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Sanchez M.C.,Complutense University of Madrid | Marin M.J.,Complutense University of Madrid | Figuero E.,Complutense University of Madrid | Llama-Palacios A.,Complutense University of Madrid | And 4 more authors.
Journal of Periodontal Research | Year: 2014

Background and Objectives: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. Material and Methods: PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. Results: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10. P. gingivalis showed a vitality reduction in the biofilm of 3 log10, while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. Conclusion: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Sanchez M.C.,Complutense University of Madrid | Llama-Palacios A.,Complutense University of Madrid | Fernandez E.,Complutense University of Madrid | Figuero E.,Complutense University of Madrid | And 5 more authors.
Dental Materials | Year: 2014

Objectives. The impact of implant surfaces in dental biofilm development is presently unknown. The aim of this investigation was to assess in vitro the development of a complex biofilm model on titanium and zirconium implant surfaces, and to compare it with the same biofilm formed on hydroxyapatite surface. Methods. Six standard reference strains were used to develop an in vitro biofilm over sterile titanium, zirconium and hydroxyapatite discs, coated with saliva within the wells of pre-sterilized polystyrene tissue culture plates. The selected species used represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The developed biofilms (growth time 1 to 120 h) were studied with confocal laser scanning microscopy using a vital fluorescence technique and with low-temperature scanning electron microscopy. The number (colony forming units/biofilm) and kinetics of the bacteria within the biofilm were studied with quantitative PCR (qPCR). As outcome variables, the biofilm thickness, the percentage of cell vitality and the number of bacteria were compared using the analysis of variance. Results. The bacteria adhered and matured with in the biofilm over the three surfaces withsimilar dynamics. Different surfaces, however, demonstrated differences both in the thickness, deposition of the extracellular polysaccharide matrix as well as in the organization of the bacterial cells. Significance. While the formation and dynamics of an in vitro biofilm model was similar irre-spective of the surface of inoculation (hydroxyapatite, titanium or zirconium), there were significant differences in regards to the biofilm thickness and three-dimensional structure. © 2014 Academy of Dental Materials.

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