Liquid-phase milk turbidimetry enables rapid phenotyping of secreted protease activity in Pseudomonas aeruginosa at less than one cent per sample

ABSTRACT Secreted proteases are major virulence determinants in Pseudomonas aeruginosa . Their production varies widely across strains and quorum-sensing (QS) backgrounds, creating the need for simple, standardized methods to quantify extracellular proteolytic activity. Here, a liquid-phase skim-milk turbidimetric assay is described to quantify total secreted protease activity in clarified culture supernatants using a Tris–CaCl₂ buffer and turbidity loss at 600 nm (A600) as the spectrophotometric endpoint. Reactions were incubated at 37°C while shaking for 3 h before measurement. Reference strains PAO1 and PA14, a QS-deficient double mutant (PAO1Δ lasR Δ rhlR ), and two additional isolates (DVL1758 and ATCC 9027) were evaluated under assay-matched growth conditions. Endpoint culture density was comparable across strains (OD600 ~ 3.4–4.0; one-way ANOVA, P > 0.05), supporting the interpretation of A600 differences as extracellular activity phenotypes. The assay resolved strain-dependent milk clearance at two practical substrate loads (2 mg and 5 mg), distinguishing low-, intermediate-, and high-proteolysis phenotypes, with the QS mutant exhibiting minimal clearance. Intralaboratory precision was acceptable for a biological assay (CV < 20% across strains and loads). Robustness across loads was supported by strong agreement between mean A600 values at 2 mg and 5 mg (Pearson r = 0.861, P = 0.0277, R ² = 0.741; excluding medium control r = 0.954, R ² = 0.910). This low-cost, widely accessible workflow enables rapid comparative profiling of secreted proteolytic phenotypes and complements more specific substrate-based or orthogonal validation assays when mechanistic resolution is required. IMPORTANCE Understanding how bacteria produce enzymes that break down proteins is key to studying infection and developing new treatments. Pseudomonas aeruginosa , an important opportunistic pathogen, secretes proteases that contribute to tissue damage and disease progression. However, measuring total protease activity across strains remains technically challenging, costly, or difficult to standardize. Here, we present a simple, low-cost liquid-phase milk turbidimetry assay that enables rapid and accessible quantification of secreted protease activity using standard laboratory equipment. This method reliably distinguishes between strains with different proteolytic phenotypes, including quorum-sensing-deficient mutants, and demonstrates good reproducibility and robustness across conditions. Because it uses widely available reagents and requires minimal processing, this assay lowers barriers for routine phenotyping and large-scale screening. Our approach provides a practical tool for studying virulence, regulatory mechanisms, and potential anti-virulence strategies in P. aeruginosa and related organisms.