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.

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.

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.

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.

Influência de Lactobacillus acidophilus sobre biofilme formado por Candida albicans in vitro e infecção em modelo de invertebrado / Interactions between Candida albicans and Lactobacillus acidophilus: biofilm formation in vitro and study in invertebrates models

As interações entre fungos e bactérias são abundantes na natureza e tem importância médica e ambiental. O desenvolvimento adequado de modelos in vitro e in vivo para caracterizar essas interações é essencial para o entendimento do desenvolvimento da doença e descoberta de novas estratégias terapêuticas. O objetivo desse estudo foi avaliar as interações entre Lactobacillus acidophilus e Candida albicans em modelos de estudo in vitro e em modelo experimental de invertebrado. No estudo in vitro, foram avaliados os efeitos de L. acidophilus 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 L. acidophilus sobre C. albicans e também os efeitos indiretos, utilizando apenas o sobrenadante da cultura de Lactobacilos. Além disso, foram testados os efeitos de L. acidophilus sobre C. albicans em diferentes fases de crescimento da cultura bacteriana (4, 6, 18 e 24 h).Para a realização do estudo in vivo, as cepas de L. acidophilus foram inoculadas juntamente com C. albicans em lagartas de Galleria mellonella para indução de infecção experimental. Os efeitos de L. acidophilus sobre a candidose experimental foram avaliados pela análise de curva de sobrevivência de G. mellonella, quantificação de UFC/mL de C. albicans e avaliação histológica da filamentação de C. albicans nos tecidos do hospedeiro. Os resultados dos testes in vitro e da contagem de UFC/mLem 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. (P ≤ 0,05). Os resultados in vitro demonstraram que a cultura de 24 h de L. acidophilus foi capaz de inibir a formação de biofilme e a filamentação por C. albicans. Esses efeitos inibitórios também foram observados quando o sobrenadante da cultura de L. acidophilus foi colocado em contato com C. albicans, sugerindo que a ação ...

Interactions between fungi and bacteria are abundant in nature and are of medical and environmental importance. The developments of adequatemodels in vitro and in vivo to characterize these interactions are essential to understand diseases and to discover new therapeutic strategies. The aim of this study was to evaluate the microbial interactions between Lactobacillus acidophilus and Candida albicans on in vitro study models and experimental models of invertebrates. In the in vitro study, we analyzed the effects of L. acidophilus on C. albicans biofilm formation as well as its filamentation ability. On both tests, the direct effects of L. acidophilus cells and indirect effects of the supernatant culture of L. acidophilus were evaluated , as well as the L. acidophilus effects on C. albicans at different bacterial culture growth stages (4, 6 18 and 24h). To perfom the in vivo study, L. acidophilus and C. albicans strains were inoculated in Galleria mellonella model to induce experimental infection. The L. acidophilus effects on experimental candidosis have been evaluated by the analysis of the survival killing curve of G. mellonella, quantify C. albicans colony forming units per millimeter (CFU/mL) and through histological evaluation of C. albicans filamentation ability on the host tissues. The in vitro test results and the CFU/mL number in G. mellonella were submitted to ANOVA and Tukey’s test. The obtained data on G. mellonella killing curves have been analyzed using the long-ranktest method. A 5% significance level has been considered on all tests. Thein vitro results showed that the L. acidophilus 24 hour culture was able to inhibit the formation of biofilm and filamentation by C. albicans. These inhibitory effects were also observed when the supernatant of the L. acidophilus culture was placed in contact with C. albicans, suggesting that the inhibitory action occurred by the secretion of bacterial substances. It was found in the in vivo study that the...

Influência de Lactobacillus acidophilus sobre biofilme formado por Candida albicans in vitro e infecção em modelo de invertebrado / Interactions between Candida albicans and Lactobacillus acidophilus: biofilm formation in vitro and study in invertebrates models

As interações entre fungos e bactérias são abundantes na natureza e tem importância médica e ambiental. O desenvolvimento adequado de modelos in vitro e in vivo para caracterizar essas interações é essencial para o entendimento do desenvolvimento da doença e descoberta de novas estratégias terapêuticas. O objetivo desse estudo foi avaliar as interações entre Lactobacillus acidophilus e Candida albicans em modelos de estudo in vitro e em modelo experimental de invertebrado. No estudo in vitro, foram avaliados os efeitos de L. acidophilus 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 L. acidophilus sobre C. albicans e também os efeitos indiretos, utilizando apenas o sobrenadante da cultura de Lactobacilos. Além disso, foram testados os efeitos de L. acidophilus sobre C. albicans em diferentes fases de crescimento da cultura bacteriana (4, 6, 18 e 24 h).Para a realização do estudo in vivo, as cepas de L. acidophilus foram inoculadas juntamente com C. albicans em lagartas de Galleria mellonella para indução de infecção experimental. Os efeitos de L. acidophilus sobre a candidose experimental foram avaliados pela análise de curva de sobrevivência de G. mellonella, quantificação de UFC/mL de C. albicans e avaliação histológica da filamentação de C. albicans nos tecidos do hospedeiro. Os resultados dos testes in vitro e da contagem de UFC/mLem 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. (P ≤ 0,05). Os resultados in vitro demonstraram que a cultura de 24 h de L. acidophilus foi capaz de inibir a formação de biofilme e a filamentação por C. albicans. Esses efeitos inibitórios também foram observados quando o sobrenadante da cultura de L. acidophilus foi colocado em contato com C. albicans, sugerindo que a ação...

Interactions between fungi and bacteria are abundant in nature and are of medical and environmental importance. The developments of adequatemodels in vitro and in vivo to characterize these interactions are essential to understand diseases and to discover new therapeutic strategies. The aim of this study was to evaluate the microbial interactions between Lactobacillus acidophilus and Candida albicans on in vitro study models and experimental models of invertebrates. In the in vitro study, we analyzed the effects of L. acidophilus on C. albicans biofilm formation as well as its filamentation ability. On both tests, the direct effects of L. acidophilus cells and indirect effects of the supernatant culture of L. acidophilus were evaluated , as well as the L. acidophilus effects on C. albicans at different bacterial culture growth stages (4, 6 18 and 24h). To perfom the in vivo study, L. acidophilus and C. albicans strains were inoculated in Galleria mellonella model to induce experimental infection. The L. acidophilus effects on experimental candidosis have been evaluated by the analysis of the survival killing curve of G. mellonella, quantify C. albicans colony forming units per millimeter (CFU/mL) and through histological evaluation of C. albicans filamentation ability on the host tissues. The in vitro test results and the CFU/mL number in G. mellonella were submitted to ANOVA and Tukey’s test. The obtained data on G. mellonella killing curves have been analyzed using the long-ranktest method. A 5% significance level has been considered on all tests. Thein vitro results showed that the L. acidophilus 24 hour culture was able to inhibit the formation of biofilm and filamentation by C. albicans. These inhibitory effects were also observed when the supernatant of the L. acidophilus culture was placed in contact with C. albicans, suggesting that the inhibitory action occurred by the secretion of bacterial substances. It was found in the in vivo study that the...

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.

Interação entre Escherichia coli e Candida albicans em biofilmes formados in vitro: análise da viabilidade celular por método colorimétrico / Interaction between Escherichia coli and Candida albicans in biofilms formed in vitro: analysis of cell viability by the colorimetric assay

As interações entre fungos e bactérias estão presentes na natureza e têm grande relevância médica e ambiental. O objetivo deste estudo foi avaliar os efeitos de Escherichia coli sobre os biofilmes de Candida albicans formados in vitro. Foram desenvolvidos biofilmes heterotípicos com a associação de C. albicans e E. coli e biofilmes monotípicos deC. albicans (grupo controle). Foi utilizada uma suspensão padronizada de C. albicans contendo 107 células.mL?1 para formação dos biofilmes. Para analisar os efeitos de E. coli no biofilme de C. albicans, foram testadas diferentes densidades celulares da suspensão de E. coli (107, 106 e 105 células.mL?1). Após 90 minutos e 24 horas da formação do biofilme, a viabilidade celular de C. albicans foi quantificada utilizando-se o ensaio colorimétrico XTT (2 metoxi 4 nitro 5 sulfofenil 5 fenilalanina carbonil 2H tetrazolium hidróxido). Os dados foram submetidos à Análise deVariância ANOVA e ao teste de Tukey, com significância de 5% (p < 0,05). Após 90 minutos da formação do biofilme, observou-se que a viabilidade celular de C. albicans dos biofilmes heterotípicos foi semelhante ao monotípico. A análise de 24 horas demonstrou que a viabilidade celular de C. albicans foi maior no biofilme monotípico emrelação aos biofilmes heterotípicos; entretanto, essa diferença não foi estatisticamente significante. Além disso, não foram observadas diferenças estatísticas entre as densidades celulares de E. coli testadas nos biofilmes heterotípicos. Conclui-se que, dentro dos parâmetros utilizados, E. coli não inibiu a formação de biofilme por C. albicans.

The interactions between fungi and bacteria are present in nature and has medical and environmental importance. The aim of this study was to evaluate the effects of interaction between ATCC strains Candida albicans and Escherichia coli through the study of biofilms in vitro. For this study were developed biofilms formed by the association between C.albicans and E.coli and monotypic biofilms of C. albicans (control group). To evaluate the effects of E. coli in biofilms of C. albicans, we tested different cell densities of E.coli suspension (107, 106 and 105 cells.mL?1). After 90 minutes and 24 hours of biofilm formation, cell viability of C. albicans was quantified using the XTT (2 methoxy 4 nitro 5 sulfophenyl 5 phenylamino carbonyl 2H tetrazolium hydroxide) colorimetric assay. Data were submitted to ANOVA and Tukey?s test, with 5% of significance. After 90 minutes of Candidal biofilm formation, we observedthat cell viability of C. albicans heterotypic biofilms was similar to monotypic biofilm (control group). For the analysis of 24 hours, it was found that cell viability of C. albicans was higher in the monotypic biofilm if compared to heterotypic biofilm, however this difference was not statistically significant. In addition, there were no statisticaldifferences between E. coli different cell densities tested in heterotypic biofilms. According to the methods used, we can conclude that E. coli did not inhibit the biofilm formation by C. albicans.

Photoinactivation of escherichia coli using xanthene dyes andlight-emitting diodes / Fotoinativação de escherichia coli utilizando corantes xantenos e diodo emissor de luz

The development of antibiotic resistance by pathogenic bacteria is currently one of the major problems in medicine. There fore, the study of new treatment modalities such as photodynamic therapy is important. The aim was to evaluate the effects of the Rose Bengal and erythrosine dye combined with a light-emitting diode (LED) on Escherichia coli. An E. coli suspension was prepared from a clinical strain and subjected to the following treatments: LED and Rose Bengal, LED and erythrosin, LED and physiological solution, and physiological solution only as control, and exposure to light for 60, 120 and 180 seconds. After incubation at 37°C for 24 h, the number of colony-forming units (CFU) was calculated and submitted to analysis of variance (ANOVA). Photodynamic therapy using Rose Bengal resulted in a reduction of 5.58 log10 in the number of CFU/mL after light exposure for 60 s and complete elimination after 180 s. However, photodynamic therapy using erythrosin only caused a slight reduction in the number of CFU/ml (0.30 log10)compared to the control group. The use of the LED alone had no toxic effect on the strain tested. In conclusion, Rose Bengal was more effective than erythrosin in photodynamic therapy against E. coli.

O desenvolvimento de resistência aos antibióticos por bactérias patogênicas é um dos maiores problemas da medicina atual. Assim, torna-se importante o estudo de novas modalidades de tratamento, como a terapia fotodinâmica. O objetivo deste estudo foi avaliar os efeitos dos fotos sensibilizadores rosa bengal e eritrosina associados a um diodo emissor de luz (LED) sobre Escherichia coli. Foi preparada uma suspensão padronizada de E. coli (106 células/mL) a partir de uma cepa clínica isolada da cavidade bucal humana. Essa cepa foi submetida aos seguintes tratamentos: laser e rosa bengal (L+RB+), laser e eritrosina (L+E+), laser e solução fisiológica (L+F-) e apenas solução fisiológica como controle (LF-) nos tempos de 60, 120 e 180 segundos de exposição à luz. Foi utilizado LED emissor de luz azul (460 nm), rosabengal e eritrosina na concentração de 50 μM. A seguir, foram realizadas culturas em ágar Infuso Cérebro-Coração para a contagem de unidades formadoras de colônias (UFC/mL) e os dados submetidos à análise de variância. A terapia fotodinâmica utilizando rosa bengal foi capaz de reduzir o número de UFC/mL de 5,58 log10 no tempo de exposição do LED de 60 seg até eliminação completa do microrganismo no tempo de 180 seg. Entretanto, a terapia fotodinâmica com eritrosina apresentou discreta redução do número de UFC/mL (0,30 log10) quando comparada ao grupo controle.O uso isolado do LED não apresentou toxicidade para as cepas testadas. Concluiu-se que o Rosa Bengal foi mais eficaz do que a eritrosina como fotossensibilizador na terapia fotodinâmica sobre Escherichia coli.