Antibiotic resistance among hypermutator carbapenem resistant Klebsiella pneumoniae

项目摘要 耐碳青霉烯类肠杆菌科细菌(CRE)是危害人类健康的主要病原菌。肺炎克雷伯菌 (CRKP)是全球最常见的CRE。应用新抗生素治疗CRKP和其他CRE感染 像头孢他啶-阿维巴坦(CZA)和美罗培南-瓦博巴坦(MVB)一样，可以提高存活率，但复发 感染是常见的，紧急抗药性是有问题的。Cre感染通常是由于菌株 在胃肠道定居。在其他细菌中，现在很明显，GI的定植是由一群 亲缘关系很近，但在基因上截然不同的菌株。这些菌株可以表现出一系列的抗生素耐药性和 生物属性，这通常不被从微生物培养中研究单个菌落所认识到。在……里面 对自然细菌种群的研究表明，~1%至5%的分离物显示出较高的自发突变率，这可能 在环境压力下赋予选择性优势，并增加种群内的多样性。是这样的 高突变(HM)菌株在慢性定植和反复感染期间在人类中最普遍， 它们最常源于DNA错配修复(MMR)基因的突变，如MutS、MutL或Muth。那里 在CRKP和CRE中，对HM的研究很少。我们认为HM在这些细菌中被低估是因为 研究没有评估长期的胃肠道定植、反复感染或临床菌株的数量。 样本。在这个项目中，我们假设1)HM CRKP可以从慢性GI患者中恢复 定植和持续/复发感染；2)MMR和其他基因突变促进体外HM，CZA 以及在体外和感染器官内对MVB的耐药性；以及3)这些突变促进了传播和接受 在体外和胃肠道定植过程中用CRKP检测携带抗生素耐药基因(ARG)的质粒。在试行中 筛选研究，我们从约30%的慢性胃肠道定植患者或持续性或慢性胃肠道疾病患者中恢复了HM CRKP 反复感染。在大多数HM CRKP菌株中发现MMR基因突变。这些突变中的一个 (MUTH V76G)被证明有助于抵抗HM、MVB和CZA，并增强转移和接受 对CRKP的组织负荷和MVB抗性的出现 静脉(IV)感染小鼠的感染器官。在这项提案的目标1中，我们将继续筛选临床 HM表型的CRKP分离株。我们将对HM分离株进行全基因组测序，并创建 以确定某些突变是否会导致HM。在目标2中，我们将评估 CZA、MVB和粘菌素耐药的HM突变在体外和静脉注射后小鼠器官内的出现 感染。然后，我们将在体外确定HM突变对携带ARG的质粒转移的影响 在小鼠胃肠道定植期间。该项目将标志着对CRE临床分离株的首次系统研究 对于HM表型。如果成功，实验将提供对HM及其关键角色的新理解 调节CRKP抗生素耐药性。这里创造的等基因HM菌株也将是评估 新型抗生素的耐药性障碍和抗生素发现的新靶点。

Project Summary Carbapenem resistant Enterobacteriaceae (CRE) are major public health threats. CR-Klebsiella pneumoniae (CRKP) are the most common CRE globally. Treatment of CRKP and other CRE infections with new antibiotics like ceftazidime-avibactam (CZA) and meropenem-vaborbactam (MVB) has improved survival, but recurrent infections are common and emergent resistance is problematic. CRE infections are usually due to strains that colonize the GI tract. Among other bacteria, it is now apparent that GI colonization is caused by a population of closely related, but genetically distinct strains. These strains can exhibit a range of antibiotic resistance and biologic attributes, which are often not appreciated by studying single colonies from microbiologic cultures. In studies of natural bacterial populations, ~1 to 5% of isolates exhibit high spontaneous mutation rates, which may confer selective advantages under environmental stress and increase diversity within the population. Such hypermutation (HM) strains are most prevalent in humans during chronic colonization and recurrent infections, and they most often stem from mutations to DNA mismatch repair (MMR) genes like mutS, mutL or mutH. There are few studies of HM among CRKP or CRE. We believe that HM is under-recognized in these bacteria because studies have not assessed long-term GI colonization, recurrent infections, or populations of strains from clinical samples. In this project, we hypothesize that 1) HM CRKP can be recovered from patients with chronic GI colonization and persistent/recurrent infections; 2) MMR and other gene mutations promote HM in vitro, and CZA and MVB resistance in vitro and within infected organs; and 3) these mutations promote transmission and receipt of antibiotic resistance gene (ARG)-bearing plasmids by CRKP in vitro and during GI colonization. In pilot screening studies, we recovered HM CRKP from ~30% of patients with chronic GI colonization, or persistent or recurrent infections. MMR gene mutations were identified in most HM CRKP strains. One of these mutations (MutH V76G) was proven to contribute to HM, MVB and CZA resistance, and enhanced transfer and acceptance of plasmids containing ARGs in vitro, and to CRKP tissue burdens and emergence of MVB resistance within infected organs of intravenously (IV)-infected mice. In aim 1 of this proposal, we will continue to screen clinical CRKP isolates for HM phenotype. We will perform whole genome sequencing on HM isolates, and create isogenic mutant strains to determine if certain mutations contribute to HM. In aim 2, we will evaluate the role of HM mutations in emergence of CZA, MVB and colistin resistance in vitro and within mouse organs following IV infection. Then, we will determine impact of HM mutations on transfer of ARG-bearing plasmids in vitro and during mouse GI colonization. This project will mark the first systematic investigations of CRE clinical isolates for HM phenotypes. If successful, experiments will provide new understanding of HM and its crucial roles in regulating CRKP antibiotic resistance. Isogenic HM strains created here will also be powerful tools for assessing resistance barriers for novel antibiotics and new targets of antibiotic discovery.