We employed high-throughput Viral Tagging and metagenomics to isolate and sequence unculturable phages targeting three AMR bacteria: Helicobacter pylori, Acinetobacter baumannii, and Haemophilus influenzae. This expanded our view of viral diversity, identifying over 100 novel phages. Many viral contigs lacked known taxonomies, highlighting current database limitations. These phages displayed broad genomic diversity and clear phylogenetic relationships, forming a valuable database for studying phage biology and antibacterial mechanisms.
Focusing on A. baumannii, we investigated infection strategies using a multi-omics approach (genomics, transcriptomics, proteomics, and metabolomics). Phage infection led to major shifts in host metabolism and a 20-fold increase in phage gene expression—many genes being ORFans with unknown function. We identified >100 phage-specific proteins/metabolites, varying by infection stage and host physiology. Notable changes included upregulation of Glutaredoxin and modulation of L-Methionine, L-Leucine, L-beta Homoproline, and Betaine. Network analyses revealed coordinated changes across omics layers, particularly in fatty acid, nucleotide, and amino acid metabolism, indicating widespread phage-driven host reprogramming.
Multiple Factor Analysis showed transcriptomics drove temporal variation, while proteomics and metabolomics aligned with infection stage and nutrient availability. Several ORFan genes, despite lacking known homologs, caused strong changes in host gene expression and physiology when expressed individually—three showed potent antibacterial activity and are under further study. This highlights the functional potential of viral “dark matter.”
Together, these data show phages rewire host physiology across biological layers. Our integrated profiling provides a robust blueprint for identifying functional phage genes and their roles in infection and therapeutic applications. Several manuscripts are in preparation, with expected major contributions to phage-host interaction and novel antibacterial discovery.
We also addressed COVID-19-related AMR by developing a phage cocktail against co-infections in pneumonia patients, targeting Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens. The cocktail showed diverse infection strategies and strong host-killing potential. Viral Tagging of these pathogens revealed high phage diversity, complicating bacterial resistance development. These findings support the therapeutic potential of the cocktail and inform future early-phase clinical trials.
