Characterization of Oral Candida spp. Biofilms in Children and Adults Carriers from Eastern Europe and South America

Abstract

Background: Candida albicans and non-Candida albicans Candida species (NCACs) are known to colonize and invade various tissues, including the oral mucosa. In this work, we aimed to characterize mature biofilms of several Candida spp. clinical isolates (n = 33) obtained from the oral mucosa of children, adults, and elders of Eastern Europe and South America.

Methods: Each strain was evaluated for its capacity to form biofilms in terms of total biomass using the crystal violet assay and for matrix components production (proteins and carbohydrates) using the BCA and phenol-sulfuric tests, respectively. The effect of different antifungals on biofilm formation was studied.

Results: in the children's group, a predominance of C. krusei (81%) was observed, while, among adults, the main species was C. albicans (59%). Most strains showed a reduced response to antimicrobial drugs when in biofilm form (p < 0.01). Moreover, it was observed that strains isolated from children produced more matrix, with higher levels of protein and polysaccharides.

Conclusions: children were more likely to be infected by NCACs than adults. More importantly, these NCACs were able to form biofilms richer in matrix components. This finding is of clinical importance, particularly in pediatric care, since stronger biofilms are highly associated with antimicrobial resistance, recurrent infections, and higher therapeutic failure.

Keywords: Candida species; aging; antifungals susceptibility; biofilm; oral candidiasis; resistance.

Figure 1

In vitro biofilm production of clinical isolates from children, adults, and elders. The black and grey bars are each from different age groups. Biofilm was quantified through staining with crystal violet (OD₅₇₀) after 48 h of incubation. Each value is the average of three independent experiments conducted in triplicate.

Figure 2

Average of in vitro biofilm (Abs/cm²) production from each species of Candida. The black bars are the mean of biofilm (Abs/cm²), which was quantified through staining with crystal violet (OD₅₇₀) after 48 h of incubation. Each value is the average of three independent experiments conducted in triplicate.

Figure 3

In vitro biofilm production of clinical isolates from children (A), adults (B), and elders (C). Biofilm was quantified through staining with crystal violet (OD₅₇₀) after 48 h of incubation. The grey bars are the biofilm biomass of each strain. Each value is the average of three independent experiments conducted in triplicate. Error bars represent the mean and standard deviations. **** p  <  0.0001, *** p  <  0.001 and * p  <  0.05 as compared to biofilm formed by the reference strain (C. albicans SC5314). Note: CA—C. albicans; CK—C. krusei; CG—C. glabrata; CI—C. intermedia; CV—C. valida.

Figure 4

Average quantity of protein (A–C) and polysaccharides (D–F) isolated from a matrix of in vitro biofilm produced by clinical isolates from children, adults, and elders. Each value is the average (protein mg/g of biofilm) ± standard deviation (SD). Note: CA—C. albicans; CK—C. krusei; CG—C. glabrata; CI—C. intermedia; CV—C. valida.

Figure 5

Proportion of protein (OD₅₆₂) and polysaccharide (OD₄₉₀) quantity from each strain from different ages. The gray bars are the polysaccharide level, and the black bars are the protein level of each strain. Each value is the average (mg/g of biofilm) of three independent experiments conducted in triplicate ± standard deviation (SD). Note: CA—C. albicans; CK—C. krusei; CG—C. glabrata; CI—C. intermedia; CV—C. valida.

Figure 6

Heatmap of the reduction (%) of biofilm formation in the presence of antifungals. Red > 80%, orange > 65%, yellow > 50%, and green < 50% reduction. Note: FLU—Fluconazole, VCN—voriconazole, AFG—anidulafungin, AmB—amphotericin B. Bold: above 50%.

Figure 7

Average of the reduction (%) of biofilm formation in the presence of antifungals in children group (A), adult group (B) and elders’ group (C). Note: FLU—Fluconazole, VCN—voriconazole, AFG—anidulafungin, AmB—amphotericin B.

Figure 8

Representative images of confocal laser scanning microscopy (CLSM) of mature biofilms of Candida spp. from children, adults, and elders, analyzed by PNA-FISH using the C. albicans PNA probe. (A): Mature biofilm of C. albicans SC5314; (B): C. glabrata BC21; (C): C. albicans BC29; (D): C. valida AN5793; (E): C. krusei 6; (F): C. albicans CAMYK 2760; (G): C. intermedia AN5310; (H): C. krusei BC06. Magnification: 600×. Laser: 488 nm (ALEXA^®-488). Arrows indicate hyphae form, and ECM means extracellular matrix. Scale bar: 100 μm.