Benchmarking biofilm assays, surface models, and detachment methods in Pseudomonas aeruginosa for reproducible downstream molecular analysis.

Pseudomonas aeruginosa is a major ESKAPE pathogen and a WHO priority pathogen, but biofilm studies remain difficult to compare because detection methods, test surfaces, strain selection, and biofilm recovery procedures vary widely across laboratories. This study evaluated three linked methodological questions: phenotypic biofilm detection, surface-associated expression of selected biofilm genes, and biofilm detachment for downstream molecular sample preparation. Eighty-four clinical isolates and the reference strain, PAO1, were screened using three phenotypic assays, and four biofilm-related genes (algR, lecA, pelA, and pslG) were assessed across six surface types in representative strains. The four biofilm removal methods were compared based on viable cell recovery and the release of cellular components. Clinical isolates displayed three distinct biofilm-forming profiles: consistently strong, surface-independent moderate/low, and highly variable producers. Surfaces such as latex and polyvinyl chloride (PVC) induced the highest expression of algR and lecA up to 3.33-fold and 4.21-fold, respectively, in strain PS120. The study revealed that surface type was the dominant factor influencing gene expression (42.6% variance), followed by strain type (17.8%) and gene identity (4.0%). Among the biofilm detachment methods, mechanical scraping provided the highest viable cell recovery (2 × 108 cfu/mL) and showed comparatively lower cellular-component release than sonication. Overall, this study provides a practical methodological framework for selecting complementary biofilm assays, reproducible surface models, and recovery methods for downstream molecular analysis of P. aeruginosa biofilms.