Bacterial pathogens continue to pose an urgent threat to public health, adapting rapidly to a changing landscape of antibiotic use, and claiming millions of lives worldwide each year. The ongoing emergence and widespread dissemination of new bacterial threats, with increased virulence, antibiotic resistance, and transmissibility, demand urgent attention on a global scale. There are critical gaps in our understanding of the adaptive strategies these pathogens deploy to i) evade the host immune system, ii) respond to therapeutic interventions, and iii) rapidly disseminate locally and globally; thereby, limiting our ability to successfully prevent and treat bacterial infections. Our long-term goal is to build upon our established track record of technological and bioinformatic innovation through the development, optimization and application of cutting-edge sequencing technologies, high-throughput molecular and multi-omic approaches and computational tools to shed new light on bacterial dynamics and their adaptive strategies. Together with our long-standing collaborations with a range of multidisciplinary world-renowned experts, and access to large, unique, systematic collections of pathogens, genomes and clinical data, we are optimally positioned to fill outstanding knowledge gaps for pressing infectious diseases. In this proposal, we will explore the adaptation and spread of high priority bacterial threats across the following three specific aims: i) We will characterize Neisseria gonorrhoeae’s genetic and transcriptional adaptation to anatomical sites of infection and host immune pressures. This will be achieved using long-read sequencing technology paired with an innovative pangenome hybrid selection technique to enrich gonorrhoeal DNA and RNA directly from patient specimens, providing insights into within-host genomic and transcriptomic diversity. ii) We will identify cryptic resistance factors in carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa, where resistance remains unexplained in >20% of cases. For a collection of clinically collected isolates lacking characterized carbapenem resistance mechanisms, we will perform bulk RNA-Seq and a novel single-cell bacterial RNA-Seq approach to elucidate differential expression and transcriptional heterogeneity driving resistance, and functionally test our predictions. iii) We will explore the epidemiological and evolutionary dynamics of plasmids, and their role in disseminating virulence and drug resistance genes. We will accomplish this by applying novel computational algorithms designed to limit the need for expensive long-read data to isolate collections spanning different populations and timescales. The expected outcome of this project is a more complete understanding of the within-host dynamics, adaptation and spread of bacterial pathogens, which will provide critical evidence for the future development of prophylaxis, treatment and infection control measures. In addition, this pro...