Antifungal effects of Streptococcus mutans extract on Candida strains susceptible and resistant to fluconazole: An in vivo study.

Previous studies showed that the crude extract obtained from Streptococcus mutans inhibited the growth of Candida albicans reference strains. In this study, we evaluated whether the antifungal effects of S. mutans extract can be extended to clinical Candida isolates, including C. albicans and non-abicans strains with different susceptibilities to fluconazole. We verified that S. mutans extract increased the survival of Galleria mellonella larvae infected with C. albicans and C. glabrata and inhibited the fungal cells in hemolymph. These antifungal effects occurred for both fluconazole-susceptible and fluconazole-resistant strains. However, larvae infected by C. krusei were not affected by S. mutans extract. LAY SUMMARY: Streptococcus mutans crude extract shows antifungal effects on clinical Candida strains susceptible and resistant to fluconazole in Galleria mellonella model.

Development of Probiotic Formulations for Oral Candidiasis Prevention: Gellan Gum as a Carrier To Deliver Lactobacillus paracasei 28.4.

Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and it is necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeted for local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food additive, with a probiotic and investigated its antifungal activity against Candida albicansLactobacillus paracasei 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1% [wt/vol]). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic-gellan gum formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacterium-impregnated gellan gum was achieved when L. paracasei 28.4 was lyophilized. The probiotic-gellan gum formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the growth of C. albicans, with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic-gellan gum formulations also had inhibitory activity against Candida sp. biofilms by reducing the number of Candida sp. cells (P < 0.0001), decreasing the total biomass (P = 0.0003), and impairing hyphae formation (P = 0.0002), compared to the control group which received no treatment. Interestingly, a probiotic formulation of 1% (wt/vol) gellan gum provided an oral colonization of L. paracasei in mice with approximately 6 log CFU/ml after 10 days. This formulation inhibited C. albicans growth (P < 0.0001), prevented the development of candidiasis lesions (P = 0.0013), and suppressed inflammation (P = 0.0006) compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.

Immunomodulatory effect of photodynamic therapy in Galleria mellonella infected with Porphyromonas gingivalis.

Porphyromonas gingivalis is an important pathogen in the development of periodontal disease. Our study investigated if the treatment with antimicrobial photodynamic therapy (aPDT) that employs a nontoxic dye, followed by irradiation with harmless visible light can attenuate the experimental infection of P. gingivalis in Galleria mellonella. Firstly, different concentrations of P. gingivalis ranging from 102 to 106 cells/larva were injected into the animal to obtain a lethal concentration. Next, the following groups of G. mellonella infected with P. gingivalis were evaluated: inoculation of the photosensitizer and application of laser (P + L+), inoculation of physiologic solution and application of laser (P-L+), inoculation the photosensitizer without laser (P + L-) and inoculation of physiologic solution without Laser (P-L-). The effects of aPDT on infection by P. gingivalis were evaluated by survival curve analysis and hemocytes count. A lethal concentration of 106 cells/larva was adopted for evaluating the effects of aPDT on experimental infection with P. gingivalis. We found that after 120 s of PDT application, the death of G. mellonella was significantly lower compared to the control groups (p = 0.0010). Moreover, the hemocyte density in the P+L+ group was increased by 9.6 × 106 cells/mL (2.62-fold increase) compared to the infected larvae with no treatment (L-P- group) (p = 0.0175). Finally, we verified that the aPDT led to a significant reduction of the number of P. gingivalis cells in G. mellonella hemolymph. In conclusion, PDT application was effective against P. gingivalis infection by increasing the survival of G. mellonella and was able to increase the circulating hemocytes indicating that PDT activates the G. mellonella immune system.

Streptococcus mutans supernatant affects the virulence of Candida albicans.

Candida albicans causes a variety of clinical manifestations through multiple virulence factors that act simultaneously to overcome the immune system and invade the host tissues. Owing to the limited number of antifungal agents available, new candidiasis therapeutic strategies are required. Previous studies have demonstrated that the metabolites produced by Streptococcus mutans lead to a decrease in the number of Candida cells. Here, for the first time, we evaluated whether the C. albicans cells that survived the pretreatment with S. mutans supernatant can modify their virulence factors and their capability to infect Galleria mellonella larvae. Streptococcus mutans supernatant (SM-S) was obtained by filtering the culture supernatant of this bacterium. Then, C. albicans cells were pretreated with SM-S for 24 h, and the surviving cells were evaluated using in vitro and in vivo assays. The C. albicans pretreated with SM-S showed a significant inhibition of hyphal growth, an altered adhesion pattern, and an impaired capability to form biofilms; however, its proteolytic activity was not affected. In the in vivo assays, C. albicans cells previously exposed to SM-S exhibited a reduced ability to infect G. mellonella and a higher amount of circulating hemocytes. Thus, SM-S could inhibit important virulence factors of C. albicans, which may contribute to the development of new candidiasis therapeutic strategies.

Streptococcus mutans Secreted Products Inhibit Candida albicans Induced Oral Candidiasis.

In the oral cavity, Candida species form mixed biofilms with Streptococcus mutans, a pathogenic bacterium that can secrete quorum sensing molecules with antifungal activity. In this study, we extracted and fractioned culture filtrate of S. mutans, seeking antifungal agents capable of inhibiting the biofilms, filamentation, and candidiasis by Candida albicans. Active S. mutans UA159 supernatant filtrate components were extracted via liquid-liquid partition and fractionated on a C-18 silica column to resolve S. mutans fraction 1 (SM-F1) and fraction 2 (SM-F2). We found anti-biofilm activity for both SM-F1 and SM-F2 in a dose dependent manner and fungal growth was reduced by 2.59 and 5.98 log for SM-F1 and SM-F2, respectively. The SM-F1 and SM-F2 fractions were also capable of reducing C. albicans filamentation, however statistically significant differences were only observed for the SM-F2 (p = 0.004). SM-F2 efficacy to inhibit C. albicans was confirmed by its capacity to downregulate filamentation genes CPH1, EFG1, HWP1, and UME6. Using Galleria mellonella as an invertebrate infection model, therapeutic treatment with SM-F2 prolonged larvae survival. Examination of the antifungal capacity was extended to a murine model of oral candidiasis that exhibited a reduction in C. albicans colonization (CFU/mL) in the oral cavity when treated with SM-F1 (2.46 log) and SM-F2 (2.34 log) compared to the control (3.25 log). Although both SM-F1 and SM-F2 fractions decreased candidiasis in mice, only SM-F2 exhibited significant quantitative differences compared to the non-treated group for macroscopic lesions, hyphae invasion, tissue lesions, and inflammatory infiltrate. Taken together, these results indicate that the SM-F2 fraction contains antifungal components, providing a promising resource in the discovery of new inhibitors for oral candidiasis.

Galleria mellonella as an experimental model to study human oral pathogens.

The oral cavity is home to a microbial community of more than 800 species. This important microbiome is formed by commensal and opportunistic bacteria, fungi and viruses. Several distinct habitats within the mouth support heterogeneous microbial communities that constitute an important link between oral and general health. The use of animal models for in vivo studies in microbial pathogenicity is well established in the scientific community. Galleria mellonella as a model host has increased in use significantly in the last few years. This invertebrate model provides studies on a large scale, serving as screens for studies on vertebrate animals, such as mice and rats. In this review, different studies of microbial genera of dental importance (Enterococcus, Candida, Lactobacillus, Porphyromonas and Streptococcus) are discussed, highlighting the use of G. mellonella as a suitable model for studying pathogenesis, efficacy of antimicrobial compounds, and immune responses.

Competitive Interactions between C. albicans, C. glabrata and C. krusei during Biofilm Formation and Development of Experimental Candidiasis.

In this study, we evaluated the interactions between Candida albicans, Candida krusei and Candida glabrata in mixed infections. Initially, these interactions were studied in biofilms formed in vitro. CFU/mL values of C. albicans were lower in mixed biofilms when compared to the single biofilms, verifying 77% and 89% of C. albicans reduction when this species was associated with C. glabrata and C. krusei, respectively. After that, we expanded this study for in vivo host models of experimental candidiasis. G. mellonella larvae were inoculated with monotypic and heterotypic Candida suspensions for analysis of survival rate and quantification of fungal cells in the haemolymph. In the groups with single infections, 100% of the larvae died within 18 h after infection with C. albicans. However, interaction groups achieved 100% mortality after 72 h of infection by C. albicans-C. glabrata and 96 h of infection by C. albicans-C. krusei. C. albicans CFU/mL values from larvae hemolymph were lower in the interacting groups compared with the monoespecies group after 12 h of infection. In addition, immunosuppressed mice were also inoculated with monotypic and heterotypic microbial suspensions to induce oral candidiasis. C. albicans CFU/mL values recovered from oral cavity of mice were higher in the group with single infection by C. albicans than the groups with mixed infections by C. albicans-C. glabrata and C. albicans-C. krusei. Moreover, the group with single infection by C. albicans had a higher degree of hyphae and epithelial changes in the tongue dorsum than the groups with mixed infections. We concluded that single infections by C. albicans were more harmful for animal models than mixed infections with non-albicans species, suggesting that C. albicans establish competitive interactions with C. krusei and C. glabrata during biofilm formation and development of experimental candidiasis.