Characterization and treatment of polymicrobial biofilms in prosthetic joint infections.
8 affiliations
- 1Clinical Microbiology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Autonomous University of Madrid (IIS-FJD, UAM), Avenida de los Reyes Católicos, 2, 28040, Madrid, Spain.
- 2Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Faculty of Engineering, Associate Laboratory in Chemical Engineering (ALiCE), University of Porto, Rua Dr. Roberto Frias s/n, 4200-465, Porto, Portugal.
- 3Centro de Astrobiología (CAB), CSIC-INTA, Calle Ajalvir, 28864, Torrejón de Ardoz, Madrid, Spain.
- 4Clinical Microbiology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Autonomous University of Madrid (IIS-FJD, UAM), Avenida de los Reyes Católicos, 2, 28040, Madrid, Spain. [email protected].
- 5CIBERINFEC-CIBER de Enfermedades Infecciosas, Calle Monforte de Lemos, 3-5, Pabellón 11, Planta 0, 28029, Madrid, Spain. [email protected].
- 6Department of Preventive Medicine, Faculty of Medicine, Public Health and Microbiology, Autonomous University of Madrid, Calle Arzobispo Morcillo, 4, 28029, Fuencarral-El Pardo, Madrid, Spain. [email protected].
- 7CIBERINFEC-CIBER de Enfermedades Infecciosas, Calle Monforte de Lemos, 3-5, Pabellón 11, Planta 0, 28029, Madrid, Spain.
- 8Faculty of Biomedical and Health Sciences, Alfonso X El Sabio University (UAX), Avenida Universidad, 1, 28691, Villanueva de La Cañada, Madrid, Spain.
Abstract
Prosthetic joint infections are complex and associated with high economic and social costs. Polymicrobial cases remain poorly understood, particularly regarding bacterial interactions and antibiotic response within mixed-species biofilms. We introduce two approaches for characterizing polymicrobial biofilms: a predictive modeling equation and fatty acid profiling. Predictive modeling estimates biofilm behavior. Fatty acid profiling provided complementary information on species dominance and possible interspecies interactions. We integrate these results with fluorescence in situ hybridization and confocal microscopy to obtain a comprehensive view of biofilm structure and composition. Using this multi-modal approach, we characterize mono-, dual-, and triple-species biofilms formed by Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. In parallel, we evaluated common anti-biofilm antibiotics, alone and in combination, across all biofilm types. The model explained 72.9% of the variance in triple-species biofilm formation (R² = 0.73). K. pneumoniae dominated all mixed biofilms in which it was present, accounting for approximately 70-80% of the bacterial population. In P. aeruginosa-S. aureus biofilms, S. aureus outnumbered P. aeruginosa (93% vs. 7%). Antibiotic efficacy varied with biofilm composition and structure. Colistin plus levofloxacin was the most effective combination, achieving reductions of up to 7.5 log₁₀ CFU/cm². S. aureus drove biofilm persistence; no treatment achieved eradication in mixed biofilms containing this species, with median reductions remaining below the 5 log₁₀ CFU/cm² threshold. Confocal microscopy revealed that S. aureus was predominantly located in the deepest biofilm layers. These results support two complementary approaches for characterizing polymicrobial biofilms through structural, predictive, and biochemical analyses and highlight the importance of biofilm architecture and interspecies interactions when evaluating antibiotic efficacy in polymicrobial infections. KEY POINTS: • Modeling and fatty acid analysis predict polymicrobial biofilm behavior • K. pneumoniae dominates biofilms, while S. aureus drives persistence • Antibiotic efficacy depends on biofilm composition and structure.
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BibTeX
@article{torrecillasadaba2026characterization,
title = {{Characterization and treatment of polymicrobial biofilms in prosthetic joint infections.}},
author = {Estíbaliz Torrecilla-Sádaba and Andreia S Azevedo and Daniel Carrizo and Isadora Maia and Laura Sánchez-García and Nuno F Azevedo and Jaime Esteban and Llanos Salar-Vidal and John Jairo Aguilera-Correa},
journal = {Applied microbiology and biotechnology},
year = {2026},
month = aug,
doi = {10.1007/s00253-026-13985-w},
url = {https://europepmc.org/article/MED/42967945},
note = {CC BY},
}
RIS
TY - JOUR
TI - Characterization and treatment of polymicrobial biofilms in prosthetic joint infections.
AU - Estíbaliz Torrecilla-Sádaba
AU - Andreia S Azevedo
AU - Daniel Carrizo
AU - Isadora Maia
AU - Laura Sánchez-García
AU - Nuno F Azevedo
AU - Jaime Esteban
AU - Llanos Salar-Vidal
AU - John Jairo Aguilera-Correa
PY - 2026
DA - 2026/08/04
JO - Applied microbiology and biotechnology
DO - 10.1007/s00253-026-13985-w
UR - https://europepmc.org/article/MED/42967945
AB - Prosthetic joint infections are complex and associated with high economic and social costs. Polymicrobial cases remain poorly understood, particularly regarding bacterial interactions and antibiotic response within mixed-species biofilms. We introduce two approaches for characterizing polymicrobial biofilms: a predictive modeling equation and fatty acid profiling. Predictive modeling estimates biofilm behavior. Fatty acid profiling provided complementary information on species dominance and possible interspecies interactions. We integrate these results with fluorescence in situ hybridization and confocal microscopy to obtain a comprehensive view of biofilm structure and composition. Using this multi-modal approach, we characterize mono-, dual-, and triple-species biofilms formed by Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. In parallel, we evaluated common anti-biofilm antibiotics, alone and in combination, across all biofilm types. The model explained 72.9% of the variance in triple-species biofilm formation (R² = 0.73). K. pneumoniae dominated all mixed biofilms in which it was present, accounting for approximately 70-80% of the bacterial population. In P. aeruginosa-S. aureus biofilms, S. aureus outnumbered P. aeruginosa (93% vs. 7%). Antibiotic efficacy varied with biofilm composition and structure. Colistin plus levofloxacin was the most effective combination, achieving reductions of up to 7.5 log₁₀ CFU/cm². S. aureus drove biofilm persistence; no treatment achieved eradication in mixed biofilms containing this species, with median reductions remaining below the 5 log₁₀ CFU/cm² threshold. Confocal microscopy revealed that S. aureus was predominantly located in the deepest biofilm layers. These results support two complementary approaches for characterizing polymicrobial biofilms through structural, predictive, and biochemical analyses and highlight the importance of biofilm architecture and interspecies interactions when evaluating antibiotic efficacy in polymicrobial infections. KEY POINTS: • Modeling and fatty acid analysis predict polymicrobial biofilm behavior • K. pneumoniae dominates biofilms, while S. aureus drives persistence • Antibiotic efficacy depends on biofilm composition and structure.
SN - 0175-7598
SN - 1432-0614
ER -