In vitro effects of commercial mouthwashes on several virulence traits of Candida albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity

Abstract

Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule.

Fig 1. Adhesion of MoWs-treated BLI C. albicans to plastic and epithelial cells.

Yeast cells, treated for 5 minutes (black columns) or 15 minutes (grey columns) with MoWs or PBS (as a control), were washed, suspended in cF12 and seeded (1 x 10⁵ cells/well) in 96-well plates where they were let adhere for 1 hour at 37°C. Then, the adhesion to abiotic surfaces (plastic bottom of the well plates—A) and adhesion to biotic surfaces (TR146-covered bottom of the well plates—B) were assessed by bioluminescence. Signals collected are expressed as relative luminescence units (RLU). The results are mean values ± standard errors of triplicates. ^#p< 0.05 or **p< 0.001 indicate significant differences between MoWs-treated vs PBS-treated BLI C. albicans. MoW1: Curasept 0.20; MoW2: Dentosan Collutorio; MoW3: Meridol Collutorio; MoW4: Elmex Sensitive Professional; MoW5: Listerine Total Care Zero; MoW7: Parodontax; MoW8: Oral B; MoW9: PlaKKontrol Protezione Totale.

Fig 2. Induction of proinflammatory cytokines secretion by TR146 cells infected with MoWs-treated C. albicans.

Yeast cells, treated for 5 minutes (black columns) or 15 minutes (grey columns) with MoWs or PBS (as negative control), were washed, suspended in cF12 and seeded (1 x 10⁵ cells/well) in 96-well plates containing a 3 days old monolayer of TR146 cells. After 24 hours incubation at 37°C, supernatants were collected and cytokines/chemokines levels were assessed by an antibody microarray. Levels of IL-1α (A) and IL-1β (B) are shown. The results are mean values ± standard errors of triplicates. **p< 0.001 indicate significant differences between MoWs-treated vs PBS-treated Candida. MoW1: Curasept 0.20; MoW2: Dentosan Collutorio; MoW3: Meridol Collutorio; MoW4: Elmex Sensitive Professional; MoW5: Listerine Total Care Zero; MoW7: Parodontax.

Fig 3. Effects of the different MoWs on C. albicans susceptibility to phagocytic cells.

Yeast cells, treated for 5 minutes (black columns) or 15 minutes (grey columns) with MoWs or PBS, as a control, were washed, suspended in cRPMI, fluorescently labelled and seeded (2 x 10⁵ cells/well) in chamber-slides containing BV2 phagocytic cells (1 x 10⁵ cells/well; E:T ratio = 1:2). After 1.5 hours incubation at 37°C, slides were fixed and fluorescent signal was acquired. The following parameters were then calculated: phagocytosis percentage (A), phagocytosis index (B) and acidic phagolysosomes percentage (C). The results are mean values ± standard errors of at least triplicate samples. ^##p< 0.001 indicate significant differences between MoWs-treated vs PBS-treated Candida. MoW1: Curasept 0.20; MoW2: Dentosan Collutorio; MoW3: Meridol Collutorio; MoW4: Elmex Sensitive Professional; MoW5: Listerine Total Care Zero; MoW7: Parodontax.

Fig 4. Morphology of C. albicans treated with the different MoWs and then exposed to phagocytic cells.

Representative fluorescent microscopy images of yeast cells, treated for 5 or 15 minutes with MoWs or PBS, as a control, washed, suspended in cRPMI, fluorescently labelled and seeded (2 x 10⁵ cells/well) in chamber-slides containing BV2 phagocytic cells (1 x 10⁵ cells/well; E:T ratio = 1:2). After 1.5 hours incubation at 37°C, slides were fixed and fluorescent signal was acquired. C. albicans cells treated with PBS, MoW 1 or MoW 4 retain their ability to undergo dimorphic transition, even when phagocytized by macrophages (representative image in panel A of MoW 4 –black arrows), therefore, only little numbers of red spots (representative image in panel B of MoW 4—white arrows) are visible. C. albicans cells treated with MoWs 2, 3, 5 and 7, are inhibited in their capacity to undergo dimorphic transition and they retain their yeast morphology (representative image in panel C of MoW 2), therefore most of them are localized inside acidic phagolysosomes (representative image in panel D of MoW 2) where they are likely inactivated. The different colours displayed by fungal cells in panels A and C indicate the fungi simply phagocytized (green Candida, stained with FITC), the fungi phagocytized and associated with acidic phagolysosomes (red Candida, stained with the acidotropic LysoTracker DND-99 dye), or the fungi not phagocytized by BV2 cells (blue Candida, stained with Uvitex 2B). Panels B and D show the same images of panels A and C respectively, using the TRITC filter, which highlights only the Candida cells (stained in red with LysoTracker DND-99 dye) localized within the acidic phagolysosomes. Every condition was assessed in at least 3 different experiments. MoW1: Curasept 0.20; MoW2: Dentosan Collutorio; MoW3: Meridol Collutorio; MoW4: Elmex Sensitive Professional; MoW5: Listerine Total Care Zero; MoW7: Parodontax.

Fig 5. Effects of MoWs on viridans streptococci and E. faecalis biofilm production.

Bacterial isolates, belonging to the species S. parasanguinis (A), E. faecalis (B), S. vestibularis (C), S. salivarius (D), S. mitis/oralis (E) and S. sanguinis (F) were treated for 1 minute with PBS or with MoWs, before being washed, suspended in BHI broth, seeded (1.5 x 10⁷ CFU/ml) in 96-well plates and incubated at 37°C for 48h to allow biofilm formation. Biofilm biomass was then assessed by CV assay and the results were expressed as percentage of biofilm production with respect to the 100% biofilm of PBS-treated bacteria. Each result is the mean value ± standard error of triplicate samples. *p< 0.05 or **p< 0.001 indicate significant differences between MoWs-treated vs PBS-treated bacteria. MoW1: Curasept 0.20; MoW2: Dentosan Collutorio; MoW3: Meridol Collutorio; MoW4: Elmex Sensitive Professional; MoW5: Listerine Total Care Zero; MoW7: Parodontax; MoW8: Oral B; MoW9: PlaKKontrol Protezione Totale.

Fig 6. Effect of MoWs-pretreated S. salivarius on C. albicans capacity to form biofilm.

S. salivarius isolate 12 was treated for 1 minute with PBS or with MoWs, before being washed, suspended in cF12 and seeded (1.5 x 10⁷ CFU/ml) together with untreated C. albicans cells (2 x 10⁶/ml) onto a TR146 epithelial cells monolayer. The plates were then incubated at 37°C and BLI C. albicans biofilm biomass was then assessed by bioluminescence at 24 hours (black columns) and 48 hours (grey columns). Each result is the mean value ± standard error of triplicate samples. *p< 0.05 indicate significant differences between MoWs-treated vs PBS-treated bacteria. MoW1: Curasept 0.20; MoW2: Dentosan Collutorio; MoW3: Meridol Collutorio; MoW4: Elmex Sensitive Professional; MoW5: Listerine Total Care Zero; MoW7: Parodontax; MoW8: Oral B; MoW9: PlaKKontrol Protezione Totale.