Anthraquinones against Cryptococcus neoformans sensu stricto: antifungal interaction, biofilm inhibition and pathogenicity in the Caenorhabditis elegans model.

Introduction. Cryptococcal biofilms have been associated with persistent infections and antifungal resistance. Therefore, strategies, such as the association of natural compounds and antifungal drugs, have been applied for the prevention of biofilm growth. Moreover, the Caenorhabditis elegans pathogenicity model has been used to investigate the capacity to inhibit the pathogenicity of Cryptococcus neoformans sensu stricto.Hypothesis. Anthraquinones and antifungals are associated with preventing C. neoformans sensu stricto biofilm formation and disrupting these communities. Antraquinones reduced the C. neoformans sensu stricto pathogenicity in the C. elegans model.Aim. This study aimed to evaluate the in vitro interaction between aloe emodin, barbaloin or chrysophanol and itraconazole or amphotericin B against growing and mature biofilms of C. neoformans sensu stricto.Methodology. Compounds and antifungal drugs were added during biofilm formation or after 72 h of growth. Then, the metabolic activity was evaluated by the MTT reduction assay, the biomass by crystal-violet staining and the biofilm morphology by confocal laser scanning microscopy. C. neoformans sensu stricto's pathogenicity was investigated using the nematode C. elegans. Finally, pathogenicity inhibition by aloe emodin, barbarloin and chrysophanol was investigated using this model.Results. Anthraquinone-antifungal combinations affected the development of biofilms with a reduction of over 60 % in metabolic activity and above 50 % in biomass. Aloe emodin and barbaloin increased the anti-biofilm activity of antifungal drugs. Chrysophanol potentiated the effect of itraconazole against C. neoformans sensu stricto biofilms. The C. elegans mortality rate reached 76.7 % after the worms were exposed to C. neoformans sensu stricto for 96 h. Aloe emodin, barbaloin and chrysophanol reduced the C. elegans pathogenicity with mortality rates of 61.12 %, 65 % and 53.34 %, respectively, after the worms were exposed for 96 h to C. neoformans sensu stricto and these compounds at same time.Conclusion. These results highlight the potential activity of anthraquinones to increase the effectiveness of antifungal drugs against cryptococcal biofilms.

Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans

Abstract Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. Objective This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. Methodology Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. Results CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. Conclusion This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries.

Yeast microbiota of free-ranging amphibians and reptiles from Caatinga biome in Ceará State, Northeast Brazil: High pathogenic potential of Candida famata / Leveduras da microbiota de anfíbios e répteis do bioma Caatinga no Ceará, Nordeste do Brasil: alto potencial patogênico de Candida famata

ABSTRACT: Studies on the fungal microbiota of reptiles and amphibians are necessary to better understand of host-microbe interactions and the establishment of fungal disease in these animals. However, these studies are limited. The present researchidentified yeasts from free-ranging reptiles and amphibians from the Caatinga biome andevaluated the virulence factors production, the antifungal susceptibility in planktonic and biofilm growth and the pathogenicity of Candida famata isolates. Twenty-nine isolates of the genera Candida, Cryptococcus and Rhodotorula were identified by phenotypic and/or molecular methods and production of hydrolytic enzymes in vitro by these genera of fungi was evaluated. In addition, susceptibility of planktonic cells and biofilms to azoles and amphotericin B was evaluated. The pathogenicity of C. famata, the most prevalent yeast species isolated, was evaluated using Caenorhabditis elegans model. C. famata was the most prevalent yeast in amphibian and reptilian microbiota. Phospholipase and protease production was observed in 18/29 and 11/29 of the yeast isolates, respectively, while 100% formed biofilms. Itraconazole presented high minimal inhibitory concentrations against C. famata and C. tropicalis. Amphotericin B reduced the biomass and metabolic activity of biofilms. C. famata induced the mortality of C. elegans. In conclusion, reptiles and amphibians are colonized by yeasts capable of producing important virulence factors, especially by Candida spp. that present low susceptibility to azoles which may result from imbalances in ecosystem. Finally, C. famata isolated from these animals presented high pathogenicity, showing the importance of the study of reptile and amphibians fungal microbiota.

RESUMO: Estudos sobre a microbiota fúngica de répteis e anfíbios são necessários para melhor compreender as interações hospedeiro-microrganismo e o estabelecimento de doenças fúngicas nesses animais. No entanto, esses estudos são limitados. O objetivo da presente pesquisa foi identificar leveduras isoladas de répteis e anfíbios do bioma Caatinga e avaliar a produção de fatores de virulência, a sensibilidade a antifúngicos no crescimento planctônico e de biofilme e a patogenicidade de Candida famata. Vinte e nove isolados dos gêneros Candida, Cryptococcus e Rhodotorula foram identificados por métodos fenotípicos e/ou moleculares e a produção de enzimas hidrolíticas in vitro por esses gêneros de fungos foi avaliada. Além disso, foi avaliada a suscetibilidade de células planctônicas e biofilmes a azólicos e anfotericina B. A patogenicidade de C. famata, a espécie de levedura isolada mais prevalente, foi avaliada usando Caenorhabditis elegans. C. famata foi a levedura mais prevalente na microbiota de anfíbios e répteis. A produção de fosfolipase e protease foi observada em 18/29 e 11/29 dos isolados de levedura, respectivamente, enquanto 100% formaram biofilmes. O itraconazol apresentou altas concentrações inibitórias mínimas contra C. famata e C. tropicalis. A anfotericina B reduziu a biomassa e atividade metabólica dos biofilmes. C. famata induziu a mortalidade de C. elegans. Em conclusão, répteis e anfíbios são colonizados por leveduras capazes de produzir importantes fatores de virulência, especialmente por cepas de Candida spp. que apresentam baixa suscetibilidade a azólicos que podem resultar de desequilíbrio no ecossistema. Por fim, C. famata isolados desses animais apresentaram alta patogenicidade, mostrando a importância do estudo da microbiota fúngica de répteis e anfíbios.