临床分离黏质沙雷菌质粒编码的16S rRNA甲基化酶基因与β-内酰胺酶基因的研究

发布时间：2018-03-13 22:19

本文选题：黏质沙雷菌　切入点：16S　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的了解201株安徽地区临床分离黏质沙雷菌药敏情况和质粒介导的16S r RNA甲基化酶检出率与基因型,为临床合理选择抗菌药物提供依据。研究临床分离黏质沙雷菌质粒介导的16S r RNA甲基化酶耐药基因与β-内酰胺酶基因的共转移情况。材料与方法菌株来源201株黏质沙雷临床分离菌株,来源于安徽省细菌耐药监测中心监测网所属34所医院(由皖南、皖中、皖北不同级别的医院组成)2005年~2012年每年9月份住院和门诊患者的各类临床标本。药敏实验质控菌株大肠埃希菌ATCC25922,转移接合试验受体菌大肠埃希菌J53 AZR,二者均为安徽省细菌耐药监控中心保存。方法1.采用MH琼脂倍比稀释法测定临床分离黏质沙雷菌株对抗菌药物的敏感性,质控菌采用E.coli ATCC 25922。2.煮沸法提取细菌的总DNA,碱裂解法提取质粒DNA;用聚合酶链反应(PCR)方法扩增质粒介导16S r RNA甲基化酶基因;PCR产物纯化测序,测序结果在Gen Bank中经Blast程序比对分析,以明确16S r RNA甲基化酶基因的基因型别以及是否突变。3.用16S r RNA甲基化酶基因阳性菌株与大肠埃希菌J53AzR行转移接合试验;对接合子进行生化鉴定并经PCR检测耐药基因,PCR扩增产物DNA测序确认;采用Muller-Hinton琼脂对比稀释法测定大肠埃希菌J53AzR与接合子对抗菌药物的最低抑菌浓度(MIC)。4.以16S r RNA甲基化酶基因阳性临床分离菌株的总DNA为模板,采用PCR方法扩增β-内酰胺酶基因;PCR产物纯化测序,测序结果在Gen Bank中经Blast程序比对,比较分析16S r RNA甲基化酶基因型别与β-内酰胺酶基因型别关系。5.以16S r RNA甲基化酶基因阳性接合子菌株质粒DNA为模板,PCR扩增β-内酰胺酶基因;PCR产物纯化测序,测序结果在Gen Bank中经Blast程序比对,明确基因型。探究16S r RNA甲基化酶基因与β-内酰胺酶基因是否共转移。结果1.201株黏质沙雷菌中,对17种抗菌药物的敏感率及耐药率可以看出,黏质沙雷菌对亚胺培南和美罗培南最敏感,敏感率高达98.51%。相比于其他三代头孢类抗菌药物,酶抑制剂复合制剂如哌拉西林-他唑巴坦和头孢哌酮-舒巴坦以及四代头孢菌素头孢吡肟的敏感性较好,分别可达76.62%、65.63%及74.13%。喹诺酮类抗菌药以加替沙星抗菌药物敏感性最高,为77.11%,其次为左氧氟沙星敏感性达75.12%。本研究中检测出135株对庆大霉素耐药、58株对阿米卡星耐药,二者耐药率分别为:67.16%及28.8%,58株对阿米卡星耐药的菌株同时对庆大霉素耐药。58株阿米卡星耐药菌株中45株来自痰,5株来自尿液,3株来自血液,3株来自分泌物,1株来自胸水,1株来自大便。2.58株同时对阿米卡星和庆大霉素耐药的黏质沙雷菌经PCR检测和DNA测序,7株为16S r RNA甲基化酶基因阳性,其中5株为arm A阳性,2株为rmt B阳性,所有菌株rmt A、rmt C、rmt D和npm A基因检测均为阴性。16S r RNA甲基化酶基因阳性菌株数占总临床分离黏质沙雷菌株数的阳性率为3.5%(7/201),在耐阿米卡星菌株中的阳性率为12.1%(7/58)。3.7株16S r RNA甲基化酶基因阳性的菌株中有6株转移接合成功,接合子经生化鉴定为大肠埃希菌。经PCR扩增及DNA测序分析证实,5株接合子携带arm A基因;1株接合子携带rmt B基因。药敏结果表明,这6株接合子与受体菌相比,对阿米卡星等五种氨基糖苷类抗生素耐药性明显提高。接合子对第四代头孢菌素以及碳青霉烯类均敏感。4.7株16S r RNA甲基化酶基因阳性的菌株经PCR检测β-内酰胺酶基因显示5株临床分离株检测出β-内酰胺酶基因,最常见的基因型别为CTX-M。且PCR产物经DNA测序表明4株CTX-M基因阳性,均为CTX-M-14型;2株TEM基因阳性,均为TEM-1型;2株OXA基因阳性,均为OXA-1型;1株SHV基因阳性,为SHV-5型。其中有3株菌株检测出二种以上β-内酰胺酶基因。5.对6株携带有16S r RNA甲基化酶基因的转移接合成功接合子,以质粒DNA为模板,经PCR检测出4株β-内酰胺酶基因,其PCR产物经DNA测序证实:3株CTX-M基因阳性,均为CTX-M-14型;2株TEM基因阳性,均为TEM-1型;1株SHV基因阳性,为SHV-5型。结论1.药敏结果显示安徽地区临床分离黏质沙雷菌对多种抗菌药物呈现不同程度耐药,耐氨基糖苷类抗菌药物的菌株呈现多重耐药现象。2.研究报道了安徽地区存在携带有质粒编码的16S r RNA甲基化酶耐药基因的黏质沙雷菌,也是国内第一次在黏质沙雷菌中检出16S r RNA甲基化酶基因。3.转移接合实验成功的接合子对氨基糖苷类抗菌药物的MIC较受体菌相比,对阿米卡星等五种氨基糖苷类抗生素耐药性明显提高。4.我国黏质沙雷菌中已经出现了质粒介导的16S r RNA甲基化酶,且多与β-内酰胺酶基因同时存在。5.质粒介导的16S r RNA甲基化酶耐药基因与β-内酰胺酶常同时存在于同一株黏质沙雷菌中,并可同时通过可接合性质粒发生共转移,造成耐药基因在不同菌属间扩散。
[Abstract]:Objective to understand the Anhui region of 201 strains of clinical isolates of Serratia marcescens susceptibility and plasmid mediated 16S R RNA methylase detection rate and genotype, provide the basis for clinical rational use of antibiotics. The transfer of clinical isolates of Serratia marcescens plasmid mediated 16S R RNA methylase gene and beta lactamase genes. Materials and methods 201 strains of Serratia strains from clinical isolates from Anhui Province, bacterial resistance monitoring network monitoring center belongs to 34 hospitals (from Anhui, Anhui, Anhui different levels of hospitals) 2005 ~2012 in September of each year of inpatient and outpatient all kinds of clinical specimens. Drug sensitivity the quality control strains of Escherichia coli ATCC25922, conjugation experiment recipient bacterium Escherichia coli J53 AZR, the two are Anhui province bacterial resistance monitoring center preservation. Methods 1. MH agar dilution method was used measuring The sensitivity of clinical isolated strains to antimicrobial agents Sarre clay, total DNA extracted by E.coli ATCC bacteria and control the bacteria 25922.2. boiling method, alkaline lysis method of extracting plasmid DNA; polymerase chain reaction (PCR) amplification of plasmid mediated 16S R RNA methylase gene; PCR sequencing in Gen pure product, Bank by Blast the program comparison and analysis of sequencing results, with 16S R RNA genotype specific methylase gene and whether mutations in the.3. joint test with 16S R RNA methylase gene positive strains of Escherichia coli and J53AzR metastasis; biochemical identification of exconjugants and detected by PCR resistance gene, PCR DNA PCR product sequencing and determination; zygotic minimal inhibitory concentrations of antibiotics of Escherichia coli J53AzR by Muller-Hinton agar dilution method compared with total DNA (MIC).4. 16S R RNA methylase gene positive clinical isolates as template And the amplification of beta lactamase genes using PCR method; purification of PCR products sequencing, sequencing results in Gen Bank by Blast program comparison, analysis and comparison of 16S R RNA methylase genotypes and beta lactamase genotypes in 16S r relationship between.5. RNA methylase gene positive strains plasmid DNA as the zygote the template, PCR amplification of beta lactamase genes; purification of PCR products sequencing, sequencing results in Gen Bank by Blast program comparison, clear genotype. R explore 16S RNA methylase gene and beta lactamase genes were transferred. Results 1.201 strains of Serratia marcescens, sensitive and resistant rates of 17 antimicrobial rate can be seen, Serratia marcescens is most sensitive to imipenem and meropenem, the sensitive rate of 98.51%. compared to the other three generation cephalosporins, enzyme inhibitor such as piperacillin tazobactam and cefoperazone - sulbactam and Good sensitivity to the four generation cephalosporin cefepime, respectively 76.62%, 65.63% and 74.13%. of quinolones with gatifloxacin antibiotic sensitivity was 77.11%, the highest, followed by levofloxacin sensitivity of 75.12%. in this study detected 135 strains of gentamicin resistant, 58 strains of Amikacin resistance, the two resistance rates were: 67.16% and 28.8%, 58 strains of Amikacin resistant strains resistant to gentamicin and Amikacin.58 strains resistant strains 45 strains from 5 strains from sputum, urine, blood from 3 strains, 3 strains from 1 strains from secretions, pleural effusion, and 1 strains from stool.2.58 strains of Serratia marcescens in Amikacin and gentamicin resistance by PCR detection and DNA sequencing, 7 strains were 16S R RNA methylase gene positive, 5 of them were arm A positive, 2 were RMT B positive, all A RMT C strain RMT, RMT, D and NPM detection of A gene were The positive rate of.16S negative R RNA methylase gene positive strains in total clinical isolates of Serratia marcescens was 3.5% (7/201), the positive rate of resistant strains in Amikacin 12.1% (7/58) strain 16S R RNA methylase gene.3.7 positive strains in 6 strains by conjugation, zygote biochemical identification of Escherichia coli. After PCR amplification and DNA sequencing analysis confirmed that 5 strains of transconjugants carrying arm A gene; 1 strains of transconjugants carrying RMT B gene. Drug sensitivity results showed that these 6 strains of zygote and compared to recipient strains of amikacin five kinds of aminoglycoside antibiotic resistance was significantly improved. Zygote were sensitive to fourth generation cephalosporins and carbapenems.4.7 strain 16S R RNA methylase gene positive strains were detected by PCR beta lactamase gene showed that 5 strains of clinical isolates detected beta lactamase gene, genotype is the most common and CTX-M. The PCR products by DNA sequencing showed that the CTX-M gene of 4 strains were positive for type CTX-M-14; TEM gene was positive in 2 strains, were type TEM-1; OXA gene was positive in 2 strains, were type OXA-1; SHV gene was positive in 1 strains, SHV-5 type. There are 3 strains detected more than two kinds of beta lactamase.5. 16S R gene transfer RNA methyltransferase genes successfully engage transconjugants against 6 strains carried by plasmid DNA as template, the PCR detection of 4 strains of beta lactamase genes, the PCR product was confirmed by DNA sequencing: 3 strains of CTX-M gene were CTX-M-14 positive, 2 strains of TEM gene positive type; that is TEM-1 type; SHV gene was positive in 1 strains, SHV-5 1.. Conclusion according to the results of drug susceptibility in Anhui clinical isolates of Serratia marcescens to antibiotics showed different degrees of resistance, resistance to aminoglycoside antibiotics showed multidrug resistance strains of.2. have reported carrying the plasmid encoding 16S R R in Anhui area Serratia marcescens NA methylase gene, is also the first time in Serratia marcescens in detection of 16S r methylase gene RNA.3. conjugation experiment successfully zygote to aminoglycoside antibiotics MIC compared with the host strain of amikacin five kinds of aminoglycoside antibiotic resistance has been significantly improved China's.4. of Serratia marcescens plasmid mediated 16S R RNA methylase, and with beta lactamase genes exist at the same time 16S R RNA methylase gene and plasmid mediated.5. beta lactamases often coexist in the same strains of Serratia marcescens in sticky matter, and at the same time the conjugative plasmid transfer occurred, resulting in diffusion resistance genes in different bacterial species.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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