Our work in this area has three broad areas of focus: (1) Development of rapid nanopore-based strategies to sequence bacterial resistance plasmids with clinical diagnostic applications; (2) Use of these and other sequencing strategies to characterize genomic mechanisms of antimicrobial resistance in clinical isolates; and (3) Study of the genomic mechanisms controlling the emergence of antimicrobial resistance in the course of acute and chronic clinical infection. These areas of focus seek both to evaluate sequencing methods that can be used practically in a clinical microbiology lab and to study the biology of antimicrobial resistance, its emergence, and its expression. Nanopore sequencing methods provide ultrafast, read-by-read data availability that may be used to support real-time infectious disease diagnostics, including rapid sequencing of bacterial isolates and resistance gene identification in the clinical microbiology lab. Work done during the current fiscal year developed a practical nanopore sequencing workflow that takes as input purified plasmid DNA and can produce full annotations of all plasmid-based resistance genes within a working day, starting with subcultured isolates. Current work is in progress to extend these methods to allow sequencing of resistance plasmids from single bacterial colonies from primary cultures. The evolution of pathogenic MDR phenotypes in the natural in vivo context of acute and chronic infection is being studied through genomic analysis of serially-collected human clinical bacterial isolates. Four initial isolate collections have been assembled along with comprehensive microbiologic characterization, with a focus on evolution of MDR phenotypes in pathogenic non-fermenting gram negative proteobacteria (Pseudomonas, Burkholderia, Achromobacter, and Bordetella). Work done during the 2018 fiscal year focused on how evolved mismatch repair deficiencies in P. aeruginosa resulting in hypermutator phenotypes may facilitate the emergence of resistance to ceftazidime-avibactam in vivo. This work included in vitro adaptive evolution experiments in combination with evolutionary genomic analysis. Other work during the 2018 fiscal year examined pathoadaptation and the emergence of resistance phenotypes in Bordetella sp. This project has focused on intra-host evolution occurring following zoonotic infection of an immunocompromised host with a non-human Bordetella species, revealing unexpected roles that unusual compound hypermutator phenotypes may play in rapid evolution of multidrug resistant isolates.
