CAREER: Environmental monitoring of antibiotic resistance using targeted long-read sequencing

世界卫生组织已将抗生素耐药性确定为人类面临的十大全球公共卫生威胁之一。抗生素耐药性（AR）可以通过携带耐药性的整个细菌的转移以及细菌之间的水平基因转移在人类、动物和环境中传播。目前迫切需要能够防止AR在全球蔓延的解决方案和干预措施。然而，对抗生素耐药菌（ARB）和抗生素耐药基因（ARGs）在社区环境中转移的途径和机制的基本理解仍然难以捉摸。本CAREER项目的总体目标是开发和验证一种适用于现场的方法，用于大规模环境监测和识别ARB和ARGs的传播途径。为了推进这一目标，首席研究员建议探索利用靶向长读DNA测序来开发一种低成本和不依赖培养的宏基因组学平台，用于在人群水平上对AR进行全球监测，该平台不需要昂贵的设备或高性能计算资源。这一项目的成功完成将为建立更有效和更具成本效益的环境监测平台奠定基础，从而在资源匮乏的环境中监测抗生素耐药性的全球传播，从而使最需要监测的社区能够进行监测，从而造福社会。进一步的社会效益将通过学生教育和培训来实现，包括在加州大学伯克利分校指导一名研究生和一名本科生。长读元基因组学（例如，纳米孔测序）的进展为开发更有效和更具成本效益的检测/平台提供了新的机会，这些检测/平台可以在其基因组背景下识别抗生素耐药基因（ARGs），目的是揭示抗生素耐药（AR）转移的机制和途径，包括细菌宿主之间的水平基因转移。然而，当前长读取AR测序的一个关键限制是，大部分测序都浪费在非信息性读取上，这反过来限制了环境样品中低丰度ARGs的深度和覆盖范围。为了解决现有AR基因组测序工具/方案的这一关键限制，该CAREER项目的首席研究员（PI）建议开发并验证一种靶向测序方法，以实现ARGs及其侧翼区域的富集，目的是促进检测宏基因组样本中低丰度ARG靶点及其基因组背景（例如细菌宿主或质粒载体）。这个项目的成功完成可能会产生变革性的影响。通过将基于crispr的富集与便携式长读测序相结合（即使用cas9引导RNA复合物切割感兴趣的基因，然后将长读测序适配器连接到被切割的基因位点），PI希望为更有效和更具成本效益的元基因组学监测平台建立基础，以监测抗生素耐药性的全球传播。为了实现该CAREER项目的教育和培训目标，PI将与加州大学伯克利分校DCAC（目的地大学咨询团）合作，为本科生和高中生开发和实施一个课堂研究模块，该模块使用便携式DNA测序技术检测学生在其社区收集的土壤样本中具有临床重要性的ARGs。此外，PI计划定制AR测序课堂研究模块，以开发和传播一个公民科学平台，使全世界的教师和学生能够通过与PARE（环境中抗生素耐药性流行）项目合作开发一个开放源代码的土壤宏基因组数据库，为全球AR监测工作做出贡献。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The World Health Organization has identified antibiotic resistance as one of the top ten global public health threats facing humanity. Antibiotic resistance (AR) can spread across humans, animals, and the environment through the transfer of whole bacteria carrying resistance as well as horizontal gene transfer between bacteria. Solutions and interventions that can prevent the global spread of AR are urgently needed. However, a fundamental understanding of the pathways and mechanisms of transfer of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in community settings has remained elusive. The overarching goal of this CAREER project is to develop and validate a field compatible method for large-scale environmental monitoring and identification of the transmission pathways of ARB and ARGs. To advance this goal, the Principal Investigator proposes to explore the utilization of targeted long-read DNA sequencing to develop a low-cost and culture-independent metagenomics platform for global monitoring of AR at the population level that would not require costly equipment or access to high performance computing resources. The successful completion of this project will benefit society by laying the foundation for more efficient and cost-effective environmental surveillance platforms to monitor the global spread of antibiotic resistance in low-resource settings thereby enabling surveillance in communities where it is often most needed. Further benefits to society will be achieved through student education and training including the mentoring of a graduate student and an undergraduate student at the University of California, Berkeley. Advances in long-read metagenomics (e.g., Nanopore Sequencing) are providing new opportunities to develop more efficient and cost-effective assays/platforms that could identify antibiotic resistant genes (ARGs) in their genomic context with the goal of unraveling the mechanisms and pathways of antibiotic resistance (AR) transfer including horizontal gene transfer between bacterial hosts. However, a critical limitation of current long-read AR sequencing is that most of the sequencing is wasted on non-informative reads, which in turns limits the depth and coverage of low-abundance ARGs in environmental samples. To address this critical limitation of existing AR genomic sequencing tools/protocols, the Principal Investigator (PI) of this CAREER project proposes to develop and validate a targeted sequencing approach to enable the enrichment of ARGs and their flanking regions with the goal of facilitating the detection of low-abundance ARG targets and their genomic context (e.g., bacterial hosts or plasmid carriers) in metagenomic samples. The successful completion of this project has potential for transformative impact. By combining CRISPR-based enrichment with portable long-read sequencing (i.e., using a Cas9-guide RNA complex to cleave genes of interest followed by the ligation of long-read sequencing adapters to the cleaved gene sites), the PI hopes to build the foundation for more efficient and cost-effective metagenomics surveillance platforms to monitor the global spread of antibiotic resistance. To implement the educational and training goals of this CAREER project, the PI will partner with the UC-Berkeley DCAC (Destination College Advising Corps) to develop and implement a classroom research module for undergraduate and high-school students that employs portable DNA sequencing technologies to detect ARGs of clinical importance in soil samples collected by the students in their communities. In addition, the PI plans to tailor the AR sequencing classroom research module to develop and disseminate a citizen science platform that will enable teachers and students worldwide to contribute to a global AR surveillance effort through the development of an open-source database of soil metagenomes in collaboration with the PARE (Prevalence of Antibiotic-Resistance in the Environment) project.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.