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.

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.

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.

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.