blaOKP β-内酰胺酶耐药基因进化研究

The blaOKP which belongs to β-lactamase gene familiy is constitutively expressed chromosomally in bacteria.In recent years, the subtpyes of balOKP gene have been found continuously and gained extensive attention by domestic and overseas scholars.Our previous study showed that blaOKP gene is not the Klebsiella pneumoniae- specific β-lactamase gene and new blaOKP subtype genes encoding β-lactamases show higher resistance to third generation cephalosporins.These findings reveal that the evolution of blaOKP gene is becoming more harmful. Therefore,further research on blaOKP gene is needed. Firstly, the spread of blaOKP gene in family Enterobacteriaceae will be investigated to know the varieties of blaOKP. PFGE(Pulsed-field gel electrophoresis) is used to analyze the homology of different strains.Consequently,the blaOKP’s features of epidemic will be found. Based on this, secondly, all the other blaOKP genes will be downloaded and aligned, and genetic background of different isolates are going to be compared. Phylogenetic analysis of the blaOKP genes will be performed. Compared with phenotypes, the trend of blaOKP evolution can be searched. Thirdly, the mechanism of presence or spread of blaOKP in different species would be discussed by gaining flanking sequences of the gene, and finding the feature of flanking sequences. Finally, extended-spectrum blaOKP genes will be site-directed mutated to seek the crucial action sites. The mechanism of β-lactam hydrolysis could be explained by analyzing the change of protein structure. Thus, through our study we can explore the mechanism of blaOKP gene evolution, providing theoretical and scientific basis for the prevention of antimicrobial resistance and the forecast for dissemination of β-lactamase.

blaOKP属β-内酰胺酶基因家族,可在细菌染色体上持续表达,近年来blaOKP基因新亚型不断被发现,受到广泛关注。项目前期研究发现blaOKP基因非肺炎克雷伯氏菌所特有,新亚型酶已表现出对第三代广谱头孢菌素抗性,其基因进化的危害性已突显。鉴于此,本课题拟调查肠杆科细菌blaOKP基因流行情况,确定基因亚型种类,PFGE技术分析流行株间同源性,揭示blaOKP基因流行规律；分析不同菌株分子遗传背景,绘制进化树,结合表型特征归纳总结,探索blaOKP基因进化趋势；同时,获取blaOKP基因侧翼序列,分析侧翼序列特征,探讨blaOKP基因在不同种细菌中分布或传播机制；以广谱型blaOKP为对象,定点突变基因,寻找关键作用位点,分析蛋白质结构变化,解析OKP酶对β-内酰胺药物水解机理。通过本研究,从基因及蛋白质水平探究blaOKP基因进化机制,为耐药性防控及β-内酰胺酶流行趋势预测提供科学依据。

blaOKP属β-内酰胺酶基因家族，可在细菌染色体上持续表达，近年来blaOKP基因新亚型不断被发现，受到广泛关注。本课题开展了不同来源细菌的耐药表型及基因型研究, PFGE技术分析流行株间同源性，揭示blaOKP基因流行规律；分析了不同菌株分子遗传背景，绘制进化树，探索blaOKP基因进化趋势；同时，获取blaOKP基因侧翼序列，分析侧翼序列特征，探讨了blaOKP基因在传播机制；以广谱型blaOKP为对象，解析OKP酶对β-内酰胺药物水解机理。研究发现分离菌株对抗生素耐药谱广，多重耐药严重，blaOKP基因检出率为5.91%。blaOKP分属两个平行进化组，blaOKP基因侧翼序列的保守性是其相对blaSHV基因进化缓慢的原因之一，等点聚焦聚焦显示，四个OKP亚型OKP酶的等电点分别为8.2，8.2，8.2及8.1，Kcat分别为650，580，550及420，Km值分别为230，245，330及400。除此之外，研究中还发现抗生素耐药基因与消毒剂、重金属耐药基因之间存在显著相关性(p<0.05), 抗生素耐药基因与消毒剂、重金属耐药基因可存在于同一移动遗传元件上，此发现为本课题的进一步深入研究提供了基础。研究窄谱β-内酰胺酶基因的流行及进化，对于探究超广谱β-内酰胺酶(ESBLs)的发生与发展具有重要作用，研究blaOKP 耐药基因进化可为 β-内酰胺酶的流行趋势预测供理论依据，为耐药性的防控供理论基础。