山东某生猪养殖县农村居民产ESBL大肠埃希菌流行现状及分子分型研究

发布时间：2018-01-17 23:28

  本文关键词：山东某生猪养殖县农村居民产ESBL大肠埃希菌流行现状及分子分型研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 超广谱β-内酰胺酶 大肠埃希菌 抗生素耐药 分子分型 农村居民

【摘要】：研究背景细菌耐药可以引起患者抗生素使用后效果不明显,医治难度增加甚至病死率上升等严重后果,已经成为全球共同面临的严峻公共卫生问题之一。超广谱β-内酰胺酶(extended-spectrum beta-lactamases,ESBL)是一类能够水解β-内酰胺类药物的酶,ESBL可通过质粒上的编码基因完成在菌株之间水平传播耐药性,是肠杆菌科细菌出现抗生素耐药的主要方式。产ESBL菌株中最常见的是大肠埃希菌,其在医院患者中的携带率为31.5%～55.8%,在山东省农村居民中的携带率可达42%。ESBL的耐药基因型主要有CTX-M、SHV和TEM,我国临床患者中流行的主要是CTX-M,尤其是CTX-M-14和CTX-M-15。山东省是全国的养猪大省,除规模化养殖外,还有数量众多的散养户,由于与养殖动物接触密切,这些养殖户居民是动物源耐药菌传播给人的重点人群。调查研究该类人群的ESBL携带情况,有助于掌握当前山东省养殖地区农村居民中产ESBL大肠埃希菌的流行现状,耐药情况及分子分型特点,进而找到耐药菌传播途径,为今后控制抗生素耐药的蔓延提供理论依据。研究目的1.了解山东省养殖地区农村居民产ESBL大肠埃希菌的流行现状;2.掌握山东省养殖地区农村居民产ESBL大肠埃希菌的药物敏感性和耐药基因特点;3.描述山东省养殖地区农村居民产ESBL大肠埃希菌的分子分型特点;4.通过分析养殖地区农村居民产ESBL大肠埃希菌的耐药基因特点和分子分型特点,探讨耐药菌传播途径,从而对今后控制抗生素耐药的蔓延提供理论依据。研究方法1.样本采集:本研究基于"IMPACT"中-瑞国际合作项目,项目选择山东省某生猪养殖县某养殖镇的12个村作为研究现场,本研究在12个村中随机抽取4个村,在每村居民中随机抽取40位成年常住居民,采集其粪便样本,并收集居民的年龄、性别和养猪情况;2.产ESBL大肠埃希菌的分离和鉴定:采用ESBL筛选性培养基直接筛选目的菌株,利用VITEK质谱仪完成菌株型别的鉴定。3.ESBL确证试验和药物敏感性试验:根据2015版CLSI指南,采用纸片法进行ESBL确证试验,采用琼脂稀释法进行药物敏感性试验。4.耐药基因检测:通过PCR扩增、毛细管电泳以及DNA测序的方法分析产ESBL大肠埃希菌所携带的耐药基因型别。5.分子分型分析:通过MLST和PFGE两种方法进行分子分型分析。主要结果1.产ESBL大肠埃希菌的携带率160位农村居民中有130人携带产ESBL的大肠埃希菌,携带率为81.3%,共分离到184株目的菌株。各村居民的携带率分别为75.0%、80.0%、77.5%和92.5%。女性和男性居民的携带率分别为84.5%和78.7%,养猪居民和非养猪居民的携带率为83.3%和80.0%,"50岁"居民、"50～59岁"居民和"≥60岁"居民的携带率分别为86.8%、87.0%和63.2%。2.产ESBL大肠埃希菌的药物敏感性(1)研究菌株对14种抗菌药物的药物敏感性,除去动物专用药物氟苯尼考,剩余13种属于11类。分析产ESBL大肠埃希菌对这11类人常用抗菌药的耐药谱,多重耐药率达91.8%,耐3-5类药物的比例为56.0%(103/184),耐6-8类药物的比例为35.9%(66/184)。养猪是导致居民对氟苯尼考重度耐药(MIC=64)的危险因素(OR=2.121,95%CI:1.034～4.353),同时也是导致居民5重耐药的危险因素(OR=1.995,95%CI:1.080～3.686)。产 ESBL 大肠埃希菌对 CTX、TET 和SXT这三种抗菌药物的耐药率超过80%,耐药率分别为100.0%、87.0%、82.1%;耐药率居于其次的是 CIP(64.7%)、FFN(56.5%)、GEN(53.8%)、PTZ(31.0%)、FO(29.9%)、AMC(20.1%)、NIT(13.0%);耐药率低于 10.0%的抗菌药物有AMI(7.1%)、CFX(4.9%)、MEM(0.0%)、TGC(0.0%)。(2)检测产ESBL大肠埃希菌中耐药基因CTX-M、SHV和TEM的携带情况,结果显示CTX-M的检出率为100.0%,共检出10种亚型,优势型是CTX-M-14,占 37.0%(68/184),其次是 CTX-M-55、CTX-M-65 和 CTX-M-15,分别占 17.4%(32/184)、16.3%(30/184)和 14.1%(26/184)。SHV 和 TEM 均未检出。3.分子分型(1)在184株产ESBL的大肠埃希菌中随机选择54株进行PFGE分子分型分析,结果这54株菌株属于35种基因指纹图谱型别,同源性在63.8%到93.0%之间。指纹图谱显示不同村居民之间、养猪居民和非养猪居民之间,所分离的产ESBL大肠埃希菌都存在同源性大于80%的现象,即菌株间存在同源性。(2)MLST结果显示,184株产ESBL的大肠埃希菌中有172株基因序列与MLST基因库中的ST序列匹配,属于81种ST(sequence types)型,其余12株未找到匹配的ST型,属于新发现,下一步将提交到MLST网站对新ST型命名。本研究中大肠埃希菌的ST型呈现多样性,型别最多的是ST10,占8.1%(14/172);其次分别为 ST38 型,占 5.8%(10/172);ST48 型,占 5.8%(10/172);ST6338 型,占3.5%(6/172);ST46型,占2.9%(5/172);ST648 型,占2.9%(5/172)。其余75种ST型仅有5株以下与其对应。结论1.山东农村养殖地区居民中产ESBL大肠埃希菌广泛流行,耐药形势比较严峻;2.山东农村养殖地区居民所携带的产ESBL大肠埃希菌分子分型表现为多样性;3.动物源性耐药基因CTX-M-55和CTX-M-65在本地区居民中广泛流行,畜禽中广泛流行的序列型ST10在本地区居民中多见,可初步证明耐药性有从畜禽向人类传播的趋势。
[Abstract]:The research background of bacterial resistance can cause antibiotic use after the effect is not obvious, even more difficult to treat increased mortality and other serious consequences, has become one of the most serious global public health problem facing. ESBLs (extended-spectrum beta-Lactamases ESBL) is a kind of ability to hydrolyze beta lactam drugs enzyme ESBL can be completed in the strain between the level of dissemination of antibiotic resistance through gene encoding plasmid, is the main way of emergence of antibiotic resistant Enterobacteriaceae. The most common ESBL producing strains in Escherichia coli, the patients in the hospital carrying rate was 31.5% ~ 55.8%, carrying resistance genes rate of 42%.ESBL in rural residents in Shandong Province, mainly CTX-M, SHV and TEM, the popular clinical patients in our country is mainly CTX-M, especially CTX-M-14 and CTX-M-15. in Shandong province is a big province of China's pig, In addition to large-scale farming, as well as a large number of farmers, due to the close contact with the animal, the animal farming households is the source of drug-resistant bacteria to human transmission of the key crowd. Carrying the investigation of this population ESBL, helps to grasp the current epidemic situation in Shandong province farming area of rural residents of middle ESBL E. coli, drug resistance and molecular characteristics, and then find the spread of antibiotic resistant bacteria, to provide a theoretical basis for future control of antibiotic resistance spread. Objective: 1. to investigate the epidemic situation of rural residents of ESBL producing Escherichia coli in Shandong province farming area; 2. rural residents in Shandong Province to master the breeding of ESBL producing Escherichia coli drug the drug sensitivity and resistance gene; molecular description of 3. residents of ESBL producing Escherichia coli in rural areas of Shandong province aquaculture area types; 4. through the analysis of rural residents ESBL large farming areas Type characteristics of Escherichia coli resistance gene and molecular components, to explore the spread of antibiotic resistant bacteria, so as to provide a theoretical basis for the future control of the spread of antibiotic resistance. Methods: This study collected 1. samples based on the "IMPACT" - Swiss international cooperation projects, the project selection in 12 villages a farming town of a pig farming county in Shandong Province as the research field, the research in 4 villages in 12 villages were randomly selected in each village were randomly selected 40 adult residents, the collection of fecal samples, and collect the residents age, gender and pig; isolation and identification of 2. ESBL producing Escherichia coli strains: screening with ESBL medium direct screening target strains, complete plant bacteria identification and drug sensitivity test of.3.ESBL was confirmed by VITEK mass spectrometry: according to the 2015 edition of the CLSI guidelines, ESBL confirmatory test by Kirby Bauer method, drug using agar dilution method The detection sensitivity test:.4. resistance gene was amplified by PCR, genotyping analysis of drug resistant genotype.5. molecule of ESBL producing Escherichia coli carried by capillary electrophoresis method and DNA sequencing analysis: molecular subtyping by MLST and PFGE two methods. The main results of the 1. ESBL producing Escherichia coli carrying rate of 160 in rural areas residents are 130 people carrying ESBL in Escherichia coli, carrying rate of 81.3%, 184 strains were isolated. The strains carrying rate were 75%, 80% village residents, 77.5% and 92.5%. in male and female residents carrying rate were 84.5% and 78.7%, pig pig residents and non residents carrying rate of 83.3% and 80%, "50 year old" residents, "50 to 59 years" and "residents aged over 60 of the residents carrying rate were 86.8%, 87% and the drug sensitivity of 63.2%.2. ESBL producing Escherichia coli (1) drugs on 14 kinds of anti bacteria drug sensitivity of bacteria ,闄ゅ幓鍔ㄧ墿涓撶敤鑽�鐗╂盁鑻�灏艰，

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