CApEsid biOfilm: a suggested pipeline for clinical phage microbiology for biofilm infections based on comparative method study

The rise of antibiotic-resistant infections, particularly those involving biofilms, presents a significant global health threat. Phage therapy, the use of bacteriophages as antimicrobial agents, offers promising solutions to this crisis. A critical component of phage therapy is the assessment of phage efficacy, in both the presence and absence of antibiotics, prior to clinical application. While considerable progress has been made using planktonic bacterial cultures, there remains an urgent need for standardized methods to evaluate phage efficacy against biofilms. In this study, we address this gap by systematically comparing ten different methods for quantifying phage activity in biofilm settings. Each method was evaluated using a panel of five anti-Pseudomonas aeruginosa phages, which were tested against both planktonic and biofilm cultures. Based on these comparisons, we propose a robust pipeline for detecting phage activity in biofilms. This pipeline, termed CApEsid biOfilm, integrates modified colony-forming unit (CFU) assays using stainless steel washers, crystal violet staining, extracellular DNA quantification using a dye, and extracellular ATP measurements. The pipeline was further validated with additional bacterial species and their respective phages. We also demonstrate its utility in detecting interactions between phages and antibiotics. Overall, this work presents a foundational pipeline that may enhance the clinical matching of phages for treating biofilm-associated infections, thereby improving the outcomes of phage therapy.