Phenotypic characterization of raw milk-associated Pseudomonas spp.: biofilm formation across different temperatures, antimicrobial resistance, and cytotoxicity.

This study investigated biofilm formation, antimicrobial resistance, and in vitro cytotoxicity in 31 Pseudomonas isolates recovered from raw milk, and assessed relationships among these traits. Biofilm formation was evaluated at 7, 15, 25, 37, and 42 °C using Congo Red Agar (CRA) and Crystal Violet (CV) assays. Antimicrobial susceptibility was determined by disk diffusion method, and cytotoxicity on Caco-2 cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Associations were assessed using Spearman's rank correlation with Benjamini-Hochberg false discovery rate (FDR) correction. The highest frequency of CRA-positive phenotypes occurred at 25 °C (90.3%), while all isolates were classified as biofilm producers by CV at this temperature; biofilm formation was also widely observed at 7 and 15 °C. Some isolates provisionally assigned to P. veronii, P. brenneri, and P. fluorescens showed strong biofilm-associated phenotypes in both CRA and CV assays at low temperatures. Cytotoxicity ranged from 0.11% to 42.8%. Multidrug resistance was detected in 90.3% of isolates, with a mean multiple antibiotic resistance (MAR) index of 0.52. CRA phenotypes were negatively correlated with cytotoxicity at 15 and 25 °C (P < 0.05). No significant associations were found between cytotoxicity and MAR index, individual antibiotic susceptibility, or quantitative CV measurements (P > 0.05). Most nominal associations between biofilm formation and individual antibiotics did not remain significant. These findings highlight substantial phenotypic diversity among raw milk-associated Pseudomonas and the ability of many isolates to maintain biofilm-forming phenotypes under dairy-relevant temperatures.