrical transformation to obtain transformant.S1 nucleic acid endonuclease analysis of 4 strains of plasmid type.4. with ERIC-PCR method to analyze LJ1, LJ2, LJ3, LJ4 strain homology LJ4 bacteria have different band type.LJ3, LJ4 does not belong to any one of the 18 found plasmid incompatible groups, LJ1 and LJ2 belong to the incompatible group Inc F II group. The carbapenem strain LJ4 carries the BLA KPC-2 type carbapenem gene, which is sequenced high flux and obtained the plasmid P CT-KPC from the LJ4 strain. It is a 151466bp ring molecule, including the plasmid skeleton of 120783bp, which encodes, replicating, transferring, maintaining and stabilizing the coding region, as well as the coding region containing two segment resistant genes. (MRR) the.P CT-KPC plasmid contains 53.81% GC content, a total of 233 identified open reading frame ORFs, and 138 encoding genes are highly similar to the known functional proteins. Plasmid LJ4C is constructed from a partial plasmid structure of P HN7A8, P KPC-LK30 and an additional tra region to form a chimerism, joining the transfer gene cluster tra and bacterial connection. The plasmid structure study found that the transformants of LJ4 strain also carried bla CTX-M-65, blafos A3, blarmt B, BLA SHV, BLA TEM1b and multiple resistant genes to spread widely between poultry and pets. The results of this study showed that the plasmid isolated from the patient was with the strain of the pet source. The plasmid structure is highly homologous, which does not exclude the possibility of homologous recombination between the sick pets and the poultry and the human source bacteria. Conclusion 1. the carbapenem bacteria producing carbapenem in our hospital are more common, such as citric acid bacilli Freund, Pseudomonas aeruginosa and Klebsiella pneumoniae. It is suggested that the multiple drug resistant strains have been detected in the clinic and should be screened out. To find out the status of carbapenem production in order to apply the.2.Carba NP test to the screening of carbapenems of multi resistant gram-negative bacilli, it is easy, fast, accurate, and can be used in clinical laboratory to promote the use of.3. with ESBLs, Fos A, RMT B, KPC plasmid and Bla ND. M-1 and Bla KPC-2 genes can be transmitted in the same type of bacteria by conjugation or transformation. It indicates that the possible.4. high throughput sequencing of these multidrug resistant genes in bacteria is rapid, comprehensive and accurate in the analysis of drug resistance genes, genetic environment and bacterial genotyping, so as to facilitate the study of the mechanism of bacterial resistance.
【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
本文编号：2126854
[Abstract]:Background with the extensive use of various antibiotics, multidrug-resistant and even pan resistant bacteria are increasing, which poses a great challenge to clinical diagnosis and treatment. It has become a worldwide problem. Carbapenems are the last line of defense for the severe infection of multiresistant gram-negative bacilli, carbapenem resistant gram-negative bacilli Carbon penicenase is the main cause of resistance to carbapenems. Carbapenenems include A, B, and D three enzymes in the classification of Ambler molecules, which are a class of beta lactamases, which can obviously hydrolyze imipenem or meropenem and other carbapenems. Most carbon green Mycophenase genes are located in transferable gene elements such as plasmids and integrons. Bacteria in the same genus and different species can obtain resistance genes through conjugation, conduction, transformation, transposing and so on, causing more bacteria to produce carbapenem. The carbapenems resistant bacteria are resistant to beta lactam antibiotics and most of them are also resistant to beta lactam antibiotics. The resistance to quinolone, aminoglycoside and other antibiotics, thus showing the characteristics of multidrug-resistant and even pan resistance. Therefore, the rapid detection of carbapenems by bacteria is of great significance in controlling the spread and infection of these bacteria. The main methods for the detection of carbapenems are the modified Hodge test (the m). Odified Hodge test) this is a phenotypic experiment. This is the phenotypic confirmatory test recommended by the American clinical laboratory standardization association (CLSI). There are also the imipenem -EDTA double paper synergistic test and the Etest MBL commercialization test, specifically for the test of carbopenicolenes in the B Group [1]. but with a group of resistant genes containing the resistant strain. The more complex and more complex, the specificity and sensitivity of the methods are challenged, and the detection methods for carbapenems are urgently needed to be innovated. The Clinical and Laboratory Standards Institute (CLSI) introduced the Carba NP test [2] in 2015 as the Enterobacteriaceae, Pseudomonas aeruginosa and the genus acomonas. The confirmatory test of the phenotypic phenotypic detection of carbapenenenes produced by bacteria was first reported in 2012 by the research team of Professor Patrice Nordmann of the French Academy of Southern forensic medicine (Prof Patrice Nordmann). Since then, the Carba NP test for the detection of carbapenem producing Pseudomonas sp. and the Carba NP test indicating the type of carbapenenenes has been reported. The test can be directly judged. The type of enzyme producing enzyme producing carbapenenenase is more simple and faster than the modified Hodge test. With the rapid development of molecular biology and bioinformatics, the whole genome sequencing technology is widely used. Whole genome sequencing is the whole sequence of unknown species of species. With more than second generation sequencing technology (NGS) and third generation sequencing technology, second generation sequencing technology can be quickly and low cost for whole genome sequencing. Its equipment suppliers are mainly 454 (Roche), SOLi D (AB) and Solexa. third generation sequencing technology began to be popularized in 2011, and their single molecule real time sequencing technology (SMRT) Different from the second generation sequencing, it was developed by Pacific Biosciences, and its sequence read long up to 3kb. high throughput sequencing technology has made human genome form a major change, especially in the field of microorganism with small genome information, which has become an identification of pathogenic microorganism, a study of pathogenic mechanism and so on. In this study, the Carba NP test recommended by CLSI was used to screen the multidrug-resistant gram-negative bacilli of suspected carbapenem production in our hospital. The multiple PCR method was used to detect the resistance genes of the screening positive results, so as to understand the prevalence of carbapenem production in Gram-negative bacilli in our hospital and to explore the Carba NP test. 4 strains of Klebsiella pneumoniae producing carbon penicylenzyme from the same patient were conjugation / conversion tests, and the differences in drug sensitivity of conjugation / transformed bacteria and parent strains were compared and the homology of 4 strains was analyzed. 1 strains of multiple resistant strains carrying bla KPC-2 were sequenced by high throughput sequencing. Through bioinformatics analysis, the drug resistance mechanism and drug resistance gene environment of Klebsiella pneumoniae were investigated. Objective 1. to understand the prevalence and molecular mechanism of multi resistant gram-negative bacilli of carbapenem production in the First Affiliated Hospital of Guangzhou Medical University and to understand the carbon production of gram-negative bacilli by Carba NP test. The effect of penicilenes on the resistance and homology of 4 strains of klebber strains from the same patient.3..4. high flux sequencing method was used to analyze the genetic environment of 1 strains of KPC positive klebber strain. Method 1. collected 59 strains of multiple tolerance of clinical isolation in the first hospital of Guangzhou Medical University, the first hospital microorganism room, October January 2010. Gram-negative bacilli of the drug, all strains were identified by VITEK2 microorganism automatic identification instrument and drug sensitivity test, screening the gram-negative bacilli resistant to more than three kinds of antimicrobial agents, and resistant to at least one carbapenene antibiotic, that is, suspected carbapenems, and the type of carbapenems, and the sensitivity of carbapenems. The epidemiological characteristics of.2. were analyzed by Carba NP test for the detection of carbapenem producing strains, and multiple PCR techniques were used to detect the related resistance genes of the positive strains of carbapenem test, to understand the main genotypes of carbapenems of gram-negative bacilli in our hospital and to explore the test of Carba NP test. The effect of gram-negative bacilli carbapenem.3. obtained 4 strains of Klebsiella pneumoniae zygote or transformant through conjugation test and electrical transformation test. The receptor bacteria carried the resistance gene bla NDM-1 or bla KPC-2 to extract plasmid DNA. The multiple resistant strain LJ1, LJ2, LJ3, LJ4 DNA were extracted by the enzyme cutting technique and analyzed the plasmid type.4. respectively. Methods the homologous and plasmid incompatible groups were analyzed, and 7 Klebsiella pneumoniae housekeeping genes were detected by PCR method. The ST typing was carried out by MLST online tools. The homologous.5. extracted transformants LJ4C total DNA was further analyzed. Illumina Miseq and Pac Bio RS II platform were used for high flux sequencing. In its PBc R pipeline, using the data of Illumina Mi Seq Reads (two generation sequencing) to carry on the error correction.RAST online tools to carry on the gene annotation, Res Finder and NCBI BLAST network tools to carry on the resistance gene analysis. Results the epidemiological and drug susceptibility results of 1.59 strains of gram-negative bacilli were analyzed. The resistance rates of.59 strains to imipenem, meropenem, and eopenem were 62.71% and 61.02% respectively. The strains of 64.41%. were mainly from 35.6% (21/59) bile 5.1% (3/59), 8.5% (5/59), ascites 5.1% (3/59), and 1 in urine. 3.6% (8/59), hydrothorax 3.4% (2/59), blood 23.7% (14/59), and drainage 5.1% (3/59). Multidrug-resistant gram-negative bacilli were mainly carried with NDM-1 gene and KPC-2 genotype strains, and carbapenem producing bacteria with citrate, Pseudomonas aeruginosa and Klebsiella pneumoniae were more common in.2.Carba NP test and multiple PCR. Analysis and comparison of the results of.Carba NP test to determine 33 strains of carbapenenenase producing carbapenenenase, respectively a class a enzyme 12, 21 B enzymes, and PCR method to detect a variety of carbapenems, including KPC (12 strains), IMP (7 strains), NDM (12 strains), VIM (3). The sensitivity and specificity of Carba NP test are 97.06% and 100%.3.3 strains Klebsiella pneumoniae respectively. Carrying resistance gene bla NDM-1, these strains can transfer drug-resistant genes through conjugation, carrying drug resistant gene bla KPC-2 strain LJ4 unsuccessfully, and use electrical transformation to obtain transformant.S1 nucleic acid endonuclease analysis of 4 strains of plasmid type.4. with ERIC-PCR method to analyze LJ1, LJ2, LJ3, LJ4 strain homology LJ4 bacteria have different band type.LJ3, LJ4 does not belong to any one of the 18 found plasmid incompatible groups, LJ1 and LJ2 belong to the incompatible group Inc F II group. The carbapenem strain LJ4 carries the BLA KPC-2 type carbapenem gene, which is sequenced high flux and obtained the plasmid P CT-KPC from the LJ4 strain. It is a 151466bp ring molecule, including the plasmid skeleton of 120783bp, which encodes, replicating, transferring, maintaining and stabilizing the coding region, as well as the coding region containing two segment resistant genes. (MRR) the.P CT-KPC plasmid contains 53.81% GC content, a total of 233 identified open reading frame ORFs, and 138 encoding genes are highly similar to the known functional proteins. Plasmid LJ4C is constructed from a partial plasmid structure of P HN7A8, P KPC-LK30 and an additional tra region to form a chimerism, joining the transfer gene cluster tra and bacterial connection. The plasmid structure study found that the transformants of LJ4 strain also carried bla CTX-M-65, blafos A3, blarmt B, BLA SHV, BLA TEM1b and multiple resistant genes to spread widely between poultry and pets. The results of this study showed that the plasmid isolated from the patient was with the strain of the pet source. The plasmid structure is highly homologous, which does not exclude the possibility of homologous recombination between the sick pets and the poultry and the human source bacteria. Conclusion 1. the carbapenem bacteria producing carbapenem in our hospital are more common, such as citric acid bacilli Freund, Pseudomonas aeruginosa and Klebsiella pneumoniae. It is suggested that the multiple drug resistant strains have been detected in the clinic and should be screened out. To find out the status of carbapenem production in order to apply the.2.Carba NP test to the screening of carbapenems of multi resistant gram-negative bacilli, it is easy, fast, accurate, and can be used in clinical laboratory to promote the use of.3. with ESBLs, Fos A, RMT B, KPC plasmid and Bla ND. M-1 and Bla KPC-2 genes can be transmitted in the same type of bacteria by conjugation or transformation. It indicates that the possible.4. high throughput sequencing of these multidrug resistant genes in bacteria is rapid, comprehensive and accurate in the analysis of drug resistance genes, genetic environment and bacterial genotyping, so as to facilitate the study of the mechanism of bacterial resistance.
【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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