Polyclonality of carbapenem resistant Enterobacteriaceae bloodstream infections

碳青霉烯类耐药克雷伯氏菌属（CRK）物种，特别是碳青霉烯类耐药肺炎克雷伯氏菌（CRKP），被全球公认为“紧迫威胁”病原体。血流和其他严重的克雷伯氏菌感染，包括那些由于CRK，通常是由一个菌株，殖民胃肠道（GI）道。新兴的全基因组序列（WGS）数据显示，胃肠道是由克隆细菌种群定殖的，其中遗传多样性随着时间的推移而发展。由于临床和研究实验室通常从阳性培养物中表征单个菌落，因此不知道血液感染由给定细菌的克隆但遗传多样性菌株引起的频率。在初步研究中，我们对10名随机选择的患者的第一次CRK阳性血培养的10个菌落进行了WGS。我们证明，10例患者中有8例的血流感染是由克隆内、遗传多样性CRK引起的，包括核心基因组中具有非同义单核苷酸多态性的菌株、染色体基因缺失、插入和其他突变以及质粒或质粒携带的基因丢失。基因型不同的CRK菌株在抗生素敏感性、荚膜多糖（CPS）含量和其他毒力相关表型方面表现出显着差异。来自2名患者的遗传变异株在小鼠血流感染中显示出显著的毒力差异。我们在这项研究中的目标是表征CRK菌株的遗传和表型多样性，从基线和纵向血培养的患者在我院回收，并牵连特定的基因，质粒和突变的抗生素耐药性和毒力。在具体目标1中，我们将通过对从阳性血培养物中选择的单个菌落进行WGS、质粒测序和核心基因组同源性和基因突变分析，确定引起基线和复发性血流感染的CRK菌株的遗传多样性。对于来自每种阳性培养物的遗传上不同的菌株，我们将在体外确定抗生素敏感性和其他表型。对于优先染色，我们将评估小鼠血流感染期间的抗生素反应和毒力。在具体目标2中，我们将验证特定基因、质粒和突变对抗生素治疗反应和毒力的影响。我们将用同基因的、实验室产生的突变体和互补的菌株静脉内感染小鼠，这些菌株的基因在患者中观察到多样性（例如，编码CPS的那些），以及质粒管理的和非管理的菌株。我们将测量接受和未接受抗生素治疗的小鼠的结果。这项研究的结果将肯定CRK阳性血培养物中遗传多样性的程度和类型，为血流感染期间CRK菌株动力学提供新的见解，并确定抗生素治疗反应和毒力的新遗传决定因素。这里收集的见解将为非CR克雷伯氏菌和其他细菌在血流和其他感染过程中的遗传多样性研究以及CRK基因或抑制抗生素反应和促进发病机制的基因靶点的机制研究奠定基础。

Carbapenem resistant Klebsiella (CRK) species, in particular carbapenem resistant Klebsiella pneumoniae (CRKP), are recognized as “urgent threat” pathogens globally. Bloodstream and other serious Klebsiella infections, including those due to CRK, usually are caused by a strain that colonizes the gastrointestinal (GI) tract. Emerging whole genome sequence (WGS) data show that the GI tract is colonized by clonal bacterial populations, in which genetic diversity develops over time. It is unknown how often bloodstream infections are caused by clonal but genetically diverse strains of a given bacterium, since clinical and research laboratories generally characterize a single colony from positive cultures. In preliminary studies, we performed WGS on 10 colonies from the first CRK-positive blood culture of 10 randomly selected patients. We demonstrated that bloodstream infections in 8 of 10 patients were caused by intraclonal, genetically diverse CRK, including strains with non-synonymous single nucleotide polymorphisms in the core genome, chromosomal gene deletions, insertions and other mutations, and plasmid or plasmid-borne gene loss. Genotypically distinct CRK strains exhibited significant differences in antibiotic susceptibility, capsular polysaccharide (CPS) content, and other virulence-associated phenotypes. Genetic variant strains from 2 patients displayed significant differences in virulence during bloodstream infections of mice. Our objectives in this study are to characterize the genetic and phenotypic diversity of CRK strains recovered from baseline and longitudinal blood cultures of patients in our hospital and to implicate specific genes, plasmids and mutations in antibiotic resistance and virulence. In specific aim 1, we will determine genetic diversity of CRK strains causing baseline and recurrent bloodstream infections by performing WGS, plasmid sequencing, and analyses of core genome phylogeny and gene mutations for individual colonies selected from positive blood cultures. For genetically distinct strains from each positive culture, we will determine antibiotic susceptibility and other phenotypes in vitro. For prioritized stains, we will assess antibiotic responses and virulence during mouse bloodstream infections. In specific aim 2, we will validate the impact of specific genes, plasmids and mutations on antibiotic treatment responses and virulence. We will infect mice intravenously with isogenic, lab-created mutant and complemented strains for genes in which diversity was observed in patients (e.g., those encoding CPS), and plasmid-curated and non-curated strains. We will measure outcomes in mice treated and not treated with antibiotics. Results from this study will affirm the extent and type of genetic diversity in CRK-positive blood cultures, afford new insights into CRK strain dynamics during bloodstream infections, and identify novel genetic determinants of antibiotic treatment responses and virulence. Insights gathered here will form a foundation for investigations of genetic diversity during bloodstream and other infections by non-CR Klebsiella and other bacteria, and for mechanistic studies of CRK genes or gene targets that dampen antibiotic responses and promote pathogenesis.