Table 1.
Design of the study of interaction between Candida species in the oral candidiasis induced in an immunosuppressed mice model.
Fig 1.
Quantification of cells in biofilms formed at the bottom of 96-well plates (in vitro study).
(A) Mean and standard deviation of C. albicans and C. krusei CFU/mL (Log) values in the following groups: monotypic formed by 200 μL of C. albicans or C. krusei; monotypic formed by 100 μL of C. albicans or C. krusei; heterotypic formed by 100 μL of C. albicans and 100 μL of C. krusei. (B) Mean and standard deviation of C. albicans and C. glabrata CFU/mL (Log) values in in the following groups: monotypic formed by 200 μL of C. albicans or C. glabrata; monotypic formed by 100 μL of C. albicans or C. glabrata; heterotypic formed by 100 μL of C. albicans and 100 μL of C. glabrata. Tukey test: different capital letters indicate a significant difference between the three groups analysed. (C) Percentage of reduction, expressed as mean values of CFU/mL, in the viability of C. albicans when associated with C. glabrata and C. krusei relative to monotypic biofilms of C. albicans.
Fig 2.
Quantification of the total mass of the biofilm formed on silicone pads (in vitro study).
(A) Analyse of C. albicans-C. krusei interaction. Mean and standard deviation of Candida CFU/mL (Log) in the following groups: monotypic formed by 1 mL of C. albicans; monotypic formed by 1 mL of C. krusei; monotypic formed by 2 mL of C. albicans; monotypic formed by 2 mL of C. krusei; heterotypic formed by 1 mL of C. albicans and 1 mL of C. krusei. (B) Analyse of C. albicans-C. glabrata interaction. Mean and standard deviation of Candida CFU/mL (Log) in the following groups: monotypic formed by 1 mL of C. albicans; monotypic formed by 1 mL of C. glabrata; monotypic formed by 2 mL of C. albicans; monotypic formed by 2 mL of C. glabrata; heterotypic formed by 1 mL of C. albicans and 1 mL of C. glabrata. ANOVA test: there are no statistically significant differences between the groups.
Fig 3.
Survival curve of G. mellonella larvae infected with Candida strains.
(A) The larvae were infected with C. albicans, C. krusei or C. glabrata (single infections); (B) The larvae were infected with C. albicans (single infection) and compared with larvae infected by C. albicans and C. glabrata (mixed infection); (C) The larvae were infected with C. albicans (single infection) and compared with larvae infected by C. albicans and C. krusei (mixed infection). Comparison of survival curves was made by Log rank test. These experiments were repeated at least twice and representative experiments are presented.
Fig 4.
Quantification of fungal cells in G. mellonella hemolymph.
Mean and standard deviation of CFU/mL (Log) values in G. mellonella hemolymph at various time periods of experimental infection. (A) C. albicans CFU/mL measurements: significant statistical differences were observed between monotypic and heterotypic groups in the 12 h period (ANOVA and Tukey’s test). C. albicans was not quantified in G. mellonella hemolymph at 18 and 24 h because all larvae had perished by those times. (B) C. krusei CFU/mL measurements: significant statistical differences were observed between groups in the 12, 18 and 24 h periods (Student t test). (C) C. glabrata CFU/mL measurements: significant statistical differences were observed between monotypic and heterotypic groups in the 24 h period (Student t test).
Fig 5.
Quantification of Candida cells adhered to the swab.
Mean and standard deviation of the CFU/mL (Log) of C. albicans in monotypic suspension, C. krusei in monotypic suspension, C. glabrata in monotypic suspension, C. albicans in heteroptypic suspension with C. krusei, C. albicans in heteroptypic suspension with C. glabrata, C. krusei in heteroptypic suspension with C. albicans, and C. glabrata in heteroptypic suspension with C. albicans. ANOVA test: there are no statistically significant differences between the groups.
Fig 6.
Quantification of fungal cells recovered from the buccal cavity of mice.
(A) Mean and standard deviation of the CFU/mL (Log) of C. albicans and C. krusei recovered from the buccal cavity of immunosuppressed mice with single and mixed infections. Student t test. *CFU/mL of C. albicans: comparison between single infection by C. albicans and mixed infection by C. albicans-C. krusei (p = 0.012); **CFU/mL of C. krusei: comparison between single infection by C. krusei and mixed infection by C. albicans-C. krusei (p = 0.004). (B) Mean and standard deviation of the CFU/mL (Log) of C. albicans and C. glabrata recovered from the buccal cavity of immunosuppressed mice with monotypic and heterotypic infections. Student t test. *CFU/mL of C. albicans: comparison between single infection by C. albicans and mixed infection by C. albicans-C. glabrata (p = 0.079); **CFU/mL of C. glabrata: comparison between single infection by C. glabrata and mixed infection by C. albicans-C. glabrata (p = 0.001).
Fig 7.
Macroscopic analysis of candidiasis lesions.
Scores and medians obtained from the macroscopic examination of the tongue dorsum of groups infected with C. albicans monospecies, C. krusei monospecies, C. glabrata monospecies, C. albicans-C. krusei multi-species and C. albicans-C. glabrata multi-species. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (p = 0.0001), with similarities between C. albicans monospecies and multi-species groups, and variations when compared with C. krusei and C. glabrata monospecies groups.
Fig 8.
Candida hyphae and yeast on the tongue dorsum of mice.
(A) Sagittal incision of the tongue dorsum of mice infected with C. albicans monospecies inoculum, displaying the presence of yeast and hyphae (↓) in keratin. PAS; original magnification: 200X. (B) Sagittal incision of the tongue dorsum of mice infected with C. albicans monospecies inoculum, displaying the presence of yeast and hyphae (↓) in keratin, and polymorphonuclear leukocytes forming intraepithelial microabscesses. PAS; magnification: 630X. C) Sagittal incision of the tongue dorsum of mice infected with C. albicans and C. krusei multi-species inoculum, displaying the presence of yeast and hyphae (↓) in keratin, and spongiosis in the epithelium. PAS; magnification: 200X. D) Sagittal incision of the tongue dorsum of mice infected with C. albicans and C. glabrata multi-species inoculum, displaying the presence of yeast and hyphae (↓) in keratin and in the epithelium. PAS; magnification: 400X.
Fig 9.
Epithelial changes caused by candidiasis lesions.
(A) Sagittal incision of the tongue dorsum of mice infected with C. albicans monospecies inoculum, displaying the presence of intraepithelial microabscesses (↓), exocytosis and spongiosis. Moderate inflammatory infiltrate is seen in lamina propria. HE; magnification: 200X. (B) Sagittal incision of the tongue dorsum of mice infected with C. krusei monospecies inoculum, displaying normal tissue appearance. HE; magnification: 200X. (C) Sagittal incision of the tongue dorsum of mice in the C. glabrata control group, displaying normal tissue appearance. HE; magnification: 100X. (D) Sagittal incision of the tongue dorsum of mice in the C. albicans and C. krusei interaction group, displaying the presence of spongiosis, basal layer duplication, hyperplasia and epithelial desquamation. Moderate inflammatory infiltrate is seen in lamina propria (↓). HE; magnification: 200X.
Fig 10.
Quantification of hyphae, yeast and epithelial changes.
(A) Scores and medians of the number of hyphae and yeast derived from single and mixed infection. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (p = 0.0009), with variations between C. albicans monospecies and multi-species groups, and similarities amid the latter. (B) Number of epithelial changes and medians observed in candidosis microscopic lesions on the tongue dorsum of mice inoculated with Candida monospecies and multi-species suspensions. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (p = 0.0013), with variations between C. albicans monospecies and multi-species groups, and similarities amid the latter.