Evolution of KPC-K pneumoniae that persist in patients on prolonged antibiotics

 描述(申请人提供)：肺炎克雷伯菌碳青霉烯酶(KPC)肺炎克雷伯菌(KPC-KP)已成为主要病原体，导致广泛耐药感染，粗死亡率可超过50%。自2008年以来，我们维护了我们中心的700株KPC-KP菌株的生物库，链接到临床和药学数据库。我们已经证明，KPC-KP菌血症和腹内感染(IAI)患者的预后范围从败血症休克导致的急性死亡，到快速治愈，到持续一年以上的复发或持续感染。正如在大多数美国中心一样，KPC-KP序列类型258(ST258)菌株在我们的患者中占主导地位。我们的初步数据表明，根据常规分子流行病学标准克隆的ST258、KPC-KP菌株具有显著的遗传异质性，包括在抗菌素耐药谱、孔蛋白功能和染色体高变区内。来自持续感染患者的纵向菌株对抗菌剂的抗药性越来越强，并在涉及内毒素和胶囊生物合成、铁运输、应激反应等过程的基因上发生突变。我们在这个项目中的主要目标是确定KPC-KP菌株引起持续感染的遗传基础。我们假设，持久性至少部分源于菌株对治疗方案和体内环境压力的进一步抗药性和毒力的减弱。我们的目标是全面定义引起持续感染的ST258、KPC-KP菌株之间的遗传差异，表征持续感染期间菌株的遗传进化，并将这些结果与抗菌素耐药性、毒力和体内基因表达相关联。为了达到这些目标，我们将追求两个具体目标。在目标1中，我们将测试从持续性感染患者纵向收集的ST258、KPC-KP菌株在体外时间杀灭试验中对多利培南和粘菌素(我们的一线治疗方案)组合的耐药性，以及在小鼠腹部感染期间的相对毒力。 根据初步数据，我们预计最后的纵向菌株将比第一个纵向菌株对多利培南-粘菌素的抗药性更强，毒力更小。在目标2中，我们将对ST258、KPC-KP菌株进行全基因组和质粒测序，使用RNA-Seq确定IAIS期间小鼠的转录本，并创建突变株，以验证测序和RNA-Seq鉴定的基因与抗性和毒力有关。我们预计，我们的结果将为KPC-KP菌株在感染期间适应治疗和环境压力的机制提供洞察力。该项目将产生关于耐药性和致病机制的新假设，并确定开发新的诊断、治疗、疫苗和治疗反应预测标记的目标。

 DESCRIPTION (provided by applicant): Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) have emerged as major pathogens, causing extensively drug resistant infections with crude mortality rates that can exceed 50%. Since 2008, we have maintained a biorepository of >700 KPC-Kp strains from our center, linked to clinical and pharmacy databases. We have shown that outcomes among patients with KPC-Kp bacteremia and intra- abdominal infections (IAI) range from acute death due to septic shock, to rapid cure, to recurrent or persistent infections that have lasted for more than a year. As is true at most U.S centers, KPC-Kp sequence type 258 (ST258) strains predominate among our patients. Our preliminary data demonstrate that ST258, KPC-Kp strains that are clonal by conventional molecular epidemiologic criteria exhibit significant genetic heterogeneity, including in antimicrobial resistance repertoire, porin function and within the chromosomal hypervariable region. Longitudinal strains from individual patients with persistent infections become increasingly resistant to antimicrobial agents, and have mutations in genes involved in lipopolysaccharide and capsule biosynthesis, iron transport, stress responses and other processes. Our major goal in this project is to identify the genetic bases by which KPC-Kp strains cause persistent infections. We hypothesize that persistence stems, at least in part, from the development of further antimicrobial resistance and attenuation of virulence by strains in response to treatment regimens and the stresses of in vivo environments. Our objectives are to comprehensively define genetic differences among ST258, KPC-Kp strains causing persistent infections, characterize the genetic evolution of strains during persistent infections, and correlate these results with antimicrobial resistance, virulence and gene expression in vivo. To achieve these objectives, we will pursue two specific aims. In aim 1, we will test longitudinally-collected ST258, KPC-Kp strains from patients with persistent infections for the emergence of resistance to the combination of doripenem and colistin (our front- line treatment regimen) during time-kill assays in vitro, and relative virulence during intra-abdominal infections of mice. Based on preliminary data, we anticipate that last longitudinal strains will be more resistant to doripenem-colistin and less acutely virulent than first longitudinal strains. In aim 2, we will perform whole genome and plasmid sequencing on ST258, KPC-Kp strains, use RNA-Seq to define transcriptomes in mice during IAIs, and create mutant strains to verify that genes identified by sequencing and RNA-Seq contribute to resistance and virulence. We anticipate that our results will afford insights into mechanisms by which KPC-Kp strains adapt to treatment and environmental pressures during infections. This project will generate new hypotheses about resistance and pathogenic mechanisms, and identify targets for the development of novel diagnostics, therapeutics, vaccines, and predictive markers for treatment responses.