肺炎克雷伯菌高毒力菌株的检测标志及毒力机制研究

Hypervirulent Klebsiella pneumoniae (HvKP) is increasingly concerned as an important pathogen of liver abscess and metastatic infections such as endophthalmitis, meningitis and sepsis. The reason that its virulence is significantly higher than classic Klebsiella pneumoniae has not been illustrated yet.There is also lack of standard method for detection of HvKP. Our study creatively put forward the detection markers of combining hypermucoviscous phenotype using string test with genetic features of virulence genes for rmpA and aerobactin using polymerase chain reaction. Pulse-field gel electrophoresis and multilocus sequence typing is used to analyze the molecular homology and clone type of HvKP causing metastatic multi-infections from mainland China.Through genome sequencing and analysis,the kind and location of virulence genes in genomes of these specific HvKP strains will be explored.We will also investigate the interaction between HvKP and phagocytes using common klebsiella pneumoniae as control strains with the help of electronic microscope and the staining technique of immunohistochemistry in order to indicate the characterisitcs of HvKP surviving, even proliferating in phagocytes. This study is of practical significance in guiding laboratory to detect and report HvKP. It will also innovatively reveal the virulence-enhanceing mechanism of HvKP. As a result, it will obviously lay foundations in understanding the hypervirulence of HvKP, avoiding multi-infections by HvKP and developing vaccine against HvKP.

高毒力肺炎克雷伯菌（Hypervirulent klebsiella pneumoniae,HvKP）是近年来受关注的、引起肝脓肿和转移感染的病原菌，导致其毒力显著高于普通肺炎克雷伯菌的机理尚不清楚，也还没有用于实验室检测的通用方法。本课题创新性地将细菌表型和基因特征结合起来，研究HvKP的高黏液表型和毒力基因联合检测标志，对实验室早期报告HvKP具有实际的指导意义；通过脉冲场凝胶电泳和多位点序列分析，明确致同一患者转移性多部位感染HvKP菌株的同源性，同时阐明源自中国大陆患者HvKP的克隆型；通过对HvKP菌株基因组测序和比较分析，了解这些菌株基因组中毒力基因的种类和分布特征，依靠电子显微镜观察和免疫组化染色技术，比较研究HvKP和普通肺炎克雷伯菌与吞噬细胞间相互作用的特点，将首次揭示HvKP毒力增强的机制，为理解HvKP的高毒力性、防止多部位感染的发生、研制抗菌疫苗奠定理论基础。

高毒力肺炎克雷伯菌（Hypervirluent klebsiella pneumoniae, HvKP）与经典菌株（Classic Klebsiella pneumoniae，CKP）不同，近年来被认为是一种新的、高毒力病原菌。我们先后对400余株肺炎克雷伯菌进行了研究。高黏液表型的HvKP占74%。利用基因扩增方法，检测了主要毒力基因（rmpA, aerobactin，ureA，fimH，wabG等)和荚膜分型基因，发现rmpA与aerobactin基因阳性率在HvKP（96.3%和92.6%）中显著高于CKP（6.2%和9.4%），HvKP以K1，K2型为主，分别占40.8%和22.2%，K57型占7.4%。检测标志“高黏液表型+毒力基因rmpA或/和aerobactin阳性+荚膜基因分型为K1/或K2/或K57基因型”可用于实验室鉴定HvKP。. 中性粒细胞对HvKP的吞噬率显著低于CKP。透射电镜观察证实中性粒细胞吞噬的HvKP数量明显少于CKP的数量，还观察到中性粒细胞细胞与细菌作用60 min，被吞噬的CKP菌体开始溶解，而HvKP菌体完整，并且可见二分裂繁殖现象。NETs捕获的HvKP数量明显少于CKP，同时在NETs捕获的CKP菌体上观察到被杀伤的“小孔”。表明HvKP同时具有抵抗中性粒细胞胞内和胞外杀伤作用。这对进一步研究HvKP的致病机制具有参考价值。. 多位点序列分型显示所有K1型HvKP为ST23型，ST11型则是CKP最常见的序列型。前者是高毒力菌株的主要流行克隆菌株。脉冲场凝胶电泳显示来自同一患者不同部位具有同源性的HvKP最常引起血液和肝脏感染，而CKP则引起血-胆和血-肺部感染较多见。此结果有助于理解HvKP和CKP在社区或医院所致感染及其传播的差异。. 对6株HvKP基因组序列分析发现，除1株菌外，5株HvKP都有一个约200kb的质粒，其中2株菌还分别包含一个80kb和100kb的质粒。进一步对编号为11492HvKP菌株的分析揭示，其毒力基因和耐药基因位于同一个质粒上，由blaCTX-M-24耐药基因整合到毒力质粒上而形成，该耐药基因存在于被称为Tn6501的新的转座子。提示新的超级细菌—多耐药的HvKP已经出现并有可能通过基因转座子在医院内引起传播，这将成为抗感染治疗和医院感染控制的难点。