Periodic Reporting for period 4 - PHARMS (Bacteriophage inhibition of antibiotic-resistant pathogenic microbes and founding for novel therapeutic strategies)
Reporting period: 2023-07-01 to 2024-12-31
Antimicrobial resistance (AMR) poses a major global threat, projected to cause more deaths than cancer, diabetes, and cardiovascular diseases by 2050 if no new therapies emerge. Only one new antibiotic class has reached the market in the past two decades, as traditional targets are exhausted and quickly resisted. Innovative therapies beyond conventional antibiotics are urgently needed. Phage therapy—a natural antibacterial strategy using bacteriophages or their derivatives—offers promise but faces key limitations: the scarcity of phage isolates and limited understanding of their molecular mechanisms. Building on our recent advances in high-throughput, host-targeted, culture-independent methods, PHARMS aims to screen all potential phages against resistant isolates, characterize their bactericidal functions, and design multifaceted phage-based therapies. By elucidating universal and pathogen-specific mechanisms, PHARMS provides a rational foundation for new therapeutic strategies against common and severe infections.
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.
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.
PHARMS promises to reshape AMR control paradigms by elucidating phage bactericidal mechanisms, currently a “black box” in microbiology. The project provides systematic insights into how phages inhibit AMR bacteria and functionally characterize candidate therapeutic genes. These efforts are expected to uncover novel mechanisms of bacterial killing and reveal how phages regulate host processes across AMR pathogens. The resulting tools may yield superior therapeutic products and inform applications beyond therapy—such as metabolic engineering, synthetic biology, and cell cycle control—reaching impact levels akin to technologies like CRISPR/Cas. Ultimately, PHARMS advances basic science while accelerating clinical readiness for phage-based antimicrobials.