产超广谱β内酰胺酶肠杆菌科细菌的表型及分子生物学研究

发布时间：2018-04-01 05:29

本文选题：超广谱β内酰胺酶　切入点：表型确证试验　出处：《中国人民解放军军事医学科学院》2009年硕士论文

【摘要】： 背景和目的 β内酰胺类抗生素的发现和使用为人类抵御细菌感染做出了巨大贡献,但随着抗生素的广泛、大量使用,细菌耐药性问题,尤其是由超广谱β内酰胺酶(extended-spectrumβ-lactamases,ESBLs)介导的β内酰胺类抗生素耐药性,导致细菌对临床常用抗生素耐药。不适当的初始抗生素治疗,可引起病人住院时间延长、费用增加,并可能导致死亡率上升。因此快速准确检测超广谱β内酰胺酶,将有助于相应感染性疾病的治疗和预防。检测超广谱β内酰胺酶的方法可分为表型鉴定法和分子生物学方法。表型鉴定法包括CLSI(Clinical and Laboratory Standards Institute)推荐的ESBLs检测法(包括筛选试验和表型确证试验),三维试验,Etest ESBLs条,Vitek ESBLs检测卡,Microscan ESBLs检测板和BD Phoenix自动化微生物系统等。分子生物学方法包括DNA探针法,寡核苷酸分型,PCR-RFLP,PCR-SSCP,连接酶反应,Real-time PCR和核酸测序法等。 Vitek-2高级专家系统通过对多种抗生素最小抑菌浓度的分析来推测ESBLs表型,通常对细菌药敏结果给出多种参考结果,这要求检测者具有丰富的经验。检测者一旦将菌株确认为产ESBLs,仪器会自动将体外测试敏感的药物修改为耐药表型,极大地限制了临床应用药物的种类。为确证Vitek-2仪器配套药敏卡AST-GN10(ref 22009)和AST-N021(ref 22030)推测产ESBLs结果的可靠性和找到一种简便实用的鉴定ESBLs的方法,我们收集高级专家系统提示产ESBLs的G~-菌(主要为大肠埃希菌和肺炎克雷伯菌),用CLSI(Clinical and LaboratoryStandards Institute)推荐的药敏纸片表型确证方法和PCR基因分型法进行研究。方法实验分三个部分。首先收集Vitek-2提示ESBLs阳性菌株采用国产药敏纸片进行表型确证试验,然后对所有确证试验阳性菌株和部分阴性菌株进行常规PCR扩增、测序、与已知基因序列比对,最后将设计引物应用到荧光染料法实时PCR扩增,建立了TEM、SHV和CTX-M型ESBLs荧光染料法实时PCR检测的方法。结果及讨论 1表型确证试验实验结果证明Vitek-2高级专家系统和药敏纸片确证试验两种方法一致性达到89.7%(78/87,仅计算CLSI纸片确证法推荐应用菌株)。对于那些受经济条件限制,无法得到Vitek-2仪器的医院,可以应用国产药敏纸片进行ESBLs检测。10株Vitek-2提示产ESBLs菌株,应用纸片法确证试验未达ESBLs阳性标准。这些差异需要分子生物学试验进一步验证。实验中发现5株阴沟肠杆菌、1株弗劳地枸橼酸杆菌仪器提示产ESBLs,纸片确证试验也阳性,但CLSI尚未把这些细菌列入产ESBLs确证试验范围,有望用分子生物学方法解决这一问题。我们同时对纸片测试提示头孢西丁耐药的菌株(Vitek提示通透性改变)用酶粗提物三维试验和药敏纸片细菌直接测试法进行质粒头孢菌素酶检测,仅一株细菌表型检测阳性,推测本院产ESBLs菌头孢西丁抗性可能主要由于细菌膜通透性改变引起。 2常规PCR反应及测序结果根据文献发表的基因序列,应用引物设计软件,在TEM、SHV和CTX-M型耐药基因保守区设计引物,对已知基因型菌株(TEM和SHV)及表型方法确证为产ESBLs的G~-菌进行PCR扩增,引物TEM、SHV、CTX-M-3、CTX-M-19均得到扩增产物,发现多数细菌携带TEM和CTX-M-1群耐药基因,部分细菌携带SHV和CTX-M-9群耐药基因。扩增结果同时提示在院内还未出现携带CTX-M-8、2、25群基因的细菌。本文设计的引物对不携带耐药基因的标准菌株扩增结果为阴性。通过对肺炎克雷伯菌(标本号为071703的)扩增产物进行基因测序并将测序结果与Pubmed公布基因比对,同源性均在98%以上。证明此四对引物可特异扩增相应基因,适合今后开展临床基因扩增研究。应用设计引物扩增,大肠埃希菌中TEM型、CTX-M-1群、CTX-M-9群基因产物阳性率分别为85.2%,63.0%,48.1%。肺炎克雷伯菌中TEM型、SHV型、CTX-M-1群、CTX-M-9群基因产物阳性率分别为64.0%,68.0%,56.0%,28.0%。TEM、SHV型ESBLs基因的鉴别主要依赖全基因测序,因此携带这些基因的细菌可能仅代表具有广谱耐药性,是否为产ESBLs菌需进一步验证。CTX-M-1群、CTX-M-9群基因在大肠杆菌、肺炎克雷伯菌、阴沟肠杆菌均有扩增,提示CTX-M型酶为院内优势ESBLs。部分菌株同时携带CTX-M-1,CTX-M-9这两种同一基因型不同亚群耐药基因,此两种基因是否存在于同一质粒或整合子上及两种基因表达的调控及相互关系值得进一步研究。应用设计的引物可同时扩增TEM/SHV耐药基因,但只在部分细菌中得到两种不同基因产物,而多数菌只得到一种扩增产物。同时应用两种CTX-M型引物扩增,只得到一种产物,不能同时得到两种不同基因产物。此种差异可能与引物设计有关(不同引物引发效率不同),也可能菌体内两种基因含量处于不同的比例,只在合适的比例时才能同时扩增。实验过程中探索多种用于PCR扩增的质粒提取方法。采用质粒提取试剂盒法、多个克隆煮沸离心法、液体增菌煮沸离心法提取的质粒作为模板,均能得到理想扩增,而多个克隆煮沸离心法最简便易行,如果和纸片法常规药敏试验使用相同的克隆制备菌液,可以节省反应时间,快速检测出产ESBLs菌。 3 SYBR法实时PCR检测应用SYBR法可实时检测基因表达情况,应用熔解曲线分析扩增产物而不是凝胶电泳观察,降低了产物扩散污染的几率,而且扩增产物熔解曲线分析保证了扩增产物的特异性,因此在科研工作中得到广泛应用。应用设计的TEM、SHV、CTX-M-3引物扩增产ESBLs菌,反应参数由普通PCR做适当修改即可在Roche Lightcycler仪器上应用,且熔解曲线分析为单个特异峰。CTX-M-19上下游引物配对扩增产物经熔解曲线分析出现两个波峰;而用简并引物CTX-M-26上游引物和CTX-M-19下游引物配合可扩增出CTX-M-9群基因产物,熔解曲线分析为单个特异峰,扩增产物电泳观察与预期片段大小一致。应用定量细菌直接煮沸法提取质粒模板的试验发现,当细菌数量在10~2/μl以上时,可以顺利进行实时荧光扩增,而达到此等数量的细菌,仅需数个细菌克隆。直接挑取细菌克隆煮沸法制备质粒模板可节省时间和费用,可以在细菌质粒耐药研究中需要少量模板时使用。应用SHV、CTX-M-3引物扩增定量细菌模板,用拟合点法可得到一条较好的扩增曲线,能对不同菌株耐药基因含量进行相对定量。但耐药基因相对定量结果对临床用药是否有指导意义,仍须进一步研究。我们应用Roche lightcycler PCR扩增仪对表型确证试验及普通PCR扩增阳性的部分菌株进行了实时PCR检测和熔解曲线分析,确定了最佳反应条件及在此试验条件下的各反应产物的熔解温度。为今后进一步设计探针法实时定量检测携带ESBLs基因菌株打下基础。
[Abstract]:Background and Purpose

The discovery and use of beta - lactam antibiotics has contributed significantly to human defense against bacterial infections , but with the widespread use of antibiotics , the problem of bacterial resistance , especially the resistance of beta - lactam antibiotics mediated by extended - spectrum beta - lactamase , which leads to prolonged hospitalization , increased costs , and may lead to an increase in mortality .

Molecular biological methods include DNA probe , oligonucleotide typing , PCR - RFLP , PCR - SSCP , ligase reaction , Real - time PCR and nucleic acid sequencing .

By analyzing the minimum inhibitory concentration of various antibiotics , the high - level expert system of VMC - 2 has been used to predict the phenotype of the spectrum , which usually gives many reference results to the results of the drug susceptibility test . In order to confirm the reliability of the results and find a simple and practical method for the identification of the products , we collect the drug sensitive cards AST - GN10 ( ref 22009 ) and AST - N021 ( ref 22030 ) , which are used to confirm the clinical application drugs . To confirm the VMC - 2 instrument kit , AST - GN10 ( ref 22009 ) and AST - N021 ( ref 22030 ) , we collect the G ~ - bacteria ( mainly E . coli and Klebsiella pneumoniae ) producing the products , and we study the phenotype confirmation method of the drug - sensitive paper sheet recommended by the Clinical and Laboratory Standards Institute and the PCR genotyping method .

method

In this paper , three parts were divided into three parts . First , the positive strains were collected by using domestic drug - sensitive paper . The positive strains and some negative strains were amplified by routine PCR , sequenced and compared with the known gene sequences . Finally , the primers were applied to real - time PCR amplification of fluorescent dye method , and the real - time PCR detection method of TEM , SHV and CTX - M - type extended - PCR was established .

Results and Discussion

1 phenotype confirmation test

The experimental results show that the consistency of the two methods is 89.7 % ( 78 / 87 , only the application strain is recommended for the validation method of the sheet validation method ) . For those with limited economic conditions , we can ' t get the hospital of VMC - 2 instrument . It is possible to use domestic drug - sensitive paper to test . Ten strains of VMC - 2 have been used to test the producing strains . The results show that 5 strains of enterobacter clostrium and 1 strain of Floridi citrate are positive for the test . However , we have not listed these bacteria in the scope of producing extended - producing strains , and it is expected to use molecular biology method to test the problem .

2 Routine PCR reactions and sequencing results

The primers were designed according to the gene sequence published in the literature , and the primers were designed in the conserved regions of TEM , SHV and CTX - M resistant genes .

The positive rates of TEM , SHV , CTX - M - 1 and CTX - M - 9 in Escherichia coli were 85.2 % , 63.0 % and 48.1 % , respectively . The positive rates of TEM , SHV , CTX - M - 1 and CTX - M - 9 were 64 . 0 % , 68 . 0 % , 56 . 0 % and 28 . 0 % respectively .

Two kinds of CTX - M primers were used to amplify only one product and two different gene products could not be obtained at the same time .

A plurality of plasmid extraction methods for PCR amplification are explored in the experiment process . The plasmid extraction kit method , a plurality of cloning boiling centrifugation methods , and a liquid enrichment boiling centrifugal method are used as a template to obtain ideal amplification , and a plurality of clone boiling centrifugation methods are most simple and feasible .

3 SYBR Method Real - time PCR Detection

SYBR method can be used to detect gene expression in real time , and melting curve is used to analyze the amplification product instead of gel electrophoresis . The probability of product diffusion pollution is reduced , and the melting curve analysis of amplification product guarantees the specificity of amplification product , so it is widely used in scientific research .

CTX - M - 19 upstream primer and CTX - M - 19 upstream primer and CTX - M - 19 downstream primer were used to amplify the CTX - M - 9 gene product .

It was found that when the number of bacteria was above 10 ~ 2 / 渭l , real - time fluorescence amplification could be carried out smoothly , and only a few bacterial clones were needed .

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378;R516

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