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Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota
Author(s) -
Sánchez M. C.,
LlamaPalacios A.,
Blanc V.,
León R.,
Herrera D.,
Sanz M.
Publication year - 2011
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/j.1600-0765.2010.01341.x
Subject(s) - biofilm , streptococcus oralis , fusobacterium nucleatum , aggregatibacter actinomycetemcomitans , actinomyces naeslundii , microbiology and biotechnology , veillonella , biology , bacteria , saliva , porphyromonas gingivalis , prevotella intermedia , chemistry , streptococcus , biochemistry , genetics
Sánchez MC, Llama‐Palacios A, Blanc V, León R, Herrera D, Sanz M. Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota. J Periodont Res 2011; 46: 252–260. © 2011 John Wiley & Sons A/S Background and Objective: There are few in vitro models available in the scientific literature for study of the structure, formation and development of the subgingival biofilm. The purpose of this study was to develop and validate an in vitro biofilm model, using representative selected bacteria from the subgingival microbiota. Material and Methods: Six standard reference strains were used to develop biofilms over sterile ceramic calcium hydroxyapatite discs coated with saliva within the wells of presterilized polystyrene tissue culture plates. The selected species represent initial ( Streptococcus oralis and Actinomyces naeslundii ), early ( Veillonella parvula ), secondary ( Fusobacterium nucleatum ) and late colonizers ( Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans ). The structure of the biofilm obtained was studied using a vital fluorescence technique in conjunction with confocal laser scanning microscopy. The biofilm bacterial kinetics were studied by terminal restriction fragment length polymorphism analysis. Results: After 12 h, initial and early colonizers were the first microorganisms detected adhering to the calcium hydroxyapatite discs. The intermediate colonizer F. nucleatum was not detected in the model until 24 h of incubation. Late colonizers A. actinomycetemcomitans and P. gingivalis could be measured inside the biofilm after 48 h. The biofilm reached its steady state between 72 and 96 h after inoculation, with bacterial vitality increasing from the hydroxyapatite surface to the central part of the biofilm. Conclusion: An in vitro biofilm model was developed and validated, demonstrating a pattern of bacterial colonization and maturation similar to the in vivo development of the subgingival biofilm.