Investigation of Horizontal Transfer of Carbapenemase Gene in Gram Negative Bacteria

革兰氏阴性菌碳青霉烯酶基因水平转移的研究

• 批准号: 10691771
• 负责人: Karen Frank
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10691771
• 关键词: Bacteria; Bacterial Genome; Biological Assay; Biology; Chromosomes; Clinical; Contracts; Data; Data Analyses; Databases; Disease Outbreaks; Elements; Enterobacteriaceae; Environment; Environmental Epidemiology; Epidemiology; Genes; Genetic; Genomics; Goals; Gram-Negative Bacteria; Horizontal Gene Transfer; Hospitals; Housekeeping; In Situ; In Vitro; Infection; Infection Control; Infrastructure; Institution; Intensive Care Units; Investigation; Klebsiella; Klebsiella pneumoniae; Location; Low Prevalence; Medical center; Molecular Epidemiology; Morbidity - disease rate; Multi-Drug Resistance; Nosocomial Infections; Organism; Paper; Patient Isolators; Patients; Pharmacologic Substance; Phenotype; Plasmids; Play; Population; Predisposition; Reagent; Refractory; Research; Research Personnel; Resistance; Role; Sampling; Sequence Analysis; Services; Sorting; Source; Surface; System; Testing; Time; Touch sensation; Vertebral column; Water; antimicrobial drug; base; carbapenem resistance; carbapenemase; clinical epidemiology; clinically significant; design; experimental study; genome analysis; genome sequencing; high throughput screening; improved; in vitro Model; in vivo; insight; member; mortality; mouse model; multi-drug resistant pathogen; novel; patient population; prevent; resistance gene; resistant strain; secondary analysis; single molecule real time sequencing; transmission process; wasting; whole genome

Bacteria that produce carbapenemases, such as the K. pneumoniae carbapenemase (blaKPC), are increasingly common multidrug resistant pathogens causing nosocomial infections and outbreaks, with high rates of morbidity and mortality worldwide. Horizontal transfer of carbapenemase-producing plasmids is believed to play a significant role in the spread of multidrug resistant K. pneumoniae and other Gram-negative bacteria. Our experiments are aimed at defining the parameters regulating the spread of carbapenem resistance. Patients can be colonized by KPC+ K. pneumoniae for extended periods of time; however, only some of these patients have evidence of blaKPC containing plasmids transferring to bacteria other than Klebsiella species based on our extensive surveillance system. Understanding any differences between in vitro potential to spread and in vivo evidence of spread will aid our attempts to prevent dissemination of the resistance phenotype. We found that investigators using in vitro models of conjugation should incorporate several parameters before drawing conclusions about a plasmids conjugative ability. Studies utilizing conjugation assays overwhelmingly use only a single assay condition. In vitro and in vivo studies of the same plasmid are often discrepant. Thus, we sought to use an even more accurate system, comparing in vitro data to in situ data, by using environmental and clinical epidemiology. Although this has been attempted previously, other studies typically use a single donor that is representative of an outbreak to a single recipient. In this study, we leverage four donor strains each with a unique molecular epidemiology, and leverage 16 patient isolate recipients not previously available for study. We generated a unique set of reagents through transformation into a plasmid-cured strain that allow for easy separation and testing of donor strain, plasmid, and recipient strain influences. Our systematic analysis showed that conjugation is heavily context-specific, and is dependent on the donor, recipient, plasmid, and the environment. The environment as a location for horizontal gene transfer has been examined and discussed, but the full extent and impact from this source of additional resistant strains is not yet known. We also sought to correlate our in vitro data to the epidemiology of blaKPC+ patients from two medical centers. We note that, surprisingly, in vitro conjugation efficiency does not correlate with observed promiscuous interspecies spread. This is in contract to conclusion of some outbreak papers that suggest horizontal transfer may be of high efficiency between different species of Enterobacteriaceae. Next, we analyzed efficient and refractory recipients by single-molecule, real-time sequencing. We used this whole genome sequence data to generate hypotheses for possible mechanisms that control conjugation, highlighting the complexity of the regulatory system for conjugation. These studies integrate epidemiology, plasmid biology, and genomics to give novel insights on clinically significant resistant Gram-negative bacteria. The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a two-year period, additional sampling was conducted from a broader range of locations, including housekeeping closets, waste water from hospital internal pipes, and external manholes. We compared these data with previously collected information from five years of patient clinical and surveillance isolates. Whole genome sequencing and analysis of 108 isolates provided comprehensive characterization of blaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with blaKPC-positive organisms, all samples from the intensive care unit pipe waste water and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were common plasmid backbones between both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings. We developed a system of tracking the spread of plasmids between bacteria by adding fluorescent tags to the plasmid and different fluorescent tags to the chromosomes. We used FACS sorting to track the transfer of the plasmids. Unfortunately, this project was terminated early in the FY22 fiscal year and it will be removed from the database in FY23, due to departure of all members of the research team for this project and the establishment of an unrelated independent project.

产生碳纤维酶的细菌，例如肺炎甲虫碳酸酶酶（BLAKPC），是越来越常见的多药耐药病原体，引起了全球发病率和死亡率，引起了医院感染和暴发。据信，产生碳青霉酶的质粒的水平转移在耐多药的K.肺炎和其他革兰氏阴性细菌的扩散中起着重要作用。我们的实验旨在定义调节碳青霉含量扩散的参数。可以通过KPC+ K.肺炎定植的患者长时间；但是，根据我们广泛的监测系统，只有其中一些患者有blakpc含有质粒转移到klebsiella物种以外的细菌的证据。了解体外潜力传播和体内传播证据之间的任何差异将有助于我们试图防止抗药性表型传播。 我们发现，使用结合体外模型的研究者在得出有关质粒共轭能力的结论之前应结合几个参数。利用共轭测定的研究仅使用单个测定条件。同一质粒的体外和体内研究通常差异。因此，我们试图通过使用环境和临床流行病学来使用更精确的系统，将体外数据与原位数据进行比较。尽管以前已经尝试过这种尝试，但其他研究通常使用代表单个接受者爆发的单个供体。在这项研究中，我们利用四个供体菌株具有独特的分子流行病学，并利用16位患者分离株的接受者以前无法进行研究。我们通过转化为质粒固定菌株，生成了一组独特的试剂，该试剂易于分离和测试供体应变，质粒和受体应变影响。 我们的系统分析表明，共轭特定于上下文，并且取决于供体，受体，质粒和环境。 已经检查和讨论了作为水平基因转移的位置的环境，但是尚不清楚这种额外抗性菌株的全部程度和影响。我们还试图将我们的体外数据与两个医疗中心的BlakPC+患者的流行病学相关联。我们注意到，令人惊讶的是，体外共轭效率与观察到的混杂间种间扩散无关。 这是结论一些爆发论文的结论，这些爆发论文表明水平转移可能是不同种类的肠杆菌科之间的高效率。接下来，我们通过单分子，实时测序分析了有效和难治的接受者。我们使用了整个基因组序列数据来生成控制共轭的可能机制的假设，从而强调了调节系统的结合。 这些研究整合了流行病学，质粒生物学和基因组学，以对临床上具有重要抗性的革兰氏阴性细菌有新的见解。 医院环境是细菌的潜在储层，其质粒赋予碳青霉烯的耐药性。我们的医院流行病学服务通常会对医院的高触摸表面，水槽和其他位置进行广泛的采样。在两年的时间里，从更广泛的位置进行了额外的抽样，包括家政壁橱，医院内管道的废水和外部人孔。我们将这些数据与先前从五年的患者临床和监视分离株中收集的信息进行了比较。对108个分离株的整个基因组测序和分析提供了BlakPC/BlandM阳性分离株的全面表征，从而实现了深入的遗传比较。令人惊讶的是，尽管患者感染了BLAKPC阳性生物体的患病率非常低，但重症监护病房的所有样品管道废水和外部人孔中含有产生碳酸磷酸盐酶的生物体（CPOS），均表明庞大，弹性的储层。我们观察到了各种各样的物种和质粒，我们注意到了CPO的环境和患者种群之间的物种和易感性谱差异。但是，两个种群之间都有常见的质粒主链，突出了可能有助于抗药性基因传播的移动元件的潜在环境储层。基于序列分析和流行病学，患者和环境分离株之间的明显关联并不常见，这表明我们机构的感染控制合理依从性合理。尽管如此，Leclercia sp。从管家环境到患者，通过这种广泛的监视发现了患者。这些数据并进一步分析了我们对医院环境中CPO的理解，并且与许多基础设施环境中的感染控制策略的设计广泛有关。 我们开发了一种系统，通过在质粒中添加荧光标签和在染色体中添加不同的荧光标签，以跟踪细菌之间质粒的扩散。 我们使用FACS排序来跟踪质粒的转移。 不幸的是，由于该项目的所有成员离开该项目，并建立了一个不相关的独立项目，因此该项目在22财政年度初被终止，该项目将从23财年的数据库中删除。