Dissecting HOCl Action in Chronic Wound Biofilms: Proteomic Insights From a Host‐Relevant Model of Pseudomonas aeruginosa

Pseudomonas aeruginosa is found in 48%–52% of chronic wound biofilms, where its resistance to antimicrobials and host immunity presents a major clinical challenge. Although hypochlorous acid (HOCl) is known to be an effective antimicrobial, its mechanism of action remains unclear because standard experimental conditions often produce a mixture of HOCl and hypochlorite (OCl⁻), making it difficult to isolate the effects of HOCl. Here, we use proteomic profiling to investigate the effects of a pure, stable HOCl gel on P. aeruginosa biofilms in a physiologically relevant chronic wound model. We applied HOCl gel (5.7 mM, pH 6) to mature P. aeruginosa biofilms established in a wound‐mimicking flow model. Proteins were analyzed using tandem mass tag (TMT)‐based quantitative proteomics, identifying 1,878 proteins. HOCl treatment significantly reduced biofilm viability and altered the abundance of 330 proteins. We observed substantial depletion of proteins involved in biosynthesis, virulence, antibiotic resistance, and biofilm formation, alongside enrichment of stress response proteins. These findings indicate a shift toward survival phenotypes and weakened pathogenicity. Our data reveal that HOCl disrupts multiple pathways essential for P. aeruginosa survival and virulence. Crucially, our experimental design eliminates confounding factors that can lead to unintentional testing of mixed HOCl and OCl⁻ species, allowing us to assess the specific effects of HOCl. These findings call for a re‐evaluation of HOCl research methodologies and reiterate the importance of realistic infection models in antimicrobial testing.