Associação da quitosana à terapia fotodinâmica para controle de Streptococcus mutans e biofilme microcosmos / Association of Photodynamic Therapy with chitosan for the control of Streptococcus mutans and microfilm biofilm

A terapia fotodinâmica (TFD) é uma técnica que combina um fotossensibilizador (FS) com luz visível e oxigênio molecular, podendo ser utilizada como agente antimicrobiano no controle de diversas doenças infecciosas, como a cárie dentária. A quitosana vem demonstrando atividade promissora quando associada à alguns fotossensibilizadores. Portanto, o objetivo desse trabalho foi investigar a associação da quitosana com o fotossensibilizador Photodithazine® (PDZ) na TFD sobre culturas planctônicas e biofilmes de S. mutans, bem como avaliar sua eficácia sobre biofilmes microcosmos orais. Inicialmente, foi preparada uma suspensão de S. mutans UA 159 padronizada em 106 células/mL. Para o estudo em culturas planctônicas, foi adicionada a suspensão de S. mutans em placas de 96 poços. Para o estudo em biofilmes, foram confeccionadas amostras de esmalte de dentes bovinos como substrato para a formação do biofilme em placas de 24 poços. As culturas planctônicas e os biofilmes foram tratados de acordo com os grupos experimentais, recebendo adição de PDZ, quitosana ou PBS, seguido pela irradiação ou pela manutenção em ambiente escuro (controle). Os efeitos dos tratamentos foram analisados por meio da contagem de UFC/mL de S. mutans em ágar Infusão Cérebro Coração (BHI) incubadas por 48h a 37°C (5% de CO2) e Microscopia Eletrônica de Varredura (MEV). Além disso, para confirmar a penetração do FS e da quitosana nas células de S. mutans foi realizado teste de absorbância. A seguir, foram estudados os efeitos da terapia fotodinâmica com PDZ associado à quitosana sobre biofilmes microcosmos de saliva formados sobre corpos-de-prova de esmalte bovino. Os efeitos da TFD sobre os biofilmes foram analisados por meio da contagem de células viáveis de micro-organismos totais em ágar BHI e estreptococos do grupo mutans em ágar Mitis Salivarius Bacitracina Sacarose (MSBS). Os dados foram analisados por ANOVA e teste de Tukey. Os resultados demonstraram que a TFD mediada por PDZ foi capaz de reduzir a contagem de células viáveis de S. mutans tanto nos testes planctônicos como nos biofilmes, assim como reduzir a contagem de micro-organismos totais e estreptococos do grupo mutans em biofilmes microcosmos orais. Esses efeitos antimicrobianos foram ainda maiores quando à quitosana foi associada à TFD. A redução do número de células viáveis foi confirmada nas imagens de MEV, nas quais pode-se observar a desestruturação das células e matriz do biofilme. Nos testes de absorção, observou-se que a quitosana aumentou a capacidade de penetração do PDZ nas células de S. mutans. Concluiu-se que a quitosana apresentou capacidade de potencializar a atividade antimicrobiana da TFD mediada por PDZ sobre S. mutans e biofilmes microcosmos orais(AU)

Photodynamic therapy (PDT) is a technique that combines a photosensitizer (FS) with visible light and molecular oxygen and can be used as an antimicrobial agent to control various infectious diseases, such as dental caries. Chitosan has shown promising activity when associated with some photosensitizers. Therefore, the objective of this study was to investigate the association of chitosan with the Photodithazine® photosensitizer (PDZ) in PDT on S. mutans planktonic cultures and biofilms, as well as evaluating its effectiveness on oral microcosm biofilms. Initially, a standardized S. mutans UA159 suspension at 106 cells/mL was prepared. For the study in planktonic cultures, the suspension of S. mutans in 96 well plates was added. For biofilm study, bovine tooth enamel samples were made as substrate for biofilm formation in 24 well plates. Planktonic cultures and biofilms were treated according to the experimental groups, receiving the addition of PDZ, chitosan or PBS, followed by laser irradiation or maintenance in a dark environment (control). The effects of treatments were analyzed by counting CFU/mL of S. mutans on Brain Heart Infusion Agar (BHI) incubated for 48h at 37 °C (5% CO2 ) and Scanning Electron Microscopy (SEM). In addition, to confirm the penetration of PS and chitosan in S. mutans UA159 cells, an absorbance test was performed. Next, the effects of photodynamic therapy with PDZ associated with chitosan on saliva microcosm biofilms formed on bovine enamel specimens were studied. The effects of PDT on biofilms were analyzed by counting viable cells of total microorganisms on BHI agar and mutans streptococci on Mitis Salivarius Bacitracin Sucrose (MSBS) agar. The data were analyzed by ANOVA and Tukey's test. The results demonstrated that PDZ-mediated PDT was able to reduce the viable cell count of S. mutans in both planktonic and biofilm tests, as well as to reduce the count of total microorganisms and mutans streptococci in oral microcosm biofilms. These antimicrobial effects were even greater when chitosan was associated with PDT. The reduction in the number of viable cells was confirmed in the SEM images, in which it is possible to observe the breakdown of the cells and the biofilm matrix. In the absorption tests, it was observed that chitosan increased the penetration capacity of PDZ in S. mutans cells. It was concluded that chitosan was able to potentiate PDZ-mediated PDT antimicrobial activity on S. mutans and oral microcosm biofilms(AU)

Two sporulated Bacillus enhance immunity in Galleria mellonella protecting against Candida albicans.

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.