Abstract
Pseudomonas aeruginosa is a major contributor to persistent chronic infections in clinical practice, owing to its robust biofilm formation capacity and frequent antimicrobial resistance acquisition. However, most current studies focus on single strains and thus overlook phenotypic differences among coexisting strains within the same host. With that in mind, we proposed a hypothesis that P. aeruginosa strains from the same patient, yet with distinct genetic backgrounds, might exhibit differing resistance profiles and virulence genes. To test this hypothesis, we selected three strains with different sequence types (STs), all isolated from the chronic wounds of a patient with long-term bilateral lower limb infections. By employing multilocus sequence typing, antimicrobial susceptibility testing, biofilm gene quantification, growth kinetics assays, Galleria mellonella virulence experiments, and phylogenetic reconstruction, we systematically evaluated the relationships between these strains'biofilm formation and virulence. The results revealed significant genetic diversity and evolutionary origin variations among the three strains. Notably, ST2584 (WYDPA-23-3) exhibited multidrug resistance (resistant to 7 of the 12 tested antibiotics) and the highest growth rate, whereas ST270 (WYDPA-23-2)-despite the downregulation of pelA, a gene linked to extracellular matrix biogenesis-demonstrated a 2.3-fold increase in biofilm formation and the highest larval lethality. By comparing multiple strains coexisting in the same host, this study further elucidates the role of P. aeruginosa biofilm in sustaining chronic infections and offers valuable guidance for optimizing clinical treatment strategies and antibiotic selection. In light of these findings, developing rapid and precise biofilm detection methods and designing innovative drugs targeting high biofilm-producing strains should be prioritized.