四/五磷酸鸟苷通过调控ESBL差异表达提高细菌耐药适应性的作用研究

The resistance level to β-lactam agents in ESBL-producing bacteria is positively correlated with the expression level of ESBL genes. Our previous study found the existence of cell-to-cell variation in expression of ESBL genes in single clonal population and the expression level of ESBL genes was increasing with increment of the extracellular ceftriaxone concentration as well as correlated with growth rate of bacteria. The heterogenous expression model of ESBL genes was identified as an important mechanism that bacteria developed to high resistance and adapted to antibiotic stresses. (p)ppGpp which is an important regulator of growth rate of bacteria mediates the expression of many genes of bacteria. We infer that (p)ppGpp can help bacteria develop to high resistance and adapt to antibiotic stresses through mediating the expression of ESBL genes. So, in this study, we intend to 1）collect ESBL-producing bacteria isolates responsible for pneumonia and construct experimental strains carrying the ESBL/GFP, then explore the relationship between concentration of (p)ppGpp and the expression level of ESBL genes; 2) introduce mutants of (p)ppGpp-deficient cells △relA/spot by mutating of relA and spot, the products of which were the (p)ppGpp synthetase, then measure the ESBL expression model of △relA/spot as well as the ability of △relA/spot to fight against the fluctuating stresses of antibiotic, meanwhile compare the differences between △relA/spot and the wild strain. This will contribute to understand the detailed mechanisms of heterogeneous expression of ESBL genes and the mechanisms of resistance of bacteria as well as put forward an innovative theory for developing new antibiotics.

病原菌超广谱β-内酰胺酶（ESBL）表达量决定其耐药水平。我们近期研究证实ESBL表达量在核酸序列完全相同的细菌间存在差异，并与细菌生长速率相关，且可随抗生素浓度变化而变化，从而提高了细菌耐药适应性，但其机制尚不明确。四/五磷酸鸟苷[(p)ppGpp] 参与调节多种基因表达，且是细菌生长速率的重要调控因子。我们推测(p)ppGpp通过调节ESBL表达量提高细菌的耐药适应性。本课题拟收集肺部感染来源产ESBL临床菌株，构建携带有ESBL/GFP荧光表达载体的实验菌株，探讨临床和实验菌株ESBL表达量与(p)ppGpp的关系；通过构建(p)ppGpp合成酶基因relA/spot双缺失菌株，研究(p)ppGpp 缺失情况下细菌ESBL表达模式及对抗生素的适应能力，并比较双缺失菌株与野生菌株的差异，以深入了解ESBL差异表达机制，为深入认识细菌耐药、寻求新的抗生素靶点提供策略和思路。

病原菌超广谱β-内酰胺酶（ESBL）表达量决定其耐药水平。我们近期研究证实ESBL表达量在核酸序列完全相同的细菌间存在差异，并与细菌生长速率相关，且提高了细菌耐药适应性，但其机制尚不明确。本研究首先收集了武汉协和医院肺部感染产ESBL大肠杆菌与肺炎克雷伯杆菌菌株及临床资料，并测定细菌携带的耐药基因，基因背景及菌株间的流行病学关系。通过构建ESBL/GFP融合基因，利用流式细胞仪检测细菌在不同生长情况下ESBL表达情况，同时测定ESBL不同表达亚群对多种抗生素（美罗培南、阿米卡星）的耐受性（最低杀菌时MDK99.99）。同时提取不同生长状态的细菌各表达亚群RNA送转录组及代谢组检测。结果显示①我院肺部感染产ESBLs大肠杆菌和肺炎克雷伯杆菌检出率高，耐药性强、在全球爆发流行的大肠杆菌ST131型及高毒力、携带多种耐药质粒的高致病性肺炎克雷伯杆菌ST11在我院流行。② 携带有ESBL/GFP的实验菌株A1指数生长期极高表达亚群细菌中VBNC状态细菌较极低表达及普通表达亚群明显增高，且该部分细菌对抗生素耐受性明显增强，持留菌比例较低表达亚群明显增高。③分析实验菌株A1在生物被膜/平台期细菌ESBL表达情况，结果提示细菌处于生物被膜/平台期中晚期时存在ESBL表达明显不同的细菌亚群，且各亚群细菌生长速率与对抗生素耐受性存在差异。④已分选指数生长期ESBL极高表达群与普通表达及极低表达群细菌RNA，及分选生物被膜/平台期细菌不同ESBL表达亚群细菌RNA，并已送转录组及代谢组(ppGpp)分析，结果近期回报。上述研究揭示了ESBL临床流行情况，系统阐述了不同生长状态下ESBL差异表达现象，该差异表达不仅与本身耐药基因介导的细菌耐药相关，更与基因水平外的细菌对抗其余抗生素的压力、有活力但不可培养细菌（VBNC）及持留菌密切相关，本研究转录组及代谢组学相关分析结果，本研究结果将深入认识VBNC及持留菌形成机制，以为控制临床慢性迁延不愈的感染及感染复发、遏制细菌耐药的发展提供新的策略。

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