{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T23:34:01Z","timestamp":1791329641603,"version":"4.3.1"},"reference-count":40,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T00:00:00Z","timestamp":1771372800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Agencia Nacional de Promocion Cientifica y Tecnologica (ANCyPT), Argentina","award":["2021-GRF-TII-00119"],"award-info":[{"award-number":["2021-GRF-TII-00119"]}]},{"name":"Fundacion Cientifica Felipe Fiorellino","award":["80\/21"],"award-info":[{"award-number":["80\/21"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Diabetic foot ulcer (DFU) infections frequently involve biofilm formation and exhibit limited responsiveness to conventional antibiotic therapy. In particular, Pseudomonas aeruginosa often participates in mono- and polymicrobial biofilms that display high tolerance to antimicrobial agents. This study evaluated the efficacy of methylene blue-mediated antimicrobial photodynamic therapy (aPDT), alone and in combination with antibiotics, against P. aeruginosa biofilms formed either as single-species or in mixed communities with Enterococcus faecalis, under conditions mimicking DFU infections. Macrocolony biofilms were challenged with amikacin alone (for single-species biofilms) or amikacin plus ampicillin (for mixed biofilms), aPDT, or sequential combinations of these treatments, and bacterial viability was quantified by colony-forming unit enumeration. Antibiotic treatment alone produced only modest reductions in P. aeruginosa viability, even at high concentrations, while aPDT using methylene blue was effective only at high photosensitizer concentrations. In contrast, sequential treatment with antibiotics followed by aPDT and a second antibiotic challenge resulted in a marked reduction in P. aeruginosa viability in both mono- and polymicrobial biofilms. Scanning electron microscopy revealed extensive structural damage in P. aeruginosa cells following combined treatments, whereas E. faecalis remained unaffected. Overall, our findings demonstrate that combining aPDT with antibiotics significantly enhances antibiofilm activity against P. aeruginosa, highlighting this approach as a promising alternative for the management of biofilm-associated DFU infections.<\/jats:p>","DOI":"10.3390\/pathogens15020226","type":"journal-article","created":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T09:22:46Z","timestamp":1771406566000},"page":"226","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Combined Effects of Amikacin and Methylene Blue-Mediated Photodynamic Therapy on Pseudomonas aeruginosa Biofilms Mimicking Mono- and Polymicrobial Diabetic Foot Ulcer Infections"],"prefix":"10.3390","volume":"15","author":[{"given":"Florencia","family":"Mariani","sequence":"first","affiliation":[{"name":"Laboratorio de Patog\u00e9nesis Bacteriana, Departamento de Investigaciones Bioqu\u00edmicas y Farmac\u00e9uticas, Centro de Estudios Biom\u00e9dicos, Biotecnol\u00f3gicos, Ambientales y Diagn\u00f3stico (CEBBAD), Universidad Maim\u00f3nides, Hidalgo 775, Buenos Aires C1405BCK, Argentina"},{"name":"Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Buenos Aires C1425FQB, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Celeste R.","family":"Costilla","sequence":"additional","affiliation":[{"name":"Laboratorio de Patog\u00e9nesis Bacteriana, Departamento de Investigaciones Bioqu\u00edmicas y Farmac\u00e9uticas, Centro de Estudios Biom\u00e9dicos, Biotecnol\u00f3gicos, Ambientales y Diagn\u00f3stico (CEBBAD), Universidad Maim\u00f3nides, Hidalgo 775, Buenos Aires C1405BCK, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6525-793X","authenticated-orcid":false,"given":"Oscar J.","family":"Oppezzo","sequence":"additional","affiliation":[{"name":"Comisi\u00f3n Nacional de Energ\u00eda At\u00f3mica, Buenos Aires C1429BNP, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8940-0873","authenticated-orcid":false,"given":"Estela M.","family":"Galvan","sequence":"additional","affiliation":[{"name":"Laboratorio de Patog\u00e9nesis Bacteriana, Departamento de Investigaciones Bioqu\u00edmicas y Farmac\u00e9uticas, Centro de Estudios Biom\u00e9dicos, Biotecnol\u00f3gicos, Ambientales y Diagn\u00f3stico (CEBBAD), Universidad Maim\u00f3nides, Hidalgo 775, Buenos Aires C1405BCK, Argentina"},{"name":"Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Buenos Aires C1425FQB, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"209","DOI":"10.2337\/dci22-0043","article-title":"Etiology, epidemiology, and disparities in the burden of diabetic foot ulcers","volume":"46","author":"McDermott","year":"2023","journal-title":"Diabetes Care"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1111\/nyas.13926","article-title":"The role of the microbiome in nonhealing diabetic wounds","volume":"1435","author":"Kalan","year":"2019","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"434","DOI":"10.12968\/jowc.2018.27.7.434","article-title":"Classification, microbiology and treatment of diabetic foot infections","volume":"27","author":"Ghotaslou","year":"2018","journal-title":"J. Wound Care"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"105942","DOI":"10.1016\/j.ijantimicag.2020.105942","article-title":"Hot topics in diabetic foot infection","volume":"55","author":"Saeed","year":"2020","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1080\/08927014.2023.2236949","article-title":"Interspecies interactions in mixed-species biofilms formed by Enterococcus faecalis and Gram-negative bacteria isolated from polymicrobial diabetic foot ulcers","volume":"39","author":"Mariani","year":"2023","journal-title":"Biofouling"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1160","DOI":"10.1111\/iwj.12777","article-title":"Microscopy visualisation confirms multi-species biofilms are ubiquitous in diabetic foot ulcers","volume":"14","author":"Johani","year":"2017","journal-title":"Int. Wound J."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Afonso, A.C., Oliveira, D., Saavedra, M.J., Borges, A., and Sim\u00f5es, M. (2021). Biofilms in Diabetic Foot Ulcers: Impact, Risk Factors and Control Strategies. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22158278"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1128\/CMR.15.2.167-193.2002","article-title":"Biofilms: Survival mechanisms of clinically relevant microorganisms","volume":"15","author":"Donlan","year":"2002","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Pouget, C., Gustave, C.A., Ngba-Essebe, C., Laurent, F., Lemichez, E., Tristan, A., Sotto, A., Dunyach-Remy, C., and Lavigne, J.P. (2021). Adaptation of Staphylococcus aureus in a medium mimicking a diabetic foot environment. Toxins, 13.","DOI":"10.3390\/toxins13030230"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1038\/s44259-024-00046-3","article-title":"Mechanisms of antimicrobial resistance in biofilms","volume":"2","author":"Liu","year":"2024","journal-title":"NPJ Antimicrob. Resist."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e00530-19","DOI":"10.1128\/JB.00530-19","article-title":"\u201cIt Takes a Village\u201d: Mechanisms Underlying Antimicrobial Recalcitrance of Polymicrobial Biofilms","volume":"202","author":"Orazi","year":"2019","journal-title":"J. Bacteriol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Ciofu, O., and Tolker-Nielsen, T. (2019). Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents\u2014How P. aeruginosa Can Escape Antibiotics. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.00913"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"102090","DOI":"10.1016\/j.pdpdt.2020.102090","article-title":"Photodynamic therapy to control microbial biofilms","volume":"33","author":"Warrier","year":"2021","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Cwalinski, T., Polom, W., Marano, L., Roviello, G., D\u2019Angelo, A., Cwalina, N., Matuszewski, M., Roviello, F., Jaskiewicz, J., and Polom, K. (2020). Methylene blue-current knowledge, fluorescent properties, and its future use. J. Clin. Med., 9.","DOI":"10.3390\/jcm9113538"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"tkab008","DOI":"10.1093\/burnst\/tkab008","article-title":"Photodynamic therapy accelerates skin wound healing through promoting re-epithelialization","volume":"9","author":"Yang","year":"2021","journal-title":"Burn. Trauma"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1111\/lam.13762","article-title":"Photodynamic therapy and combinatory treatments for the control of biofilm-associated infections","volume":"75","author":"Ribeiro","year":"2022","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1111\/j.1524-475X.2008.00434.x","article-title":"In vitro multispecies Lubbock chronic wound biofilm model","volume":"16","author":"Sun","year":"2008","journal-title":"Wound Repair Regen."},{"key":"ref_18","unstructured":"CLSI (2018). Performance Standards for Antimicrobial Susceptibility Testing, Clinical and Laboratory Standards Institute. [29th ed.]."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1111\/php.12613","article-title":"A possible phenomenon of persistence in Pseudomonas aeruginosa treated with methylene blue and red light","volume":"92","author":"Hogert","year":"2016","journal-title":"Photochem. Photobiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00103-13","DOI":"10.1128\/mBio.00103-13","article-title":"Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm","volume":"4","author":"Serra","year":"2013","journal-title":"mBio"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.2217\/fmb.12.76","article-title":"Biofilm-specific antibiotic resistance","volume":"7","author":"Mah","year":"2012","journal-title":"Future Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e71077","DOI":"10.1002\/hsr2.71077","article-title":"The Global Challenge of Antimicrobial Resistance: Mechanisms, Case Studies, and Mitigation Approaches","volume":"8","author":"Nazir","year":"2025","journal-title":"Health Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1007\/s00253-024-13009-5","article-title":"In vitro study: Methylene blue-based antibacterial photodynamic inactivation of Pseudomonas aeruginosa","volume":"108","author":"Zada","year":"2024","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1016\/j.pdpdt.2018.03.009","article-title":"Exploring different photodynamic therapy parameters to optimize elimination of Enterococcus faecalis in planktonic form","volume":"22","author":"Soares","year":"2018","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Rubilar-Huenchuman, M., Ortega-Villanueva, C., Gonz\u00e1lez, I.A., and Palavecino, C.E. (2024). The Effect of Photodynamic Therapy on Enterococcus spp. and Its Application in Dentistry: A Scoping Review. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16060825"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1111\/j.1600-0765.2008.01187.x","article-title":"The antibacterial effect of photodynamic therapy in dental plaque-derived biofilms","volume":"44","author":"Fontana","year":"2009","journal-title":"J. Periodontal. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.pdpdt.2015.08.008","article-title":"Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms","volume":"13","author":"Prochnow","year":"2016","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1111\/php.13056","article-title":"Hydrogen Peroxide Enhances the Antibacterial Effect of Methylene Blue-based Photodynamic Therapy on Biofilm-forming Bacteria","volume":"95","author":"Yang","year":"2019","journal-title":"Photochem. Photobiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1007\/978-3-319-09782-4_8","article-title":"Antimicrobial photodynamic therapy for treatment of biofilm-based infections","volume":"831","author":"Biel","year":"2015","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Bueno-Silva, B., Parma-Garcia, J., Frigo, L., Su\u00e1rez, L.J., Macedo, T.T., Uyeda, F.H., Melo, M.A.R.D.C., Sacco, R., Mour\u00e3o, C.F., and Feres, M. (2024). Antimicrobial Activity of Methylene Blue Associated with Photodynamic Therapy: In Vitro Study in Multi-Species Oral Biofilm. Pathogens, 13.","DOI":"10.3390\/pathogens13040342"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.jphotobiol.2016.02.036","article-title":"Synergistic antimicrobial effect of photodynamic therapy and ciprofloxacin","volume":"158","author":"Ronqui","year":"2016","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"101810","DOI":"10.1016\/j.pdpdt.2020.101810","article-title":"Photodynamic therapy using methylene blue, combined or not with gentamicin, against Staphylococcus aureus and Pseudomonas aeruginosa","volume":"31","author":"Ballesta","year":"2020","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"106307","DOI":"10.1016\/j.micpath.2023.106307","article-title":"Photodynamic therapy for ESKAPE pathogens: An emerging approach to combat antimicrobial resistance (AMR)","volume":"183","author":"Mathur","year":"2023","journal-title":"Microb. Pathog."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"113941","DOI":"10.1016\/j.addr.2021.113941","article-title":"Photodynamic and antibiotic therapy in combination against bacterial infections: Efficacy, determinants, mechanisms, and future perspectives","volume":"177","author":"Feng","year":"2021","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2671","DOI":"10.1021\/acsinfecdis.5c00369","article-title":"Synergistic Paradigms in Infection Control: A Review on Photodynamic Therapy as an Adjunctive Strategy to Antibiotics","volume":"11","author":"Soares","year":"2025","journal-title":"ACS Infect. Dis."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"21146","DOI":"10.1038\/s41598-022-25546-y","article-title":"Physicochemical mechanisms of bacterial response in the photodynamic potentiation of antibiotic effects","volume":"12","author":"Soares","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"112499","DOI":"10.1016\/j.jphotobiol.2022.112499","article-title":"Synergic dual phototherapy: Cationic imidazolyl photosensitizers and ciprofloxacin for eradication of in vitro and in vivo E. coli infections","volume":"233","author":"Aroso","year":"2022","journal-title":"J. Photochem. Photobiol. B."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Zangirolami, A.C., Yerra, K.R., Yakovlev, V.V., Blanco, K.C., and Bagnato, V.S. (2024). Combined Antibiotic and Photodynamic Therapies in Pseudomonas aeruginosa: From Synergy to Antagonism. Antibiotics, 13.","DOI":"10.3390\/antibiotics13121111"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1080\/17460441.2019.1588880","article-title":"Omics of antimicrobials and antimicrobial resistance","volume":"14","author":"Chernov","year":"2019","journal-title":"Expert. Opin. Drug Discov."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"101906","DOI":"10.1016\/j.ctcp.2024.101906","article-title":"Efficacy and safety of red and infrared light in the adjunctive treatment on diabetic foot ulcers: A systematic review and meta-analysis","volume":"57","author":"Zhang","year":"2024","journal-title":"Complement. Ther. Clin. Pract."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/2\/226\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T09:28:34Z","timestamp":1771406914000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/2\/226"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,18]]},"references-count":40,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["pathogens15020226"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15020226","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,18]]}}}