本文關(guān)鍵詞：山東某生豬養(yǎng)殖縣農(nóng)村居民產(chǎn)ESBL大腸埃希菌流行現(xiàn)狀及分子分型研究　出處：《山東大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 超廣譜β-內(nèi)酰胺酶 大腸埃希菌 抗生素耐藥 分子分型 農(nóng)村居民

【摘要】：研究背景細(xì)菌耐藥可以引起患者抗生素使用后效果不明顯,醫(yī)治難度增加甚至病死率上升等嚴(yán)重后果,已經(jīng)成為全球共同面臨的嚴(yán)峻公共衛(wèi)生問(wèn)題之一。超廣譜β-內(nèi)酰胺酶(extended-spectrum beta-lactamases,ESBL)是一類(lèi)能夠水解β-內(nèi)酰胺類(lèi)藥物的酶,ESBL可通過(guò)質(zhì)粒上的編碼基因完成在菌株之間水平傳播耐藥性,是腸桿菌科細(xì)菌出現(xiàn)抗生素耐藥的主要方式。產(chǎn)ESBL菌株中最常見(jiàn)的是大腸埃希菌,其在醫(yī)院患者中的攜帶率為31.5%～55.8%,在山東省農(nóng)村居民中的攜帶率可達(dá)42%。ESBL的耐藥基因型主要有CTX-M、SHV和TEM,我國(guó)臨床患者中流行的主要是CTX-M,尤其是CTX-M-14和CTX-M-15。山東省是全國(guó)的養(yǎng)豬大省,除規(guī)模化養(yǎng)殖外,還有數(shù)量眾多的散養(yǎng)戶(hù),由于與養(yǎng)殖動(dòng)物接觸密切,這些養(yǎng)殖戶(hù)居民是動(dòng)物源耐藥菌傳播給人的重點(diǎn)人群。調(diào)查研究該類(lèi)人群的ESBL攜帶情況,有助于掌握當(dāng)前山東省養(yǎng)殖地區(qū)農(nóng)村居民中產(chǎn)ESBL大腸埃希菌的流行現(xiàn)狀,耐藥情況及分子分型特點(diǎn),進(jìn)而找到耐藥菌傳播途徑,為今后控制抗生素耐藥的蔓延提供理論依據(jù)。研究目的1.了解山東省養(yǎng)殖地區(qū)農(nóng)村居民產(chǎn)ESBL大腸埃希菌的流行現(xiàn)狀;2.掌握山東省養(yǎng)殖地區(qū)農(nóng)村居民產(chǎn)ESBL大腸埃希菌的藥物敏感性和耐藥基因特點(diǎn);3.描述山東省養(yǎng)殖地區(qū)農(nóng)村居民產(chǎn)ESBL大腸埃希菌的分子分型特點(diǎn);4.通過(guò)分析養(yǎng)殖地區(qū)農(nóng)村居民產(chǎn)ESBL大腸埃希菌的耐藥基因特點(diǎn)和分子分型特點(diǎn),探討耐藥菌傳播途徑,從而對(duì)今后控制抗生素耐藥的蔓延提供理論依據(jù)。研究方法1.樣本采集:本研究基于"IMPACT"中-瑞國(guó)際合作項(xiàng)目,項(xiàng)目選擇山東省某生豬養(yǎng)殖縣某養(yǎng)殖鎮(zhèn)的12個(gè)村作為研究現(xiàn)場(chǎng),本研究在12個(gè)村中隨機(jī)抽取4個(gè)村,在每村居民中隨機(jī)抽取40位成年常住居民,采集其糞便樣本,并收集居民的年齡、性別和養(yǎng)豬情況;2.產(chǎn)ESBL大腸埃希菌的分離和鑒定:采用ESBL篩選性培養(yǎng)基直接篩選目的菌株,利用VITEK質(zhì)譜儀完成菌株型別的鑒定。3.ESBL確證試驗(yàn)和藥物敏感性試驗(yàn):根據(jù)2015版CLSI指南,采用紙片法進(jìn)行ESBL確證試驗(yàn),采用瓊脂稀釋法進(jìn)行藥物敏感性試驗(yàn)。4.耐藥基因檢測(cè):通過(guò)PCR擴(kuò)增、毛細(xì)管電泳以及DNA測(cè)序的方法分析產(chǎn)ESBL大腸埃希菌所攜帶的耐藥基因型別。5.分子分型分析:通過(guò)MLST和PFGE兩種方法進(jìn)行分子分型分析。主要結(jié)果1.產(chǎn)ESBL大腸埃希菌的攜帶率160位農(nóng)村居民中有130人攜帶產(chǎn)ESBL的大腸埃希菌,攜帶率為81.3%,共分離到184株目的菌株。各村居民的攜帶率分別為75.0%、80.0%、77.5%和92.5%。女性和男性居民的攜帶率分別為84.5%和78.7%,養(yǎng)豬居民和非養(yǎng)豬居民的攜帶率為83.3%和80.0%,"50歲"居民、"50～59歲"居民和"≥60歲"居民的攜帶率分別為86.8%、87.0%和63.2%。2.產(chǎn)ESBL大腸埃希菌的藥物敏感性(1)研究菌株對(duì)14種抗菌藥物的藥物敏感性,除去動(dòng)物專(zhuān)用藥物氟苯尼考,剩余13種屬于11類(lèi)。分析產(chǎn)ESBL大腸埃希菌對(duì)這11類(lèi)人常用抗菌藥的耐藥譜,多重耐藥率達(dá)91.8%,耐3-5類(lèi)藥物的比例為56.0%(103/184),耐6-8類(lèi)藥物的比例為35.9%(66/184)。養(yǎng)豬是導(dǎo)致居民對(duì)氟苯尼考重度耐藥(MIC=64)的危險(xiǎn)因素(OR=2.121,95%CI:1.034～4.353),同時(shí)也是導(dǎo)致居民5重耐藥的危險(xiǎn)因素(OR=1.995,95%CI:1.080～3.686)。產(chǎn) ESBL 大腸埃希菌對(duì) CTX、TET 和SXT這三種抗菌藥物的耐藥率超過(guò)80%,耐藥率分別為100.0%、87.0%、82.1%;耐藥率居于其次的是 CIP(64.7%)、FFN(56.5%)、GEN(53.8%)、PTZ(31.0%)、FO(29.9%)、AMC(20.1%)、NIT(13.0%);耐藥率低于 10.0%的抗菌藥物有AMI(7.1%)、CFX(4.9%)、MEM(0.0%)、TGC(0.0%)。(2)檢測(cè)產(chǎn)ESBL大腸埃希菌中耐藥基因CTX-M、SHV和TEM的攜帶情況,結(jié)果顯示CTX-M的檢出率為100.0%,共檢出10種亞型,優(yōu)勢(shì)型是CTX-M-14,占 37.0%(68/184),其次是 CTX-M-55、CTX-M-65 和 CTX-M-15,分別占 17.4%(32/184)、16.3%(30/184)和 14.1%(26/184)。SHV 和 TEM 均未檢出。3.分子分型(1)在184株產(chǎn)ESBL的大腸埃希菌中隨機(jī)選擇54株進(jìn)行PFGE分子分型分析,結(jié)果這54株菌株屬于35種基因指紋圖譜型別,同源性在63.8%到93.0%之間。指紋圖譜顯示不同村居民之間、養(yǎng)豬居民和非養(yǎng)豬居民之間,所分離的產(chǎn)ESBL大腸埃希菌都存在同源性大于80%的現(xiàn)象,即菌株間存在同源性。(2)MLST結(jié)果顯示,184株產(chǎn)ESBL的大腸埃希菌中有172株基因序列與MLST基因庫(kù)中的ST序列匹配,屬于81種ST(sequence types)型,其余12株未找到匹配的ST型,屬于新發(fā)現(xiàn),下一步將提交到MLST網(wǎng)站對(duì)新ST型命名。本研究中大腸埃希菌的ST型呈現(xiàn)多樣性,型別最多的是ST10,占8.1%(14/172);其次分別為 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種ST型僅有5株以下與其對(duì)應(yīng)。結(jié)論1.山東農(nóng)村養(yǎng)殖地區(qū)居民中產(chǎn)ESBL大腸埃希菌廣泛流行,耐藥形勢(shì)比較嚴(yán)峻;2.山東農(nóng)村養(yǎng)殖地區(qū)居民所攜帶的產(chǎn)ESBL大腸埃希菌分子分型表現(xiàn)為多樣性;3.動(dòng)物源性耐藥基因CTX-M-55和CTX-M-65在本地區(qū)居民中廣泛流行,畜禽中廣泛流行的序列型ST10在本地區(qū)居民中多見(jiàn),可初步證明耐藥性有從畜禽向人類(lèi)傳播的趨勢(shì)。
[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墿姘熻嫰灝艱，

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