Efeitos do sobrenadante da cultura de Streptococcus mutans sobre os fatores de virulência de C. albicans: estudos in vitro e in vivo / Effects of the culture supernatant of Streptococcus mutans on the virulence factors of C. albicans: in vitro and in vivo studies

Candida albicans possui capacidade de causar uma ampla variedade de manifestações clínicas devido a múltiplos fatores de virulência que agem simultaneamente para vencer o sistema imune e invadir os tecidos do hospedeiro. Estudos recentes demonstraram que o crescimento de C. albicans pode ser inibido por metabólitos produzidos por Streptococcus mutans, que estão presentes no sobrenadante da cultura bacteriana. Assim, o objetivo desse estudo foi investigar se o sobrenadante da cultura de S. mutans, além de inibir o crescimento de C. albicans, é capaz de atenuar os mecanismos de virulência desse fungo. Inicialmente, uma suspensão padronizada de S. mutans (107 células/mL) foi incubada em caldo BHI a 37ºC por 4 h em 5% de CO2. O crescimento em BHI foi filtrado em membrana de 0,22 µm, obtendo-se o sobrenadante da cultura de S. mutans livre de células. A seguir, uma suspensão padronizada de C. albicans (107 células/mL) foi adicionada ao sobrenadante filtrado da cultura de S. mutans em caldo BHI, sendo incubada a 37ºC por 24 h. Para os grupos controle, a suspensão de C. albicans foi colocada em caldo BHI ou YPD esterilizados e incubada nas mesmas condições. Após o período de incubação, o crescimento de C. albicans foi centrifugado e lavado para obtenção de uma suspensão padronizada de C. albicans contendo as células sobreviventes da exposição ao sobrenadante de S. mutans. Essa suspensão de C. albicans foi então utilizada nos testes in vitro e in vivo para determinação dos fatores de virulência desse micro-organismo. No estudo in vitro, foi investigada a atividade proteolítica extracelular de C. albicans, bem como sua capacidade de filamentação, adesão e formação de biofilmes (1:30, 6, 24 e 48 h). Para o estudo in vivo, foi utilizado o modelo de Galleria mellonella, analisando-se a curva de sobrevivência, o número de células fúngicas e hemócitos na hemolinfa de larvas infectadas por C. albicans. Para a análise estatística foi utilizado ANOVA, teste de Tukey, Kruskal-Wallis e Log-rank, com nível de significância de 5%. Verificou-se que as células de C. albicans expostas ao sobrenadante de S. mutans apresentaram redução na filamentação, formação de biofilmes e patogenicidade em G. mellonella em relação ao controle. A exposição ao sobrenadante de S. mutans também mudou o padrão de aderência de C. albicans. Entretanto, o sobrenadante de S. mutans não reduziu a atividade proteolítica de C. albicans. Concluiu-se que o sobrenadante de S. mutans apresentou capacidade de inibir importantes mecanismos de virulência de C. albicans, podendo ser uma fonte de novos agentes antifúngicos a ser explorada

Candida albicans has the ability to cause a wide variety of clinical manifestations due to multiple virulence factors that act simultaneously to overcome the immune system and invade host tissues. Recent studies have shown that the growth of C. albicans can be inhibited by metabolites produced by Streptococcus mutans. Thus, the objective of this study was to investigate whether the culture supernatant of S. mutans is able to attenuate the virulence mechanisms of this fungus. Initially, a standardized suspension of S. mutans (107 cells/mL) was incubated in BHI broth at 37ºC for 4 h in 5% CO2. The growth in BHI was filtered through a 0.22 µm membrane, obtaining the cell free culture supernatant of S. mutans. Then, a standardized suspension of C. albicans (107 cells/mL) was prepared and added to the supernatant of the culture of S. mutans in BHI broth, being incubated at 37ºC for 24 h. For the control groups, the suspension of C. albicans was placed in sterile BHI or YPD broth and incubated under the same conditions. After the incubation period, the growth of C. albicans was centrifuged and washed to obtain a standardized suspension of C. albicans containing the surviving cells from exposure to the S. mutans supernatant. This suspension of C. albicans was then used to perform in vitro and in vivo tests to determine the virulence factors of this microorganism. In the in vitro study, the extracellular proteolytic activity of C. albicans was investigated, as well as its capacity for filamentation, adhesion and biofilm formation (1:30, 6, 24 and 48 h). For the in vivo study, the Galleria mellonella model was used, analyzing the survival curve, the number of fungal cells and hemocytes in the hemolymph of larvae infected with C. albicans. For statistical analysis, ANOVA, Tukey's test, Kruskal-Wallis and Log-rank were used, with a significance level of 5%. It was found that the cells of C. albicans exposed to the supernatant of S. mutans showed a reduction in filament, formation of biofilms and pathogenicity in G. mellonella in relation to the control. Exposure to the S. mutans supernatant also changed the pattern of adherence of C. albicans. However, the S. mutans supernatant did not reduce the proteolytic activity of C. albicans. It was concluded that the supernatant of S. mutans had the capacity to inhibit important mechanisms of virulence of C. albicans, being able to be a source of new antifungal agents to be explored.

Inhibitory effect of probiotic Lactobacillus supernatants from the oral cavity on Streptococcus mutans biofilms.

Probiotics can release bioactive substances that can inhibit the growth and biofilm formation of pathogenic microorganisms such as Streptococcus mutans. In this context, we evaluated whether the supernatants of Lactobacillus strains isolated from caries-free subjects can inhibit S. mutans, one of the most important bacteria for dental caries. First, the supernatants of 22 Lactobacillus strains were screened for antibacterial activity against S. mutans in planktonic cultures. All 22 Lactobacillus strains studied (100%) showed antibacterial activity. Thereafter, the Lactobacillus strains with the greatest reductions in the planktonic S. mutans cultures were tested on biofilms. The L. fermentum 20.4, L. paracasei 11.6, L. paracasei 20.3 and L. paracasei 25.4 strains could significantly reduce the number of S. mutans cells in biofilms formed in hydroxyapatite (p < 0.05). This reduction was also confirmed by scanning electron microscopy analysis and was not caused by the decreased pH value in the medium (p > 0.05). In addition, the supernatants of these probiotic strains could also reduce the total biomass of S. mutans biofilms (p < 0.05). In conclusion, most of the Lactobacillus strains tested have some antibacterial activity against S. mutans. L. fermentum 20.4, L. paracasei 11.6, L. paracasei 20.3 and L. paracasei 25.4 produce bioactive substances that caused a significant reduction in S. mutans biofilms.

Atividade antifúngica do extrato bruto e frações de Streptococcus mutans sobre Candida albicans em modelos de estudo in vivo / Antifungal activity of crude extract and fractions of Streptococcus mutans on Candida albicans in models of in vivo study

Estudos realizados in vitro tem demonstrado que Streptococcus mutans podem produzir metabólitos capazes de inibir Candida albicans, tornando interessante a identificação e desenvolvimento de novas substâncias para o tratamento da candidose bucal. Assim, o objetivo desse estudo foi extrair, fracionar e identificar as substâncias produzidas por S. mutans e avaliar seus efeitos sobre a patogenicidade de C. albicans e na resposta imunológica em modelos de estudo in vivo. As substâncias do sobrenadante da cultura de S. mutans foram extraídas com acetato de etila e posteriormente fracionadas em coluna de sílica derivatizada C-18 (150 g, Φ = 3,5 cm) utilizando diferentes soluções de MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) como eluente, obtendo cinco diferentes frações (SM-F1, SM-F2, SMF3, SM-F4 e SM-F5). A identificação das substâncias contidas no extrato bruto e frações foi realizada por cromatografia gasosa acoplada a espectrometria de massas. Foram testados os efeitos do extrato bruto e frações do sobrenadante da cultura de S. mutans sobre a candidose experimental induzida em modelo invertebrado de Galleria mellonella e em camundongos imunossuprimidos. Para a escolha da concentração a ser testada nos modelos in vivo foi realizada a determinação da Concentração Inibitória Mínima do extrato bruto e frações sobre C. albicans. No modelo de infecção experimental com G. mellonella, os efeitos do extrato e frações foram analisados pelos testes de curva de sobrevivência, quantificação de UFC/mL de C. albicans na hemolinfa e determinação da densidade hemocitária das larvas de G. mellonella. O extrato, assim como as frações com melhores resultados em modelo de invertebrado foram selecionados para o estudo de candidose bucal em camundongos. Nesse modelo experimental, o desenvolvimento de candidose foi avaliado pelos testes de recuperação e determinação de UFC/mL de C. albicans da cavidade bucal, análise macroscópica e microscópica do dorso da língua. Os dados obtidos foram analisados estatisticamente pelo Programa Graph Pad Prism 5.0, com nível de significância de 5%. Na determinação da concentração inibitória mínima, apenas o extrato bruto e a fração SM-F2 demonstraram efeito sobre C. albicans, sendo 10 mg/mL e 15mg/mL, respectivamente. No modelo de G. mellonella não houve aumento da sobrevida das larvas com a utilização profilática do extrato, ocorrendo 100% de morte nas primeiras 24 h em ambos os grupos com infecção. No entanto, ao realizarmos o tratamento pós-infecção com o extrato bruto houve um aumento da sobrevida em 18,75 a 25%. Como não houve efeito profilático para o extrato bruto, as frações foram administradas apenas terapeuticamente, verificando-se aumento da sobrevida das larvas com as fração SM-F1 e SM-F2. Na contagem de UFC/mL de C. albicans na hemolinfa das larvas, foi verificada diferença estatisticamente significante apenas com o extrato bruto e fração SM-F2 após 12 h de infecção Em relação a densidade hemocitária, os grupos tratados com extrato bruto e frações (SM-F1 e SM-F2) apresentaram maior número de hemócitos circulantes na hemolinfa em relação ao grupo apenas infectado com C. albicans. Portanto, as frações SM-F1 e SM-F2 foram escolhidas para os testes em camundongos. Nesse modelo de estudo, verificou-se que o extrato bruto, SM-1 e SM-F2 foram capazes de reduzir significamente o número de UFC/mL de Candida na cavidade bucal e as lesões de candidose no dorso da língua, sendo esses efeitos mais proeminentes para SM-F2. Assim, concluiu-se que as frações SM-F1 e SM-F2 do extrato de S. mutans contêm substâncias antifúngicas com ação terapêutica sobre a candidose experimental, podendo ser alvos de novas estratégias terapêuticas para a candidose bucal(AU)

In vitro studies have shown that Streptococcus mutans can produce metabolites capable of inhibiting Candida albicans, becoming interesting the identification and development of new substances for the treatment of oral candidiasis. Thus, the objective of this study was to extract, fractionate and identify the substances produced by S. mutans and evaluate their effects on the pathogenicity of C. albicans and on the immune response in in vivo study models. Substances from the S. mutans culture supernatant were extracted with ethyl acetate and subsequently fractionated on a C-18 derivatized silica column (150 g, Φ = 3.5 cm) using different solutions of MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) as eluent, obtaining five different 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 crude extract products and the fractions of the supernatant of the S. mutans culture were assessed on experimental candidiasis induced in invertebrate model of Galleria mellonella and in immunosuppressed mice. For a choice of the concentration to be tested in the in vivo models, a determination of the Minimum Inhibitory Concentration (MIC) of the crude extract and fractions on C. albicans was performed. In the model of experimental infection with G. mellonella, the effects of extract and fractions were analyzed by the survival curve test, quantification of CFU/mL of C. albicans in hemolymph and determination of haemocyte density of G. mellonella larvae. The extract, as well as fractions with better results in invertebrate model were selected for the study of oral candidiasis in mice. In this experimental model, the development of candidiasis was evaluated by the tests of recovery and determination of CFU/mL of C. albicans from the mice's oral cavity, macroscopic and microscopic examination of the tongue dorsum. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. In the determination of the minimal inhibitory concentration, only the crude extract and the SM-F2 fraction showed effect on C. albicans, with 10 mg/mL and 15 mg/mL, respectively. In the model of G. mellonella there was no increase in the larvae survival with the prophylactic use of the extract, occurring 100% of death in the first 24 h in both groups with infection. However, when we performed the post infection treatment with the crude extract there was an increase in survival from 18.75 to 25%. As there was no prophylactic effect for the crude extract, the fractions were administered only therapeutically, verifying increased survival of the larvae with the SM-F1 and SM-F2 fraction. In the CFU/mL count of C. albicans on larval hemolymph, a statistically significant difference was observed only with crude extract and SM-F2 fraction after 12 h of infection. In relation to hemocyte density, the groups treated with crude extract and fractions (SM-F1 and SM-F2) had a higher number of hemocytes circulating in the hemolymph compared to the group only infected with C. albicans. Therefore, the fractions SM-F1 and SMF2 were chosen for the tests in mice. In this study model, it was verified that the crude extract, SM-F1 and SM-F2 were able to significantly reduce the number of CFU/mL of Candida in oral cavity and lesions of candidiasis of the tongue dorsum, these effects were more prominent for SM-F2. Thus, it was concluded that the SM-F1 and SM-F2 fractions of the S. mutans extract contain antifungal substances with therapeutic action on experimental candidiasis, being able to be targets of new therapeutic strategies for oral candidiasis(AU)

Study of Microbial Interaction Formed by "Candida krusei" and "Candida glabrata": "In Vitro" and "In Vivo" Studies.

Recently, the non-albicans Candida species have become recognized as an important source of infection and oral colonization by association of different species in a large number of immunosuppressed patients. The objective of this study was to evaluate the interactions between C. krusei and C. glabrata in biofilms formed in vitro and their ability to colonize the oral cavity of mouse model. Monospecies and mixed biofilms were developed of each strain, on 96-well microtiter plates for 48 h. These biofilms were analyzed by counting colony-forming units (CFU/mL) and by determining cell viability, using the XTT hydroxide colorimetric assay. For the in vivo study, twenty-four mice received topical applications of monospecie or mixed suspensions of each strain. After 48 h, yeasts were recovered from the mice and quantified by CFU/mL count. In the biofilm assays, the results for the CFU/mL count and the XTT assay showed that the two species studied were capable of forming high levels of in vitro monospecie biofilm. In mixed biofilm, the CFU of C. krusei increased (p=0.0001) and C. glabrata decreased (p=0.0001). The metabolic activity observed in XTT assay of mixed biofilm was significantly reduced compared with a single C. glabrata biofilm (p=0.0001). Agreeing with CFU in vitro count, C. glabrata CFU/mL values recovered from oral cavity of mice were statistically higher in the group with single infection (p=0.0001) than the group with mixed infection. We concluded that C. krusei inhibits C. glabrata and takes advantage to colonize the oral cavity and to form biofilms.

Study of Microbial Interaction Formed by "Candida krusei" and "Candida glabrata": "In Vitro" and "In Vivo" Studies

Abstract Recently, the non-albicans Candida species have become recognized as an important source of infection and oral colonization by association of different species in a large number of immunosuppressed patients. The objective of this study was to evaluate the interactions between C. krusei and C. glabrata in biofilms formed in vitro and their ability to colonize the oral cavity of mouse model. Monospecies and mixed biofilms were developed of each strain, on 96-well microtiter plates for 48 h. These biofilms were analyzed by counting colony-forming units (CFU/mL) and by determining cell viability, using the XTT hydroxide colorimetric assay. For the in vivo study, twenty-four mice received topical applications of monospecie or mixed suspensions of each strain. After 48 h, yeasts were recovered from the mice and quantified by CFU/mL count. In the biofilm assays, the results for the CFU/mL count and the XTT assay showed that the two species studied were capable of forming high levels of in vitro monospecie biofilm. In mixed biofilm, the CFU of C. krusei increased (p=0.0001) and C. glabrata decreased (p=0.0001). The metabolic activity observed in XTT assay of mixed biofilm was significantly reduced compared with a single C. glabrata biofilm (p=0.0001). Agreeing with CFU in vitro count, C. glabrata CFU/mL values recovered from oral cavity of mice were statistically higher in the group with single infection (p=0.0001) than the group with mixed infection. We concluded that C. krusei inhibits C. glabrata and takes advantage to colonize the oral cavity and to form biofilms.

Resumo Recentemente, as espécies não albicans tem se tornado uma importante fonte de infecção e de colonização oral pela associação de espécies em um grande número de pacientes imunossuprimidos. O objetivo desse estudo foi avaliar a interação entre C. krusei e C. glabrata em biofilmes formados in vitro e sua capacidade em colonizar a cavidade oral em modelo de camundongo. Biofilmes monoespécies e mistos foram formados em placas de 96 poços por 48 h. Esses biofilmes foram analisados pela contagem de UFC/mL e pela determinação da viabilidade celular, usando ensaio de XTT. Para o estudo in vivo, vinte e quatro camundongos receberam aplicações tópicas de suspensões monoespécies e mistas de cada espécie. Após 48 h, as leveduras foram recuperadas dos camundongos e quantificadas por UFC/mL. Nos ensaios de biofilme, os resultados da contagem de UFC/mL e do ensaio de XTT mostraram que as duas espécies estudadas foram capazes de formar grande quantidade de biofilme monoespécie in vitro. Nos biofilmes mistos, a UFC/mL de C. krusei aumentou (p=0,0001) e de C. glabrata diminuiu (p=0,0001). A atividade metabólica observada no ensaio de XTT nos biofilmes mistos foi significantemente reduzida comparada com o biofilme formado apenas de C. glabrata (p=0,0001). Concordado com as contagens in vitro, os valores de UFC/mL de C. glabrata recuperados da cavidade oral dos camundongos foram estatisticamente maior no grupo com infecção simples (p=0,0001) do que do grupo com infecção mista. Nós concluímos que C. krusei inibe C. glabrata e possui vantagem em colonizar a cavidade oral e formar biofilmes.

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions.

Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata and Candida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (10(7) cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a single C. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions

Abstract Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata andCandida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (107 cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a singleC. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experimental model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain.