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Evaluation of multi-species biofilms formed on different substrata and exposed to salivary concentrations of antimicrobial agents: microorganisms quantification and biofilm tridimensional morphology

Abstract

The formation and accumulation of biofilm on substrates in the oral cavity is the main etiological factor of oral diseases. Studies evaluating multispecies biofilm are scarce and little is known of the three-dimensional organization of the species present in the biofilm and the changes that could occur even in the presence of antimicrobials excreted in saliva. The aim of this study is to evaluate multispecies biofilm formed on substrates in the oral cavity when exposed to salivary concentrations of different antimicrobial agents. Specimens of three substrates will be used in the experiment, hydroxyapatite, titanium and acrylic resin. After the surface roughness measurement and sterilization by ethylene oxide, the specimens will be placed in 24-well plate for the acquired pellicle formation. In each well will be added fluid universal medium (FUM) containing inoculum of the 6 microorganisms: Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus, Streptococcus oralis and Candida albicans. The plates will be incubated anaerobically at 37° C to complete the formation of a biofilm for 72 h. Then the specimens will be immersed in FUM medium containing one of the antimicrobial agents (azithromycin, metronidazole or fluconazole) for 24 h. The measurement of viable cells will be performed by removing the biofilm from the substrates by sonication (7 watts, 30 seconds) in phosphate buffer solution (PBS). This solution will be serially diluted in PBS and plated on the culture medium Blood Columbia agar, Mitis Salivarius agar, Fastidious Anaerobe agar and Biggy agar. After the incubation, the counts of colony forming units (CFU) will be performed with a stereomicroscope and expressed as CFU/mm2. The visualization of the species in different colors in the biofilm will be performed by fluorescence in situ hybridization (FISH) and the observation under confocal laser scanning microscope. CFU counts and confocal microscopy will be held in the biofilm (baseline) and after immersion in the antimicrobial agent. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DE LUCENA-FERREIRA, SILVIA CARNEIRO; RICOMINI-FILHO, ANTONIO PEDRO; DA SILVA, WANDER JOSE; CURY, JAIME APARECIDO; DEL BEL CURY, ALTAIR ANTONINHA. Influence of daily immersion in denture cleanser on multispecies biofilm. CLINICAL ORAL INVESTIGATIONS, v. 18, n. 9, p. 2179-2185, DEC 2014. Web of Science Citations: 12.
CAUALCANTI, I. M. G.; RICOMINI FILHO, A. P.; LUCENA-FERREIRA, S. C.; DA SILVA, W. J.; PAES LEME, A. F.; SENNA, P. M.; DEL BEL CURY, A. A. Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma. ARCHIVES OF ORAL BIOLOGY, v. 59, n. 7, p. 695-703, JUL 2014. Web of Science Citations: 13.

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