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.

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.

Atividade antifúngica de metabólitos produzidos por Streptococcus mutans sobre Candida albicans / Antifungal activity of metabolites produced by Streptococcus mutans on Candida albicans

RESUMO A formação de biofilmes e produção de hifas por Candida albicans são importantes fatores de virulência, principalmente, para a aderência à mucosa e invasão tecidual. A busca por metabólitos secundários produzidos por S. mutans é de suma importância, pois poderá fornecer novas estratégias terapêuticas no combate às infecções por Candida, possibilitando o desenvolvimento de medicamentos capazes de inibir os mecanismos de patogenicidade das espécies desse gênero. Desse modo, o objetivo desse estudo foi obter o extrato bruto e frações a partir do sobrenadante da cultura de 4 h de S. mutans (UA159) e avaliar seus efeitos sobre os mecanismos de patogenicidade de C. albicans (ATCC18804) por meio de estudos in vitro. Após o cultivo de S. mutans, o extrato bruto foi obtido via partição líquido-líquido com acetato de etila (3x) e posteriormente fracionado em coluna de sílica derivatizada C-18 eluída com gradiente MeOH:H2O, fornecendo cinco frações (SM-F1, SM-F2, SM-F3, SM-F4 e SM-F5). A identificação das substâncias contidas no extrato bruto e frações foi realizada utilizando cromatografia gasosa acoplada a espectrometria de massas (CGEM), sendo encontradas as seguintes substâncias: ácido octanóico e uridina no extrato bruto, ácido propanóico, (3R)-3-Methyl-1,4-bis(trimethylsilyl)piperazine-2,5- dione, pirimidina, gulose e oleamida na SM-F1 e ácido nicotínico e triptofano na SM- F2. O extrato bruto e as frações foram submetidos aos ensaios de bioatividade sobre a formação de hifas de C. albicans e analisados por microscopia óptica e microscopia eletrônica de varredura (MEV). O extrato bruto e a fração SM-F2, que resultou em maior inibição das hifas, foram investigados na expressão de genes de C. albicans envolvidos no mecanismo de filamentação (CPH1, EFG1, HWP1, UME6 e YWP1) por PCR quantitativo em tempo real (RT-qPCR). Além disso, o potencial de inibição do extrato bruto, frações SM-F1 e SM-F2 foi avaliado sobre a formação de biofilmes de C. albicans, analisados pela quantificação de células viáveis (UFC/mL) e MEV Os resultados demonstraram inibição significativa na formação de hifas de C. albicans pelo extrato bruto e principalmente, pela SM-F2, com alterações significativas na expressão de todos os genes analisados. O extrato bruto e SM-F2 regularam negativamente a expressão dos genes CPH1, EFG1, HWP1 e UME6, em contrapartida, regularam positivamente a expressão do gene YWP1, envolvido no mecanismo de dispersão das leveduras. Nas análises de UFC/mL, os resultados demonstraram redução nas contagens de C. albicans nos biofilmes formados quando em contato com o extrato bruto, frações SM-F1 e SM-F2 em todas as concentrações testadas, sendo que o extrato bruto reduziu totalmente as células de C. albicans na concentração 15 mg/mL. Os resultados do nosso estudo demonstraram que o extrato bruto e frações de S. mutans apresentaram efeitos inibitórios sobre importantes mecanismos de virulência de C. albicans, fornecendo evidências que S. mutans produz substâncias com ação antifúngica, tornando-o promissor na busca de novos compostos antimicrobianos para prevenção e tratamento das candidoses humanas(AU)

The biofilm formation and hyphae production by Candida albicans are important virulence factors, mainly for mucosal adhesion and tissue invasion. The search for secondary metabolites produced by S. mutans is of great importance, since it may provide new therapeutic strategies against Candida infections, enabling the development of drugs that inhibit the mechanisms of pathogenicity of the species of this genus. Thus, the objective of this study was to obtain the crude extract and fractions from the supernatant of the 4 h culture of S. mutans (UA159) and evaluate their effects on the mechanisms of pathogenicity of C. albicans (ATCC18804) through in vitro studies. After culture of S. mutans, the crude extract was obtained via liquid-liquid partition with ethyl acetate (3x) and then fractionated on a C-18 derivatized silica column eluted with MeOH:H2O gradient, providing five fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The following substances were found: octanoic acid and uridine in crude extract, propanoic acid, (3R) -3-methyl -1,4-bis (trimethylsilyl) piperazine-2,5-dione, pyrimidine, gulose and oleamide in SM-F1 and nicotinic acid and tryptophan in SM-F2. The crude extract and fractions were submitted to bioactivity assays on hyphae formation by C. albicans and analyzed by optical microscopy and scanning electron microscopy (SEM). The crude extract and SM-F2 fraction, which resulted in highest inhibition of hyphae, were investigated in the expression of C. albicans genes involved in the filamentation mechanism (CPH1, EFG1, HWP1, UME6 and YWP1) by quantitative real-time PCR (RT-qPCR). In addition, the potential of inhibition of the crude extract, SM-F1 and SM-F2 fractions in biofilms of C. albicans were evaluated by the quantification of viable cells (CFU/mL) and SEM. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. The results demonstrated a significant inhibition on C. albicans hyphae formation by the crude extract and mainly by SMF2, with significant alterations in the expression of all genes analyzed. The crude extract and SM-F2 negatively regulated the expression of the CPH1, EFG1, HWP1 and UME6 genes, in contrast, positively regulated the expression of the YWP1 gene, involved in the mechanism of yeast dispersion. In the CFU/mL analyzes, the results showed a reduction in the counts of C. albicans in the biofilms formed when in contact with the crude extract, SM-F1 and SM-F2 fractions in all tested concentrations and the crude extract totally reduced C. albicans cells at 15 mg/mL concentration. The results of our study demonstrated that the crude extract and fractions of S. mutans presented inhibitory effects on important mechanisms of virulence of C. albicans, providing evidence that S. mutans produces substances with antifungal action, making it promising in the search for new compounds antimicrobials for the prevention and treatment of human candidoses(AU)

Recent Advances in the Use of Galleria mellonella Model to Study Immune Responses against Human Pathogens.

The use of invertebrates for in vivo studies in microbiology is well established in the scientific community. Larvae of Galleria mellonella are a widely used model for studying pathogenesis, the efficacy of new antimicrobial compounds, and immune responses. The immune system of G. mellonella larvae is structurally and functionally similar to the innate immune response of mammals, which makes this model suitable for such studies. In this review, cellular responses (hemocytes activity: phagocytosis, nodulation, and encapsulation) and humoral responses (reactions or soluble molecules released in the hemolymph as antimicrobial peptides, melanization, clotting, free radical production, and primary immunization) are discussed, highlighting the use of G. mellonella as a model of immune response to different human pathogenic microorganisms.

Lactobacillus paracasei 28.4 reduces in vitro hyphae formation of Candida albicans and prevents the filamentation in an experimental model of Caenorhabditis elegans.

The objective of this study was to evaluate the influence of microbe-microbe interactions to identify a strain of Lactobacillus that could reduce the filamentation of Candida albicans ATCC 18804 using in vitro and in vivo models. Thus presenting a probiotic effect against the fungal pathogen. First, we analyzed the ability of 25 clinical isolates of Lactobacillus to reduce filamentation in C. albicans in vitro. We found that L. paracasei isolate 28.4 exhibited the greatest reduction of C. albicans hyphae (p = 0.0109). This reduction was confirmed by scanning electron microscopy analysis. The influence of C. albicans filamentation was found to be contributed through reduced gene expression of filament associated genes (TEC1 and UME6). In an in vivo study, prophylactic provisions with L. paracasei increased the survival of Caenorhabditis elegans worms infected with C. albicans (p = 0.0001) by 29%. Prolonged survival was accompanied by the prevention of cuticle rupture of 27% of the worms by filamentation of C. albicans, a phenotype that is characteristic of C. albicans killing of nematodes, compared to the control group. Lactobacillus paracasei isolate 28.4 reduced the filamentation of C. albicans in vitro by negatively regulating the TEC1 and UME6 genes that are essential for the production of hyphae. Prophylactic provision of Lactobacillus paracasei 28.4 protected C. elegans against candidiasis in vivo. L. paracasei 28.4 has the potential to be employed as an alternative method to control candidiasis.