Microbial interactions are necessarily associated with the development of polymicrobial oral biofilms. The objective of this study was to determine the coaggregation of eight strains of Candida albicans with Actinomyces naeslundii and Streptococcus mutans. In autoaggregation assays, C. albicans strains were grown in RPMI-1640 and artificial saliva medium (ASM) whereas bacteria were grown in heart infusion broth. C. albicans, A. naeslundii and S. mutans were suspended to give 10(6), 10(7) and 10(8) cells mL(-1) respectively, in coaggregation buffer followed by a 1 h incubation. The absorbance difference at 620 nm (ΔAbs) between 0 h and 1 h was recorded. To study coaggregation, the same protocol was used, except combinations of microorganisms were incubated together. The mean ΔAbs% of autoaggregation of the majority of RPMI-1640-grown C. albicans was higher than in ASM grown. Coaggregation of C. albicans with A. naeslundii and/or S. mutans was variable among C. albicans strains. Scanning electron microscopy images showed that A. naeslundii and S. mutans coaggregated with C. albicans in dual- and triculture. In conclusion, the coaggregation of C. albicans, A. naeslundii and S. mutans is C. albicans strain dependent.
Oral biofilms comprise of extracellular polysaccharides and polymicrobial microorganisms. The objective of this study was to determine the effect of polymicrobial interactions of Candida albicans, Actinomyces naeslundii, and Streptococcus mutans on biofilm formation with the hypotheses that biofilm biomass and metabolic activity are both C. albicans strain and growth medium dependent. To study monospecific biofilms, C. albicans, A. naeslundii, and S. mutans were inoculated into artificial saliva medium (ASM) and RPMI-1640 in separate vials, whereas to study polymicrobial biofilm formation, the inoculum containing microorganisms was prepared in the same vial prior inoculation into a 96-well plate followed by 72 hours incubation. Finally, biofilm biomass and metabolic activity were measured using crystal violet and XTT assays, respectively. Our results showed variability of monospecies and polymicrobial biofilm biomass between C. albicans strains and growth medium. Based on cut-offs, out of 32, seven RPMI-grown biofilms had high biofilm biomass (HBB), whereas, in ASM-grown biofilms, 14 out of 32 were HBB. Of the 32 biofilms grown in RPMI-1640, 21 were high metabolic activity (HMA), whereas in ASM, there was no biofilm had HMA. Significant differences were observed between ASM and RPMI-grown biofilms with respect to metabolic activity (P <01). In conclusion, biofilm biomass and metabolic activity were both C. albicans strain and growth medium dependent.
Microbial infection has been shown to involve in oral carcinogenesis; however, the underlying mechanisms remain poorly understood. The present study aimed to characterize the growth of oral microorganisms as both monospecies and polymicrobial biofilms and determine the effects of their products on oral keratinocytes. Candida albicans (ALC3), Actinomyces naeslundii (AN) and Streptococcus mutans (SM) biofilms or a combination of these (TRI) were grown in flow-cell system for 24 h. The biofilms were subjected to fluorescent in situ hybridization using species-specific probes and analysed using confocal laser scanning microscopy. The effluent derived from each biofilm was collected and incubated with malignant (H357) and normal (OKF6) oral keratinocytes to assess extracellular matrix adhesion, epithelial-mesenchymal transition (EMT) and cytokines expression. Incubation of OKF6 with ALC3 and TRI effluent significantly decreased adhesion of the oral keratinocyte to collagen I, whereas incubation of H357 with similar effluent increased adhesion of the oral keratinocyte to laminin I, significantly when compared with incubation with artificial saliva containing serum-free medium (NE; P < 0.05). In OKF6, changes in E-cadherin and vimentin expression were not consistent with EMT although there was evidence of a mesenchymal to epithelial transition in malignant oral keratinocytes incubated with AN and SM effluent. A significant increase of pro-inflammatory cytokines expression, particularly interleukin (IL)-6 and IL-8, was observed when H357 was incubated with all biofilm effluents after 2- and 24-h incubation when compared with NE (P < 0.05). In conclusion, C.albicans, A.naeslundii and S.mutans form polymicrobial biofilms which differentially modulate malignant phenotype of oral keratinocytes.