革蘭陰性菌中金屬β-內(nèi)酰胺酶基因的分子進化和耐藥特征雙聚類分析
發(fā)布時間：2018-08-16 16:41
【摘要】：目的了解沈陽地區(qū)臨床上耐β-內(nèi)酰胺類抗生素革蘭陰性菌的耐藥特征，，結(jié)合生物信息學(xué)技術(shù)，利用最新的貝葉斯推斷和馬氏鏈蒙特卡羅模型（MCMC）對金屬β-內(nèi)酰胺酶（MβLs）基因進行分子進化分析，從而將生物信息學(xué)技術(shù)與微生物學(xué)進行交叉融合，為傳統(tǒng)技術(shù)難以解決的細菌進化問題提供了嶄新的研究視角。 方法收集了2012~2013年沈陽沈海醫(yī)院臨床分離的革蘭陰性菌113株，對這些菌株進行菌種鑒定和藥物敏感試驗，用雙聚類算法對菌株耐藥特征進行分析。然后借助Perl語言和BioPerl模塊編輯程序，實現(xiàn)金屬β-內(nèi)酰胺酶基因的大規(guī)模下載和本地數(shù)據(jù)文件整合成FASTA文件兩大功能，構(gòu)建了該基因的數(shù)據(jù)集。利用貝葉斯推斷和馬氏鏈-蒙特卡羅模型對該基因進行分子進化分析。 結(jié)果菌種鑒定結(jié)果顯示所收集的菌株由肺炎克雷伯菌、大腸埃希菌、銅綠假單胞桿菌和鮑曼不動桿菌等13種細菌組成，對18種抗菌藥物的藥敏試驗表明這些菌株整體的耐藥率較低。在不同種屬中，肺炎克雷伯菌耐藥率較低，大腸埃希菌、銅綠假單胞桿菌和鮑曼不動桿菌的耐藥率則較高。藥敏試驗結(jié)果的雙聚類分析顯示，本實驗所有的菌株主要被聚為I、II、III三大類，歸為I類的菌株耐藥率普遍很高，歸為II類的菌株的數(shù)量最多，菌株耐藥率普遍較低，歸為III類的菌株數(shù)量較少，菌株的耐藥率介于I和II類之間。對于II大類，根據(jù)所耐抗菌藥物不同也分為三小類，分別用II-A、II-B、II-C表示。利用PCR方法檢測耐藥基因，blaIMP陽性率為14.8%，blaVIM陽性率為0.9%，測序后BLAST比對分析顯示blaIMP陽性產(chǎn)物為IMP-1型，blaVIM陽性產(chǎn)物為VIM-2型。 通過貝葉斯推斷和MCMC模型分別對VIM基因型和總MβLs基因型數(shù)據(jù)集進行分子進化分析。最大分支樹結(jié)果顯示VIM基因型序列被分為三個簇，VIM基因型的堿基替代速率是1.127×E-3次/位點/每年，不同簇之間的最近共同祖先出現(xiàn)的時間（TMRCA）從10年到13年不等�？侻βLs基因型序列同樣被分為三個簇，其堿基替代速率是1.025×E-2次/位點/每年，不同簇之間的最大進化祖先出現(xiàn)的時間從15年到22年不等. 以貝葉斯輪廓線圖（Bayesian skyline plot）方法動態(tài)分析MβLs的進化特征，在過去的20年間，VIM基因型進化突變率在2000年開始有一個明顯的峰值，在2003年達到最高水平，隨后平緩下降。雖然進化突變率曲線有所下降，但是仍處在較高水平�？侻βLs基因型進化突變率隨時間的推移變化不大，并有略微下降。貝葉斯輪廓曲線在2000年開始有一個上升，在2003年達到峰值，隨后平緩下降。雖然曲線有所下降，但是進化的突變率還較高，這說明總MβLs基因型的進化壓力仍較大。金屬β-內(nèi)酰胺酶基因的遺傳多樣性與抗菌藥物的選擇性壓力有一定的正相關(guān)性，即耐藥基因的遺傳多樣值越高，抗菌藥物的選擇壓力越大。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R3416
本文編號：2186568
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R3416
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相關(guān)期刊論文 前3條
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