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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 \u00b0C and 25 \u00b0C by crystal violet staining. After 48 h, 72% and 65% of Pseudomonas strains formed biofilms at 6 \u00b0C and 25 \u00b0C, respectively, with higher biomass production at 6 \u00b0C. 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) &gt; 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.<\/jats:p>","DOI":"10.3390\/foods14071105","type":"journal-article","created":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T04:48:18Z","timestamp":1742791698000},"page":"1105","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Evaluation of Biofilm Production and Antibiotic Resistance\/Susceptibility Profiles of Pseudomonas spp. Isolated from Milk and Dairy Products"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-3990-1824","authenticated-orcid":false,"given":"Iv\u00e1n","family":"Briega","sequence":"first","affiliation":[{"name":"Departamento de Tecnolog\u00eda de Alimentos, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria (INIA), CSIC, Carretera de La Coru\u00f1a km 7, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5180-766X","authenticated-orcid":false,"given":"Sonia","family":"Garde","sequence":"additional","affiliation":[{"name":"Departamento de Tecnolog\u00eda de Alimentos, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria (INIA), CSIC, Carretera de La Coru\u00f1a km 7, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4212-4928","authenticated-orcid":false,"given":"Carmen","family":"S\u00e1nchez","sequence":"additional","affiliation":[{"name":"Departamento de Tecnolog\u00eda de Alimentos, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria (INIA), CSIC, Carretera de La Coru\u00f1a km 7, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3810-6989","authenticated-orcid":false,"given":"Eva","family":"Rodr\u00edguez-M\u00ednguez","sequence":"additional","affiliation":[{"name":"Departamento de Tecnolog\u00eda de Alimentos, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria (INIA), CSIC, Carretera de La Coru\u00f1a km 7, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6988-5618","authenticated-orcid":false,"given":"Antonia","family":"Picon","sequence":"additional","affiliation":[{"name":"Departamento de Tecnolog\u00eda de Alimentos, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria (INIA), CSIC, Carretera de La Coru\u00f1a km 7, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5487-9856","authenticated-orcid":false,"given":"Marta","family":"\u00c1vila","sequence":"additional","affiliation":[{"name":"Departamento de Tecnolog\u00eda de Alimentos, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria (INIA), CSIC, Carretera de La Coru\u00f1a km 7, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,22]]},"reference":[{"key":"ref_1","unstructured":"Cohen, J., Powderly, W.G., and Opal, S.M. 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