Genomic- informed development of a cheap, portable assay to rapidly profile drug-resistant bacterial infections - Project Summary Antimicrobial-resistant (AMR) infections are a global health crisis, causing nearly 5 million deaths annually, with the highest burden in low- and middle-income countries (LMICs). Carbapenem-resistant Enterobacterales (CRE) are among the most dangerous AMR pathogens because they are resistant to nearly all antibiotics. CRE acquire this resistance mainly through two mechanisms: i) carbapenemase production and ii) changes in their cell structure (porin deficiency) that prevent antibiotics from entering the bacteria. Although porin deficiency causes about 15% of CRE, the exact impact of different porin mutations—especially in regions like Latin America—remains poorly understood. Additionally, the lack of rapid, affordable diagnostic tools in these areas delays effective treatment, leading to higher mortality, especially in cases of bloodstream infections. To address these challenges, I developed BADLOCK, a CRISPR-based diagnostic test that can rapidly identify bacterial species and AMR genes from blood cultures. BADLOCK is affordable, portable, and designed for resource-limited settings, aligning with World Health Organization recommendations. Preliminary data show it can accurately detect common bacteria and resistance genes, making it a promising tool for use in LMICs. This proposal focuses on characterizing porin mutations and expanding BADLOCK’s capabilities. I will sequence the genomes of a completed cohort of over 500 Enterobacterales isolates from Peru to identify porin mutations (among other AMR determinants), assess their contribution to resistance through genomic comparisons with other CRE cohorts, and conduct lab experiments to confirm the role of these mutations. CRISPR guides will be developed using novel computational strategies to target diverse species, common AMR genes, and validated porin mutations, thereby improving BADLOCK’s utility. Finally, it will be piloted in Peruvian hospitals to evaluate its performance in real-world settings. This project will improve diagnostic capacity in resource-limited settings and create the first catalog of isogenic strains with porin mutations. Additionally, it will provide critical insights into resistance mechanisms and support more effective management of bacterial infections in LMICs. As an infectious disease physician with an extensive background in bacterial genomics, I am well-poised to carry out these research goals. However, this proposal includes an ambitious training and career development plan that will further enhance the molecular biology and global health research skills necessary to launch my career as an independent, R01-funded investigator. The robust scientific and educational resources of Harvard Medical School and the Broad Institute provide an excellent training environment, and I have built a mentorship team consisting of world leaders in bacterial genomics, infectious disease diagnostics, and global health – all of whom are committed to supporting me in my success as an independent researcher.
