Genomic variation in epidemic carbapenem-resistant K.pneumoniae

 描述(由申请人提供) 耐碳青霉烯类肠杆菌科(CRE)，尤其是产碳青霉烯酶(KPC)肺炎克雷伯菌(KPC-KP)在美国和世界范围内已成为一种重要的医院获得性致病菌。引人注目的是，绝大多数KPC-KP分离物属于单一的序列类型ST258。然而，这种流行病克隆的分子进化仍然知之甚少，导致其流行病学成功的遗传因素仍然未知。最近的比较基因组学研究表明，KP ST258菌株在质粒和染色体结构上都发生了显著的变化，特别是在K抗原编码的衣壳多糖合成(Cps)基因操纵子上。与在其他致病菌(如肺炎链球菌)中所描述的相似，Cps-Having区域的重组似乎是ST258基因多样化的主要驱动力。到目前为止，我们已经在KP ST258菌株中发现了6个不同的cps操纵子(cps-1到-6)，并鉴定了几个与ST258相关的含有blaKPC的质粒。值得注意的是，我们的初步数据显示，ST258分支(不同的cps组)的变异不仅限于cps区域，而且还涉及多个基因座，这些基因含有与定殖、抗性和毒力有关的基因。一个重要的担忧是，这种流行克隆可能会通过染色体重组和/或质粒转移获得更强的毒力，演变为高毒力、多药耐药(HV-MDR)毒株，并使目前抗击这一危机的努力进一步复杂化。令人担忧的是，我们的初步结果显示，与来自其他循环分支的菌株相比，某些新的KP ST258分支(即含有新的cps操纵子)具有更高的毒力。因此，我们假设可变的染色体区域和/或获得性质粒改变了宿主-病原体的相互作用，从而可能导致毒力增强的菌株的出现。我们的目标是鉴定这些基因组因子和质粒内容，并表征它们与ST258毒力相关的生物学作用，并检测质粒的传递性。在这一应用中，我们将调查遗传重组对KP ST258遗传变异的影响程度。通过对来自不同CPS组(CPS-1至CPS-6)的代表性KP分离株及其可能的供体毒株(可能是ST258中CPS替换的来源的独特STS)的全基因组测序，我们将系统地评估该流行病克隆的大小基因组变异，并阐明它们的分子进化历史(目标1)。接下来，通过使用体外和体内模型，我们将调查导致胶囊编码机制和其他生物重要基因座替换的重组是否会导致HV-MDR分支的出现(目标2a)。此外，我们将检验ST258遗传背景对KPC质粒具有更高的转移效率和稳定性的假设，这可能在一定程度上解释了这个KPC流行克隆(目标2b)的成功。这项研究产生的结果将产生关于KP基因组变异的内容和特征的丰富信息，并可能识别新的毒力因子。确定影响KP菌株致病潜力的基因组变异将提供潜在的诊断和治疗解决方案。

 DESCRIPTION (provided by applicant) Carbapenem-resistant Enterobacteriaceae (CRE), especially Klebsiella pneumoniae carbapenemase (KPC)- producing K. pneumoniae (KPC-Kp), have emerged as a significant hospital-acquired pathogen causing considerable morbidity and mortality in the United States and worldwide. Strikingly, a large majority of KPC-Kp isolates belong to a single sequence type, ST258. However, the molecular evolution of this epidemic clone is still very poorly understood and the genetic factors contributing to its epidemiological success remain unknown. Recent comparative genomic studies indicate that Kp ST258 strains are undergoing significant diversification in both plasmid and chromosome structures, especially at the K-antigen encoded capsular polysaccharides synthesis (cps) gene operons. Similar to what has been described in other pathogenic bacteria (e.g. Streptococcus pneumoniae), the recombination of cps-harboring region appears to be a major driving force in the genetic diversification in ST258. To date, we have found six different cps operons (cps-1 to -6) in Kp ST258 strains and identified several ST258 associated blaKPC-harboring plasmids. Notably, our preliminary data showed the variations in ST258 clades (different cps groups) are not limited to the cps regions, but also involve multiple loci that harbor genes contributing to colonization, resistance and virulence. One significant concern is that this epidemic clone may acquire enhanced virulence via chromosomal recombination and/or plasmid transfer, evolving as a highly virulent, multidrug resistant (HV-MDR) strain and further complicating current efforts to battle this crisis. Alarmingly, our preliminary results showed that certain "new" Kp ST258 clades (i.e. harboring novel cps operons) had higher virulence in comparison to strains from other circulating clades. Thus, we hypothesize that variable chromosomal regions and/or acquired plasmids alter host-pathogen interactions that may lead to the emergence of strains of enhanced virulence. Our objective is to identify these genomic factors and plasmid contents, and characterize their biological role associated with ST258 virulence, and examine plasmid transmissibility. In this application, we will investigate the extent to which genetic recombination has influenced genetic variation in Kp ST258. Through whole genome sequencing (WGS) of representative Kp isolates from different cps groups (cps-1 to cps-6) as well as their putative donor strains (unique STs that are likely the source of the cps replacement in ST258), we will systematically evaluate both large and small scale genomic variations in this epidemic clone and elucidate their molecular evolution history (Aim 1). Next, by using in vitro and in vivo models, we will investigate whether recombination that results in replacement of capsule-encoding machinery as well as other biologically important loci results in the emergence of HV-MDR clades (Aim 2a). In addition, we will test the hypothesis that ST258 genetic background has higher transfer efficiency and stability for KPC plasmids, which may partly explain the success of this KPC epidemic clone (Aim 2b). The results generated in this study will yield a wealth of information regarding the content and characteristics of Kp genomic variations and will likely identify novel virulence factors. The identification of genomic variations that impact the disease-causing potential of Kp strains will provide potential diagnostic and therapeutic solutions.