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

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.