革兰阴性菌中金属β-内酰胺酶基因的分子进化和耐药特征双聚类分析

发布时间：2018-08-16 16:41

【摘要】：目的了解沈阳地区临床上耐β-内酰胺类抗生素革兰阴性菌的耐药特征，，结合生物信息学技术，利用最新的贝叶斯推断和马氏链蒙特卡罗模型（MCMC）对金属β-内酰胺酶（MβLs）基因进行分子进化分析，从而将生物信息学技术与微生物学进行交叉融合，为传统技术难以解决的细菌进化问题提供了崭新的研究视角。方法收集了2012~2013年沈阳沈海医院临床分离的革兰阴性菌113株，对这些菌株进行菌种鉴定和药物敏感试验，用双聚类算法对菌株耐药特征进行分析。然后借助Perl语言和BioPerl模块编辑程序，实现金属β-内酰胺酶基因的大规模下载和本地数据文件整合成FASTA文件两大功能，构建了该基因的数据集。利用贝叶斯推断和马氏链-蒙特卡罗模型对该基因进行分子进化分析。结果菌种鉴定结果显示所收集的菌株由肺炎克雷伯菌、大肠埃希菌、铜绿假单胞杆菌和鲍曼不动杆菌等13种细菌组成，对18种抗菌药物的药敏试验表明这些菌株整体的耐药率较低。在不同种属中，肺炎克雷伯菌耐药率较低，大肠埃希菌、铜绿假单胞杆菌和鲍曼不动杆菌的耐药率则较高。药敏试验结果的双聚类分析显示，本实验所有的菌株主要被聚为I、II、III三大类，归为I类的菌株耐药率普遍很高，归为II类的菌株的数量最多，菌株耐药率普遍较低，归为III类的菌株数量较少，菌株的耐药率介于I和II类之间。对于II大类，根据所耐抗菌药物不同也分为三小类，分别用II-A、II-B、II-C表示。利用PCR方法检测耐药基因，blaIMP阳性率为14.8%，blaVIM阳性率为0.9%，测序后BLAST比对分析显示blaIMP阳性产物为IMP-1型，blaVIM阳性产物为VIM-2型。通过贝叶斯推断和MCMC模型分别对VIM基因型和总MβLs基因型数据集进行分子进化分析。最大分支树结果显示VIM基因型序列被分为三个簇，VIM基因型的碱基替代速率是1.127×E-3次/位点/每年，不同簇之间的最近共同祖先出现的时间（TMRCA）从10年到13年不等。总MβLs基因型序列同样被分为三个簇，其碱基替代速率是1.025×E-2次/位点/每年，不同簇之间的最大进化祖先出现的时间从15年到22年不等. 以贝叶斯轮廓线图（Bayesian skyline plot）方法动态分析MβLs的进化特征，在过去的20年间，VIM基因型进化突变率在2000年开始有一个明显的峰值，在2003年达到最高水平，随后平缓下降。虽然进化突变率曲线有所下降，但是仍处在较高水平。总MβLs基因型进化突变率随时间的推移变化不大，并有略微下降。贝叶斯轮廓曲线在2000年开始有一个上升，在2003年达到峰值，随后平缓下降。虽然曲线有所下降，但是进化的突变率还较高，这说明总MβLs基因型的进化压力仍较大。金属β-内酰胺酶基因的遗传多样性与抗菌药物的选择性压力有一定的正相关性，即耐药基因的遗传多样值越高，抗菌药物的选择压力越大。
[Abstract]:Objective to investigate the clinical characteristics of 尾 -lactam resistant gram-negative bacteria in Shenyang area, and to study the bioinformatics technique. The molecular evolution of metal 尾 -lactamases (M 尾 -Ls) gene was analyzed by using the latest Bayesian inference and Markov chain Monte Carlo model (MCMC), so that bioinformatics and microbiology were cross-fused. It provides a new research perspective for the problem of bacterial evolution, which is difficult to solve by traditional technology. Methods 113 strains of Gram-negative bacteria were collected from Shenyang Shenhai Hospital from 2012 to 2013. Strain identification and drug sensitivity tests were carried out. The drug resistance characteristics of these strains were analyzed by double cluster algorithm. Then, with the help of Perl language and BioPerl module editing program, the functions of large-scale downloading of metal 尾 -lactamase gene and integration of local data file into FASTA file are realized, and the data set of this gene is constructed. Bayesian inference and Markov chain Monte Carlo model were used to analyze the molecular evolution of the gene. Results the strains collected were composed of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. The susceptibility test of 18 antimicrobial agents showed that the overall resistance rate of these strains was low. The resistance rate of Klebsiella pneumoniae was lower than that of Escherichia coli Pseudomonas aeruginosa and Acinetobacter baumannii. The results of double cluster analysis showed that all the strains in this experiment were mainly divided into three groups, I. I. the resistance rate of the strains classified as class I was generally high, the number of strains classified as class II was the most, and the resistance rate of strains was generally lower. The number of strains classified as III was relatively small, and the drug resistance rate of the strains was between class I and II. For class II, there are also three subgroups according to the antimicrobial resistance, which are expressed by II-An II-BX II-C, respectively. PCR method was used to detect the positive rate of the drug resistant gene, the positive rate of VIM was 0.90.After sequencing, the BLAST analysis showed that the positive product of blaIMP was IMP-1 type VIM positive product, and the positive rate of blaIMP was VIM-2 type. The molecular evolution of VIM genotypes and total M 尾 -Ls genotypes were analyzed by Bayesian inference and MCMC model, respectively. The results of the largest branch tree showed that the base substitution rate of the VIM genotypes divided into three cluster vim genotypes was 1.127 脳 E-3 times / locus per year, and the (TMRCA) of the most recent common ancestor among different clusters ranged from 10 to 13 years. The total M 尾 -Ls genotypes were also divided into three clusters with a base substitution rate of 1.025 脳 E ~ (-2) times / locus per year, and the time of the largest evolutionary ancestors among different clusters ranged from 15 to 22 years. The evolutionary characteristics of M 尾 -Ls were dynamically analyzed by Bayesian contour (Bayesian skyline plot) method. The evolutionary mutation rate of vim genotype began to peak in 2000, reached its highest level in 2003, and then decreased slightly. Although the evolutionary mutation rate curve has decreased, it is still at a high level. The evolutionary mutation rate of total M 尾 -Ls genotype changed little with time, and decreased slightly. The Bayesian contour curve began to rise in 2000, peaked in 2003, and then declined slightly. Although the curve decreased, the mutation rate of evolution was still high, which indicated that the evolutionary pressure of total M 尾 -Ls genotype was still high. The genetic diversity of metallo- 尾 -lactamase gene was positively correlated with the selective pressure of antimicrobial agents, that is, the higher the value of genetic diversity of drug resistance genes, the greater the selection pressure of antimicrobial agents.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R3416

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