PMI TechNote: Bacteriophages-From the Petri Dish to Conquering the Nooks and Crannies of Difficult-to-Treat Infections.

Bacterial infections present a challenge in clinical settings due, in part, to their remarkable diversity in physiological context and treatment resistance. Bacteriophage viruses (phages) offer renewed therapeutic promise, yet their performance in vivo often differs from expectations based on standard laboratory assays. Biofilms, with their dense extracellular matrix and spatial heterogeneity, restrict phage access, diffusion, and replication. Anaerobic niches and metabolically dormant bacterial subpopulations at the biofilm core pose additional hurdles, often rendering bacteria tolerant to both antibiotics and phages. These physiological constraints are rarely captured during phage isolation, where agar-based plaque assays, oxygen-rich conditions, and rapidly growing hosts inadvertently deselect phages adapted to biofilms, anaerobic zones, or dormant states. Understanding how phage-bacterial interactions vary across these niches and how antibiotics synergize with or antagonize phage activity is essential for designing therapies with improved effectiveness beyond the Petri dish. This TechNote synthesizes key challenges for phage infection dynamics in biofilms, anaerobic environments, and dormant populations, highlighting the need for isolation and screening strategies that better reflect the physiology of clinical infections.