Psychrotrophic Pseudomonas spp. contaminate milk and dairy products, producing heat-resistant enzymes and pigments that reduce their shelf life. Identifying spoiling strains is essential for tracing contamination and improving preservation. This study examines 208 Pseudomonas spp. isolates from Spanish dairy products, assessing their genetic diversity and spoilage potential in vitro and under simulated storage. Pulsed-field gel electrophoresis (PFGE) identified 108 distinct strains, clustered into 70 groups (≥ 80 % similarity), showing high diversity. Gene sequencing (ileS or rpoD) classified the strains into 20 species, with P. fluorescens (19 %), P. fragi (16 %), P. lundensis (12 %), and P. shahriarae (6 %) being predominant. P. shahriarae, P. atacamensis, P. salmasensis, P. solani, and P. canadensis were isolated from milk or dairy products for the first time. In fresh cheese, 89 % of Pseudomonas spp. caused discoloration (fluorescent yellow, creamy, orange, and blue) after 7 days of cold storage. In refrigerated milk, 48 % of strains showed significant proteolysis after 5 days, with OPA (o-Phthaldialdehyde-based method) values > 0.274, especially some P. fluorescens, P. gessardii, P. fulva, P. shahriarae, Pseudomonas spp., and P. koreensis strains. After simulated UHT treatment and accelerated storage, 75 % of strains retained thermostable proteolytic activity, especially P. fluorescens, P. proteolytica, P. shahriarae, and P. fulva strains. The aprX gene, coding for a thermostable protease, was present in 57 % of strains, suggesting other proteases may also be produced. Overall, the isolated Pseudomonas spp. led to different spoilage patterns during milk and fresh cheese storage, emphasizing the need for specific strain identification to improve preservation strategies.

Dairy-borne Pseudomonas spp., known for causing spoilage, may also exhibit antibiotic resistance and form biofilms, enhancing their persistence in dairy environments and contaminating final products. This study examined biofilm formation and antibiotic resistance in 106 Pseudomonas spp. strains isolated from milk, whey, and spoiled dairy products. Phylogenetic analysis (based on partial ileS sequences) grouped most strains within the P. fluorescens group, clustering into the P. fluorescens, P. gessardii, P. koorensis, and P. fragi subgroups. Biofilm formation in polystyrene microplates was assessed at 6 °C and 25 °C by crystal violet staining. After 48 h, 72% and 65% of Pseudomonas strains formed biofilms at 6 °C and 25 °C, respectively, with higher biomass production at 6 °C. High biofilm producers included most P. fluorescens, P. shahriarae, P. salmasensis, P. atacamensis, P. gessardii, P. koreensis, and P. lundensis strains. The adnA gene, associated with biofilm formation, was detected in 60% of the biofilm producers, but was absent in P. fragi, P. lundensis, P. weihenstephanensis, and P. putida. Antibiotic susceptibility was tested using the disk diffusion method. All strains were susceptible to amikacin and tobramycin; however, 73% of the strains were resistant to aztreonam, 28% to imipenem and doripenem, 19% to ceftazidime, 13% to meropenem, and 7% to cefepime. A multiple antibiotic resistance index (MARI) > 0.2 was found in 30% of the strains, including multidrug-resistant (n = 15) and extensively drug-resistant (n = 3) strains. These findings highlight Pseudomonas spp. as persistent contaminants and antibiotic resistance reservoirs in dairy environments and products, posing public health risks and economic implications for the dairy industry.