基于组学与微流控芯片技术的肺炎克雷伯菌ESBL耐药表型发生与转换机制的研究

The extended spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (KP) is a major cause for treatment failure of infectious disease. However, the molecular mechanism of its phenotypic occurrence and transformation of ESBL gene in KP have not been completely understood so far. It was found that a certain amount of KP strains harboring ESBL gene were still sensitive to board-spectrum β-lactam antibiotics without resistant phenotype in our pilot study. In addition, some KP strains developed apparent heterogeneity after application of antimicrobial reagents in different concentration. The existence of "silence", variation and transformation of ESBL in their phenotypes in different environment is potentially modulated by a certain of pivotal regulating elements. In the current proposing project, we anticipate to deeply investigate the mechanism of the ESBL phenotypic "silence" in the ESBL genotype﹙+﹚/phenotype﹙-﹚KP strains, to surveil the pivotal regulating elements associated with ESBL gene expression, to decode and verify the molecular mechanism of phenotypic occurrence and transformation of ESBL gene with the application of microfluid chip and multi-omics (such as pangenomics, transcriptomics and proteinomics) analysis. This promising project may put forward an innovative theory for controlling the emerging and dissemination of KP antibiotic resistance.

肺炎克雷伯菌（Klebsiella pneumoniae, KP）产超广谱β内酰胺酶（Extended-spectrum β-lactamase, ESBL）是引起临床感染治疗失败的重要原因，但其ESBL耐药表型发生与转换的分子机制仍不明确。我们前期研究发现部分携带ESBL耐药基因的KP菌株仍对抗生素敏感，同时KP耐药表型在不同抗菌药物浓度梯度下存在明显异质性。这种ESBL基因在不同环境下表型"沉默"与差异表达，可能是其表型在某种关键元件的调控下相互转换的结果。本项目拟对携带ESBL耐药基因而表型阴性KP菌株，在基因组学、转录组学和蛋白组学分析的基础上，结合微流控芯片技术，研究ESBL基因﹙+﹚/表型﹙-﹚KP菌株表型"沉默"机制，筛选调控ESBL基因表达的关键元件，揭示和解析KP菌株ESBL耐药表型发生和转化的关键环节和分子机制，并对之加以验证，为控制KP耐药性的发生及传播提供新思路。

肺炎克雷伯菌（Klebsiella pneumoniae, KP）产超广谱β内酰胺酶（Extended-spectrum β-lactamase, ESBL）是引起临床感染治疗失败的重要原因，但其ESBL耐药表型发生与转换的分子机制仍不明确。我们通过对本项目组收集的ESBL表型阴性的细菌进行耐药基因PCR扩增及表型确证实验，在国内首次发现了ESBL基因阳性表型阴性的菌株。进一步通过构建不同启动子及其相关序列与blaSHV基因的重组质粒，明确启动子及其相关位点突变是ESBL型基因blaSHV-5，-12耐药表型转换的原因。此外，本研究在开展过程中还有如下发现：（1）发现了新的Kluyvera菌属新物种：胆道克吕沃菌。并且进一步发现细菌共生现象可能是细菌对抗抗生素的一种应对机制。（2）在一株多重耐药肺炎克雷伯杆菌中发现了一个同时携带多种耐药基因和多种重金属耐药基因的IncHI2型质粒，并且完成了质粒全基因组测序。（3）发现了一株变栖克雷伯菌X39,证实菌株X39是一株既可以感染动物和人，也可以侵染植物并在植物中定植的细菌。（4）通过对13株不同耐药级别的肺炎克雷菌进行全基因组测序，从基因组水平阐明了细菌耐药传播的机制。以上研究结果可以为控制肺炎克雷伯菌耐药性的发生及传播提供新思路，为利用CRISPR-Cas9基因编辑系统编辑耐药性肺炎克雷伯菌ESBL耐药基因，实现多重耐药肺炎克雷伯菌向敏感肺炎克雷伯菌转化提供依据。