Genomic variation in epidemic carbapenem-resistant K.pneumoniae

 描述（由申请人提供） 碳青霉烯类耐药肠杆菌科（CRE），尤其是产碳青霉烯酶（KPC）的肺炎克雷伯菌。肺炎链球菌（KPC-Kp）已经成为一种重要的医院获得性病原体，在美国和世界范围内引起相当大的发病率和死亡率。引人注目的是，绝大多数KPC-Kp分离株属于单一序列类型ST 258。然而，这种流行性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。最近的比较基因组研究表明，Kp ST 258菌株正在经历显着的多样化，在质粒和染色体结构，特别是在K-抗原编码的荚膜多糖合成（cps）基因操纵子。与其他病原菌（如肺炎链球菌）中所描述的相似，携带cps的区域的重组似乎是ST 258遗传多样性的主要驱动力。迄今为止，我们已经在Kp ST 258菌株中发现了六种不同的cps操纵子（cps-1至-6），并鉴定了几种ST 258相关的携带blaKPC的质粒。值得注意的是，我们的初步数据显示ST 258进化枝（不同cps组）中的变异不限于cps区域，还涉及多个含有有助于定殖、抗性和毒力的基因的基因座。一个重要的问题是，这种流行性克隆可能通过染色体重组和/或质粒转移获得增强的毒力，演变为高毒力，多药耐药（HV-MDR）菌株，并进一步复杂化目前的努力，以应对这一危机。令人震惊的是，我们的初步结果表明，某些“新的”Kp ST 258进化枝（即携带新的cps操纵子）与来自其他循环进化枝的菌株相比具有更高的毒力。因此，我们假设可变染色体区域和/或获得的质粒改变宿主-病原体相互作用，可能导致出现增强的毒力菌株。我们的目标是确定这些基因组因子和质粒内容物，并表征其与ST 258毒力相关的生物学作用，并检查质粒的传递性。在本申请中，我们将研究遗传重组对Kp ST 258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分离株及其推定供体菌株（可能是ST 258中cps替换来源的独特ST）的全基因组测序（WGS），我们将系统地评估该流行性克隆中的大规模和小规模基因组变异，并阐明其分子进化史（目标1）。接下来，通过使用体外和体内模型，我们将研究导致胶囊编码机制以及其他生物学上重要的基因座的替换的重组是否导致HV-MDR进化枝的出现（Aim 2a）。此外，我们还将检验ST 258遗传背景对KPC质粒具有更高的转移效率和稳定性的假设，这可能部分解释这种KPC流行性克隆（Aim 2b）的成功。在这项研究中产生的结果将产生丰富的信息的内容和特点的Kp基因组变异，并可能会发现新的毒力因子。鉴定影响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.