Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota.
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.


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.


CONCLUSIONS
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.
ETEP Research Group, University Complutense of Madrid, Madrid, Spain.
M C
MC
Sánchez
A
A
Llama-Palacios
V
V
Blanc
R
R
León
D
D
Herrera
M
M
Sanz
D000190:Actinomyces / Q000254:growth & development / Q000502:physiology; D016976:Aggregatibacter actinomycetemcomitans / Q000254:growth & development / Q000502:physiology; D001422:Bacterial Adhesion / Q000502:physiology; D018407:Bacterial Physiological Phenomena; D001431:Bacteriological Techniques; D018441:Biofilms / Q000254:growth & development*; D003470:Culture Media; D003773:Dental Plaque / Q000382:microbiology*; D017886:Durapatite; D005453:Fluorescence; D016967:Fusobacterium nucleatum / Q000254:growth & development / Q000502:physiology; D005881:Gingiva / Q000382:microbiology*; D006801:Humans; D050296:Microbial Viability; D018613:Microscopy, Confocal; D012150:Polymorphism, Restriction Fragment Length; D016966:Porphyromonas gingivalis / Q000254:growth & development / Q000502:physiology; D012463:Saliva / Q000382:microbiology; D019533:Streptococcus oralis / Q000254:growth & development / Q000502:physiology; D013997:Time Factors; D014678:Veillonella / Q000254:growth & development / Q000502:physiology
D003470:Culture Media; D017886:Durapatite