Avaliação da eficácia da DNase associada a terapia fotodinâmica na inativação de biofilmes de isolados clínicos de Candida albicans resistente a fluconazol
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2021-06-30
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Universidade Estadual Paulista (Unesp)
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A terapia fotodinâmica antimicrobiana (TFD) promove a inativação dos microrganismos em cultura planctônica, entretanto, biofilmes são menos sensíveis à essa terapia. A resistência dos biofilmes de Candida spp. é multifatorial e está associada ao efeito protetor da matriz extracelular do biofilme (MEC) que impede a penetração do agente fotossensibilizador (FS) e de fármacos. O objetivo deste estudo foi avaliar a eficácia da enzima DNase I associada à TFD, mediada por Photodithazine® (200 mg/L) e Luz LED (660 nm; 50 J/cm2), sobre biofilmes de isolados clínicos de C. albicans resistentes ao fluconazol (CaR). Biofilmes de 48 horas foram formados a partir das cepas de CaR [ATCC 96901; e dois isolados clínicos (R14 e R70)]. A DNase foi utilizada com objetivo de degradar a MEC dos biofilmes e potencializar o efeito da TFD. Os biofilmes foram expostos a pré-incubação com DNase por 5 min, seguida da aplicação do fotossensibilizador (P) e da Luz (L), de forma isolada ou em associação, originando 8 grupos de tratamento (P+L+, P-L+, P+L-, P-L-, P-L-DNase, P+L+DNase, P+L-DNase e P-L+DNase; n=12). A eficácia dos tratamentos foi avaliada através dos seguintes métodos: contagem de unidades formadoras de colônias (UFC), quantificação de: peso seco total e insolúvel, proteínas solúveis e insolúveis, polissacarídeos solúveis em água (WSP) e em álcali (ASP), e DNA extracelular (eDNA). Os dados foram submetidos ao teste ANOVA three-way com pós teste de Bonferroni. O tratamento com DNase em associação à TFD (P+L+DNase) apresentou redução de 1.92; 1.65 e 1.29 em log10 da viabilidade celular para as cepas ATCC 96901, R14 e R70, respectivamente. Também foi observado redução dos componentes da MEC dos biofilmes, como WSP e eDNA, bem como sua biomassa insolúvel quando comparados com o grupo controle (P-L-). O peso seco total e a quantificação de proteínas não apresentaram diferença estatística entre os tratamentos. Todas as cepas apresentaram o mesmo padrão de comportamento quando tratadas com DNase, e os componentes da MEC foram afetados, potencializando a eficácia da TFD.
Antimicrobial photodynamic therapy (PDT) promotes microorganisms’ inactivation in planktonic cultures; however, the same effect does not occur in biofilms. The resistance of Candida spp. biofilms is multifactorial and it has been associated to the protective effect of biofilm’s matrix extracellular (MEC) that prevents the penetration of photosensitizing agent (PA) and drugs. The objective of this study was to evaluate the effectiveness of DNase I enzyme associated with PDT, mediated to Photodithazine® (200 mg/L) and LED light (660 nm; 50 J/cm2 ), on biofilms of fluconazole-resistant C. albicans’ clinical isolates (CaR). 48h-old biofilms were formed from the strains of CaR [ATCC96901; and two clinical isolates (R14 and R70)]. DNase was used in the aim of degrading the MEC of the biofiilms and potentializing the effect of PDT. Biofilms were exposed to pre-incubation with DNase for 5 minutes, followed by photosensitizer aplication (P) and the ligh (L), either in isolated or associated form, giving rise for 8 groups of treatment (P+L+, P-L+, P+L-, P-L-, P-L-DNase, P+L+DNase, P+L-DNase and P-L+DNase; n=12). The effectiviness of treatments was evaluated trough the following methods: colony forming unit (CFU) counting, quantification of: total and isoluble dry-weight, soluble and insoluble proteins, water-soluble polysaccharides from (WSP) and alkali-soluble polysaccharides (ASP), and extracellular DNA (eDNA). The data were analyzed by three-way ANOVA test with Bonferroni's post-test. The DNase treatment associated with PDT (P+L+DNase) showed a reduction of 1.92; 1.65 e 1.29 in log10 of cell viability to ATCC 96901, R14 and R70 strains, respectively. It was also noticed a reduction of biofilms MEC’s components, like WSP and eDNA, as well as its insoluble biomass when it’s been compared with control group (P-L-). The total dry-weight and the proteins quantification showed no statistical diference between treatments. All of the strains showed the same standard behavior when treating with DNase, and MEC’s components were affected, improving PDT’s effectiveness.
Antimicrobial photodynamic therapy (PDT) promotes microorganisms’ inactivation in planktonic cultures; however, the same effect does not occur in biofilms. The resistance of Candida spp. biofilms is multifactorial and it has been associated to the protective effect of biofilm’s matrix extracellular (MEC) that prevents the penetration of photosensitizing agent (PA) and drugs. The objective of this study was to evaluate the effectiveness of DNase I enzyme associated with PDT, mediated to Photodithazine® (200 mg/L) and LED light (660 nm; 50 J/cm2 ), on biofilms of fluconazole-resistant C. albicans’ clinical isolates (CaR). 48h-old biofilms were formed from the strains of CaR [ATCC96901; and two clinical isolates (R14 and R70)]. DNase was used in the aim of degrading the MEC of the biofiilms and potentializing the effect of PDT. Biofilms were exposed to pre-incubation with DNase for 5 minutes, followed by photosensitizer aplication (P) and the ligh (L), either in isolated or associated form, giving rise for 8 groups of treatment (P+L+, P-L+, P+L-, P-L-, P-L-DNase, P+L+DNase, P+L-DNase and P-L+DNase; n=12). The effectiviness of treatments was evaluated trough the following methods: colony forming unit (CFU) counting, quantification of: total and isoluble dry-weight, soluble and insoluble proteins, water-soluble polysaccharides from (WSP) and alkali-soluble polysaccharides (ASP), and extracellular DNA (eDNA). The data were analyzed by three-way ANOVA test with Bonferroni's post-test. The DNase treatment associated with PDT (P+L+DNase) showed a reduction of 1.92; 1.65 e 1.29 in log10 of cell viability to ATCC 96901, R14 and R70 strains, respectively. It was also noticed a reduction of biofilms MEC’s components, like WSP and eDNA, as well as its insoluble biomass when it’s been compared with control group (P-L-). The total dry-weight and the proteins quantification showed no statistical diference between treatments. All of the strains showed the same standard behavior when treating with DNase, and MEC’s components were affected, improving PDT’s effectiveness.
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Fotoquímioterapia, Candida albicans, Resistência antifúngica a fármacos, Photochemoterapy, Candida albicans, Antifungal drug resistance