Antibiotic resistance among hypermutator carbapenem resistant Klebsiella pneumoniae

项目摘要 耐碳青霉烯类肠杆菌科（CRE）是主要的公共卫生威胁。CR-肺炎克雷伯菌 CRKP是全球最常见的CRE。用新抗生素治疗CRKP和其他CRE感染 如头孢他啶-阿维巴坦（CZA）和美罗培南-戊硼巴坦（MVB）可改善生存率，但复发 感染是常见的，并且出现的耐药性是有问题的。CRE感染通常是由于菌株 寄生在胃肠道在其他细菌中，现在很明显，胃肠道定植是由一群 密切相关，但基因不同的菌株。这些菌株可以表现出一系列抗生素抗性， 生物学属性，这通常不被研究来自微生物培养物的单菌落所理解。在 在自然细菌种群的研究中，约1%至5%的分离株表现出高自发突变率，这可能 在环境压力下赋予选择优势，并增加种群内的多样性。等 高突变（HM）菌株在慢性定殖和复发感染期间在人类中最普遍， 它们通常源于DNA错配修复（MMR）基因的突变，如mutS、mutL或mutH。那里 在CRKP或CRE中HM的研究很少。我们相信HM在这些细菌中被低估了， 研究尚未评估长期GI定植、复发性感染或来自临床的菌株群体， 样品在这个项目中，我们假设1）HM CRKP可以从慢性GI患者中恢复， 定植和持续性/复发性感染; 2）MMR和其他基因突变在体外促进HM， 体外和感染器官内的MVB抗性;以及3）这些突变促进传播和接受 抗生素耐药基因（ARG）质粒的CRKP在体外和在GI定植。在试点 在筛选研究中，我们从约30%的慢性GI定植或持续或 反复感染。在大多数HM CRKP菌株中鉴定出MMR基因突变。其中一种突变 (MutH V76 G）被证明有助于HM、MVB和CZA抗性，并增强转移和接受 体外含ARG的质粒，以及CRKP组织负荷和MVB耐药性的出现， 静脉（IV）感染小鼠的感染器官。在本提案的目标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， 等基因突变菌株，以确定某些突变是否有助于HM。在目标2中，我们将评估 IV给药后在体外和小鼠器官内出现CZA、MVB和粘菌素耐药性的HM突变 感染然后，我们将确定HM突变对携带ARG质粒的体外转移的影响， 在小鼠胃肠道定植期间。该项目将标志着CRE临床分离株的首次系统性研究 对于HM表型。如果成功的话，实验将提供对HM的新理解，以及它在人类疾病中的关键作用。 调节CRKP抗生素耐药性。这里创造的同基因HM菌株也将是评估 新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.