Synergistic growth, metabolic products, and matrix quantification in dual-species biofilms of oral pathogens

Authors

  • Evelyn Giuliana Velásquez-Espedilla Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Dentística, Endodontia e Materiais Odontológicos
  • Victor Feliz Pedrinha Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Dentística, Endodontia e Materiais Odontológicos
  • Maricel Rosario Cardenas Cuellar Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Dentística, Endodontia e Materiais Odontológicos
  • Pedro Luis Busto Rosim Universidade Estadual Paulista (UNESP), Faculdade de Odontologia de Araraquara, Departamento de Dentística Restaurador
  • Daniela Alejandra Cusicanqui Méndez Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Coletiva
  • Thiago Cruvinel Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Coletiva
  • Flaviana Bombarda de Andrade Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Dentística, Endodontia e Materiais Odontológicos

DOI:

https://doi.org/10.1590/1678-7765-2025-0887

Keywords:

Biofilms, Dental caries, Endodontics, Enterococcus faecalis, Extracellular polysaccharides, Matrix, Streptococcus mutans

Abstract

Biofilms exhibit virulence traits that promote microbial protection, such as an extracellular matrix composed of exopolysaccharides (EPS), and contribute to host tissue damage via acid production. Objective  This study evaluated bacterial viability, biovolume, matrix (β-polysaccharides), and EPS (α-polysaccharides) formation in monospecies and mixed biofilms of Streptococcus mutans and Enterococcus faecalis, as well as pH and lactic acid production. Methodology  Biofilms were grown on dentin blocks for 7 days, and aliquots were collected for colony-forming unit (CFU/mL) counts. Subsequently, biofilms were stained with LIVE/DEAD®, Calcofluor White®, and Alexa Fluor 647–dextran conjugate® to assess bacterial viability, matrix formation, and EPS formation, respectively, using confocal laser scanning microscopy (CLSM). Lactic acid production was determined by enzymatic spectrophotometry, and the pH of the culture medium was measured using a pH meter. Kruskal–Wallis and Dunn’s tests were used for pH, viability, biovolume, matrix, and EPS analyses, while one-way ANOVA followed by Tukey’s test was applied for CFU/mL counts and lactic acid production (α = 0.05). Results  Dual-species biofilms showed higher viability and biovolume than S. mutans monospecies biofilms (p < 0.05), whereas the other parameters did not differ among groups (p > 0.05). Conclusion  Dual-species biofilms of S. mutans and E. faecalis may represent a suitable model for investigating antimicrobial strategies, as their association increased viability and biovolume, which are relevant features in cariology and endodontics.

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Published

2026-07-20

Issue

Vol. 34 (2026)

Section

Original Articles

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How to Cite

Velásquez-Espedilla, E. G., Pedrinha, V. F., Cardenas Cuellar, M. R., Rosim, P. L. B., Cusicanqui Méndez, D. A., Cruvinel, T., & Andrade, F. B. de. (2026). Synergistic growth, metabolic products, and matrix quantification in dual-species biofilms of oral pathogens. Journal of Applied Oral Science, 34, e2025-0887. https://doi.org/10.1590/1678-7765-2025-0887