Rapid assays for diagnostic typing of Klebsiella capsules and rational design of therapeutic phage cocktails

PROJECT 2: RAPID ASSAYS FOR DIAGNOSTIC TYPING OF KLEBSIELLA CAPSULES AND RATIONAL DESIGN OF THERAPEUTIC PHAGE COCKTAILS Project Summary/Abstract Inter-strain variation within pathogenic bacterial species presents a major challenge to the effective deployment of phage therapy as a solution to the growing antimicrobial resistance (AMR) crisis. Klebsiella pneumoniae (Kp), a key contributor to AMR gene dissemination, exemplifies this challenge with its extensive genetic and phenotypic diversity, particularly in its capsular polysaccharides (K-antigens), which serve as the principal determinants of phage susceptibility. The lack of scalable, standardized methods to characterize inter-strain variation hinders the development of rationally designed phage cocktails that can effectively target diverse clinical Kp strains. This project aims to develop robust, high-throughput assays that comprehensively characterize strain-to-strain variability and its impact on phage efficacy. In Aim 1, we will develop a multiplexed PCR-based DNA microarray to rapidly and precisely identify Kp capsule types across diverse clinical strains. The proposed assay will capture the full genetic diversity of K-antigen loci, providing a scalable and cost-effective method to molecularly "type" Kp strains without the need for whole-genome sequencing or culture. This approach will facilitate rapid stratification of strains based on their phage susceptibility profiles, addressing a critical gap in current clinical phage therapy workflows. In Aim 2, we will establish pooled functional assays to dissect the genomic factors underlying inter-strain differences in phage susceptibility. By introducing a dual-barcoded transposon system into a panel of genetically diverse Kp strains and expressing thousands of phage accessory genes (AGs) in each strain, we will create a highly multiplexed experimental framework to study the complex interactions that define phage host-range. The resulting high-dimensional dataset will be used to train AI models to predict strain-specific phage compatibility, providing actionable insights for personalized phage therapy. In Aim 3, we will develop an organoid-based infection model to assess how strain variation influences phage efficacy in a physiologically relevant human lung environment. We will perform pooled phage sensitivity screens to evaluate how exposure to host-specific factors alters capsule expression and phage susceptibility, revealing strain-dependent adaptation mechanisms that could impact phage therapeutic success in clinical settings. By directly addressing inter-strain variation at multiple levels-genetic, phenotypic, and environmental-our work will provide standardized tools to systematically catalog and predict phage susceptibility across diverse clinical isolates. The proposed assays will establish a foundational framework for precision phage therapy, applicable not only to Kp but also to other ESKAPE pathogens of critical concern.