Photodynamic inactivation of planktonic cultures of Streptococcus mutans using erythrosine irradiated by LED / Inativação fotodinâmica de culturas planctônicas de Streptococcus mutans usando eritrosina irradiada por LED

Objective: The aim of this in vitro study was to evaluate the efficacy of photodynamic inactivation (PDI) with erythrosine (E), using a light-emitting diode (LED) on planktonic cultures of Streptococcus mutans. Material and Methods: A Streptococcus mutans strain (UA 159) was used to prepare the suspensions containing 107 cells/mL, which was tested under different experimental conditions: a) LED irradiation in the presence of erythrosine as a photosensitizer (E+L+); b) LED irradiation only (P-L+); c) treatment with erythrosine only (E+L-); and d) no LED irradiation or photosensitizer (P) treatment, which served as a control group (P-L-). After treatment, strains were seeded onto MSBS agar for determination of the number of colony-forming units (CFU/mL). Results: The results were submitted to analysis of variance and the Tukey test (p < 0.05). No reduction in the number of CFU/mL was observed in the treatment group with erythrosine (E+L+) when compared to the control (P-L-). Conclusion: PDI using erythrosine did not reduce the number of CFUs per millimeter within the parameters in this study. (AU)

Objetivo: o objetivo deste estudo in vitro foi avaliar a eficácia da inativação fotodinâmica (PDI) com a eritrosina (E), usando diodo de emissão de luz azul (LED) em culturas planctônicas de Streptococcus mutans. Material e métodos: a cepa de Streptococcus mutans (UA 159) foi usada para o preparo das suspensões padrões contendo 107 células/mL, as quais foram testadas em diferentes condições experimentais a) irradiação com LED em presença da eritrosina como fotossensibilizador (E+L+); b) irradiação com LED apenas (F-L+); c) tratamento com eritrosina apenas (E+L-); e d) tratamento sem irradiação com LED ou fotossensibilizador (F), que serviu como grupo controle (F-L-). Após o tratamento, as cepas foram semeadas em ágar MSBS para determinação do número de unidades formadoras de colônias (UFC/mL). Resultados: os resultados foram submetidos à análise de variância e teste de Tukey (p < 0.05). Não foi observada redução no número de UFC/mL no grupo de tratamento com eritrosina (E+L+) quando comparado ao grupo controle (F-L-). Conclusão: a PDI usando etritrosina e LED não reduziu o número de UFCs por milímetro com os parâmetros utilizados neste estudo.(AU)

Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans.

Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.

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.

Different extracts of Zingiber officinale decrease Enterococcus faecalis infection in Galleria mellonella.

Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 µL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.

Different Extracts of Zingiber officinale Decrease Enterococcus faecalis Infection in Galleria mellonella

Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 μL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.

Extratos seco, fresco e glicólico de Zingiber officinale foram obtidos para avaliar suas ações por meio de ensaio de sobrevivência em G. mellonella contra infecção por Enterococcus faecalis. Oitenta larvas foram divididas em: 1) Suspensão de E. faecalis (controle); 2) E. faecalis + extrato fresco de Z. officinale (FEO); 3) E. faecalis + extrato seco de Z. officinale (DEO); 4) E. faecalis + extrato glicólico de Z. officinale (GEO); 5) Solução tampão fosfato salina (PBS). Para o grupo de controle, 5 µL de inóculo de suspensão padronizada (107 células/mL) de E. faecalis (ATCC 29212) foi injetado na última proleg esquerda de cada lagarta. Para os grupos com tratamento, após a injeção de E. faecalis, os extratos foram injetados na última proleg direita. Após as injeções, as lagartas foram armazenadas a 37 °C e o número de animais mortos foi registrado diariamente em 168 h (7 dias) para analisar a curva de sobrevivência. As lagartas foram consideradas mortas quando elas não mostraram qualquer movimento após o toque. A infecção por E. faecalis levou à morte de 85% das lagartas após 168 h. Não obstante, nos grupos de tratamento com associação dos extratos, houve um aumento nas taxas de sobrevivência de 50% (GEO), 61% (FEO) e 66% (DEO) das lagartas. Em todos os grupos com tratamento, as lagartas apresentaram um aumento na sobrevivência, com diferença estatisticamente significativa em relação ao grupo controle (p=0,0029). Não houve diferença estatisticamente significativa entre os tratamentos com os diferentes extratos (p=0,3859). Pode concluir-se que os extratos testados mostraram atividade antimicrobiana contra a infecção por E. faecalis, aumentando a sobrevivência das lagartas de G. mellonella.

Biofilms of Candida albicans serotypes A and B differ in their sensitivity to photodynamic therapy.

Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 µM) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L + P+), laser application alone (L + P-), photosensitizer application alone (L-P+), and application of saline as control (L-P-). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p < 0.05). The two strains studied were sensitive to PDT (L + P+), with a log reduction of 0.49 for serotype A and of 2.34 for serotype B. Laser application alone only reduced serotype B cells (0.53 log), and the use of the photosensitizer alone had no effect on the strains tested. It can be concluded that in vitro biofilms of C. albicans serotype B were more sensitive to PDT.

Avaliação dos efeitos de Streptococcus mutans sobre formação de biofilme e morfogênese de Candida albicans in vitro e estudo experimental em Galleria mellonella / Evaluation of the effects of Streptococcus mutans on biofilm formation and morphogenesis of Candida albicans in vitro and experimental study in Galleria mellonella

As interações entre fungos e bactérias estão presentes na natureza e tem grande importância médica e ambiental. O desenvolvimento adequado de modelos in vitro e in vivo para caracterizar essas interações é essencial para o entendimento da patogênese das doenças e descoberta de novas estratégias terapêuticas. O objetivo desse estudo foi avaliar as interações microbianas entre Streptococcus mutans e Candida albicans em modelos de biofilmes formados in vitro e em modelo experimental de Galleria mellonella. No estudo in vitro, foram avaliados os efeitos de S. mutans sobre a formação de biofilme por C. albicans e sobre a capacidade de filamentação de C. albicans. Em ambos os testes, foram avaliados os efeitos diretos das células de S. mutans sobre C. albicans e também os efeitos indiretos de S. mutans sobre C. albicans, utilizando apenas o sobrenadante da sua cultura. Além disso, foram testados os efeitos de S. mutans sobre C. albicans em diferentes fases de crescimento da cultura bacteriana (4, 6, 18 e 24 h). Para a realização do estudo in vivo, pela primeira vez na literatura, foram inoculados S. mutans juntamente com C. albicans em lagartas de G. mellonella para indução de infecção experimental. Os efeitos de S. mutans sobre a candidose experimental em G. mellonella foram avaliados pela análise da curva de sobrevivência, pelo estudo da cultura da hemolinfa para quantificação de C. albicans e através da avaliação histológica da presença de hifas de C. albicans nos tecidos do hospedeiro. Os resultados dos testes in vitro e da contagem de UFC/mL em G. mellonella foram submetidos à Análise de Variância e teste de Tukey. Os dados obtidos na curva de sobrevivência de G. mellonella foram analisados pelo método de Log-rank. Em todos os testes, foi considerado nível de significância de 5%. Os resultados in vitro demonstraram que as células de S. mutans favoreceram a formação de biofilme por C. albicans, entretanto quando C. albicans foi colocada em contato apenas com ...

Interactions between fungi and bacteria are present in nature and has great medical and environmental importance. The development of adequate models in vitro and in vivo to characterize these interactions are essential for understanding the pathogenesis of diseases and discovery of new therapeutic strategies. The aim of this study was to evaluate the microbial interactions between Streptococcus mutans and Candida albicans in vitro and in an experimental model of Galleria mellonella. In the in vitro study, the effects of S. mutans on biofilm formation and on the filamentation ability by C. albicans were evaluated. On both tests, we evaluated the direct effects of S. mutans cells and indirect effects of the supernatant culture of S. mutans on C. albicans. Furthermore, the effects of the S. mutans were tested on C. albicans at different stages of growth of the bacterial culture (4, 6, 18 and 24 h). In vivo study, were inoculated S. mutans together with C. albicans in larvae of G. mellonella for induction of experimental infection. The effects of S. mutans on experimental candidiasis in G. mellonella was evaluated by analysis of the survival curve, the study of the culture of hemolymph for quantification of C. albicans and by histological evaluation the formation of hyphae of C. albicans in the host tissues. The results of in vitro tests and number of CFU/mL in G. mellonella were submitted to ANOVA and Tukey's test. The data obtained in the survival of G. mellonella were analyzed using the log- rank test. In all tests, were adopted significance level 5%. In vitro results showed that cells of S. mutans favored biofilm formation by C. albicans, however when C. albicans was put in contact with the only supernatant of a culture of S. mutans, there was a reduction in biofilm formation and inhibition of the morphological transition of C. albicans. In the in vivo study, it was found that the injection of S. mutans in larva e previously infected by C. albicans ...

Comparison of the hemolytic activity between C. albicans and non-albicans Candida species.

The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37 ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

Comparison of the hemolytic activity between C. albicans and non-albicans Candida species

The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

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.

Photodynamic inactivation of Staphylococcus aureus and Escherichia coli biofilms by malachite green and phenothiazine dyes: an in vitro study.

OBJECTIVES: The organization of biofilms in the oral cavity gives them added resistance to antimicrobial agents. The action of phenothiazinic photosensitizers on oral biofilms has already been reported. However, the action of the malachite green photosensitizer upon biofilm-organized microorganisms has not been described. The objective of the present work was to compare the action of malachite green with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms. METHODS: The biofilms were grown on sample pieces of acrylic resin and subjected to photodynamic therapy using a 660-nm diode laser and photosensitizer concentrations ranging from 37.5 to 3000 µM. After photodynamic therapy, cells from the biofilms were dispersed in a homogenizer and cultured in Brain Heart Infusion broth for quantification of colony-forming units per experimental protocol. For each tested microorganism, two control groups were maintained: one exposed to the laser radiation without the photosensitizer (L+PS-) and other treated with the photosensitizer without exposure to the red laser light (L-PS+). The results were subjected to descriptive statistical analysis. RESULTS: The best results for S. aureus and E. coli biofilms were obtained with photosensitizer concentrations of approximately 300 µM methylene blue, with microbial reductions of 0.8-1.0 log(10); 150 µM toluidine blue, with microbial reductions of 0.9-1.0 log(10); and 3000 µM malachite green, with microbial reductions of 1.6-4.0 log(10). CONCLUSION: Greater microbial reduction was achieved with the malachite green photosensitizer when used at higher concentrations than those employed for the phenothiazinic dyes.