Synergism of Streptococcus mutans and Candida albicans Reinforces Biofilm Maturation and Acidogenicity in Saliva: An In Vitro Study

Abstract

Early childhood caries, a virulent-form of dental caries, is painful, difficult, and costly to treat that has been associated with high levels of Streptococcus mutans (Sm) and Candida albicans (Ca) in plaque-biofilms on teeth. These microorganisms appear to develop a symbiotic cross-kingdom interaction that amplifies the virulence of plaque-biofilms. Although biofilm studies reveal synergistic bacterial-fungal association, how these organisms modulate cross-kingdom biofilm formation and enhance its virulence in the presence of saliva remain largely unknown. Here, we compared the properties of Sm and Sm-Ca biofilms cultured in saliva by examining the biofilm structural organization and capability to sustain an acidic pH environment conducive to enamel demineralization. Intriguingly, Sm-Ca biofilm is rapidly matured and maintained acidic pH-values (~4.3), while Sm biofilm development was retarded and failed to create an acidic environment when cultured in saliva. In turn, the human enamel slab surface was severely demineralized by Sm-Ca biofilms, while there was minimal damage to the enamel surface by Sm biofilm. Interestingly, Sm-Ca biofilms exhibited an acidic environment regardless of their hyphal formation ability. Our data reveal the critical role of symbiotic interaction between S. mutans and C. albicans in human saliva in the context of pathogenesis of dental caries, which may explain how the cross-kingdom interaction contributes to enhanced virulence of plaque-biofilm in the oral cavity.

Intervening in Symbiotic Cross-Kingdom Biofilm Interactions: a Binding Mechanism-Based Nonmicrobicidal Approach

“…Microbial stocks were stored at 280°C in tryptic soy broth containing 50% glycerol before use. All strains were grown to mid-exponential phase (optical densities at 600 nm [OD 600 ] of 0.8 [C. albicans] and 1.0 [S. mutans], respectively) in ultrafiltered (10 kDa molecular mass cutoff; Millipore, Billerica, MA, USA) yeast-tryptone extract broth containing 2.5% tryptone and 1.5% yeast extract (UFYTE; pH 5.5 and 7.0 for C. albicans and S. mutans, respectively) with 1% (wt/vol) glucose at 37°C and 5% CO 2 , as described previously (24,40). Cells were harvested by centrifugation (6,000 Â g, 10 min, 4°C).…”

“…Human tooth enamel blocks (4 mm  4 mm) were prepared and coated with sterile clarified whole saliva (sTE). Cultures of ;2  10 6 CFU/mL of S. mutans and ;2  10 4 CFU/mL of C. albicans were grown on sTE in saliva supplemented with 1% sucrose (wt/ vol), as detailed elsewhere (24). Briefly, biofilms were formed on enamel blocks mounted vertically at 37°C in 5% CO 2 for 114 h. Biofilms were treated with PBS or b-mannanase as described in the "In vitro biofilm model" section.…”

“…Then, biofilms were gently removed and the enamel slabs were collected for topography and surface roughness measurement. The surface topography and roughness of the enamel surface were analyzed by a nondestructive confocal contrasting method using Zeiss LSM 800 with a C Epiplan-Apochromat 50Â (numerical aperture, 0.95) nonimmersion objective (24,25). The images were processed using ConfoMap (Zeiss) to create 3D topography rendering and measure the surface properties in 3D.…”

“…S5). Then, we inspected the impact on enamel surface integrity by the treated biofilms both visually and quantitatively using confocal surface topographical analysis (24,25). A smooth and flat surface was observed from the intact surface prior to biofilm formation (Fig.…”

The effectiveness of apple, sugar-free chewing gum and Rolly Brush® in the reduction of plaque index: crossover randomized clinical trial

“…5 One of the main acid-producing pathogen that causes dental caries found in biofilms is Streptococcus mutans along with opportunistic fungal pathogen Candida albicans. 6,7 Preventive treatments, especially for children and teenagers, include better eating habits limiting the ingestion of sugar and promotion of oral hygiene. The most effective method for HDB control is mechanical cleaning through tooth brushing with fluoride with fluoride toothpaste and auxiliary methods of oral hygiene.…”