Endemic and emerging species of viruses, bacteria, and fungi pose a continuous threat to public health. Pandemics may arise from microbial evasion of population immunity, available therapeutics, and detection methods. In addition to managing the ecological drivers of spillover, we must have greater scrutiny of potential pathogens currently circulating in humans, animals, and the environment. Monitoring for unknown pathogens and pathogenic mechanisms is a particular challenge that new, genomics-driven diagnostic methodologies have the potential to address. My thesis work explores and provides proof of concept for two such approaches. First, I contributed to development and bench validation of a novel hybrid capture based next-generation sequencing diagnostic panel for all known viral tick-borne pathogens, Targeted Identification of Tick-borne Analytes by hybrid-capture NGS for RNA (TITAN-RNA). Systematic testing with simulated novel viruses and field samples revealed a 10% tolerance for evenly distributed mutations, or 27% tolerance for natural viral divergence. Its analytic performance is amenable for quantitative viral pathogen-agnostic clinical testing in humans and animals, and it detected putatively novel segmented Flavi-like viruses in an invasive tick vector, Haemaphysalis longicornis. Second, I investigated a modified group 1 capsular locus associated with multidrug resistance from surveillance of over 4,000 pathogenic clinical isolates of E. coli from sick dogs across the US and Canada. Using targeted mutagenesis and antibiotic susceptibility testing under different conditions, I assessed the mechanistic hypothesis that this locus had been repurposed as an antibiotic trap. Transcriptomic analysis revealed co-expression of both modified Group 1 and Group 4 capsular loci in select isolates, providing new insight into capsule regulation. The contribution of various β-lactamases was assessed in relation to cephalosporin resistance. We also corroborated that the colanic acid associated WzaCA can function as a porin for antibiotic import, conferring antibiotic susceptibility and a potential new avenue for therapeutic intervention.