Efeito da N-acetilcisteína em biofilme de Candida albicans
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Data
2019-03-11
Autores
Nunes, Thaís Soares Bezerra Santos
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Candida albicans (Ca) é o principal fungo patógeno humano, responsável por infecções como a candidose orofaríngea e a candidemia. Essas infecções estão fortemente associadas com a formação de biofilmes. O uso abusivo de antifúngicos tem levado ao desenvolvimento de resistência fúngica. As terapias direcionadas aos biofilmes, principalmente contra a matriz extracelular (MEC), são relevantes para o desenvolvimento de novos tratamentos mais eficazes. Um dos agentes que tem demonstrado ação antibiofilme é a N-acetilcisteína (NAC). Assim, o objetivo deste estudo foi avaliar a ação da NAC em biofilmes in vitro de C. albicans susceptível (CaS) e resistente ao fluconazol (CaR). Culturas planctônicas de CaS e CaR foram cultivadas e submetidas ao teste de susceptilidade à NAC. Foram determinadas também a curva de inativação de ambas as cepas sob ação da NAC, seu efeito na formação do biofilme e no biofilme maduro. Por fim, foi avaliada a ação NAC na composição da MEC do biofilme, por meio da quantificação de peso seco total e do precipitado, polissacarídeos solúveis em água (WSP) e álcali (ASP), proteínas do sobrenadante e do precipitado e DNA extracelular (eDNA). Os dados obtidos foram analisados descritivamente e pelos testes ANOVA/Welch e Tukey/Gomes-Howell ou Kruskal-Wallis (=0,05). A concentração inibitória mínima da NAC com redução de 90% (CIM90) foi de 25 mg/mL para ambas as cepas. Apesar de não ter sido encontrado nenhum valor de concentração fungicida mínima (CFM), a NAC reduziu (p≤ 0,001) a viabilidade fúngica em 1,81 a 4,06 log10 em ambas as cepas avaliadas. Na curva de inativação, apenas as concentrações ≥ CIM reduziram o crescimento fúngico durante 24 h, essa ação fungistática foi mais evidente entre 8-10h. Concentrações < CIM aumentaram o crescimento fúngico. Em biofilmes em formação, concentrações > CIM (100 e 50 mg/mL) inibiram a viabilidade (redução de 1,41 a 2,77 log10) e a biomassa (redução de 39 a 88%), e para CaS a NAC 25 e 12,5 mg/mL também reduziu biomassa em 77 e 34%, respectivamente. Esse efeito inibitório também foi observado em diferentes fases da formação do biofilme (24, 12 e 6 h). Já em biofilmes maduros, apenas a NAC 100 mg/mL reduziu a viabilidade (1,12 a 2,30 log10) em ambas as cepas, e em CaR 50 e 25 mg/mL de NAC reduziram 1,26 e 0,64 log10, respectivamente. Na biomassa, a NAC 100 mg/mL reduziu 69-72% para ambas as cepas, e em CaR a NAC 50 e 25 mg/mL também reduziu em 64 e 48%, respectivamente. NAC 12,5 mg/mL também aumentou (p< 0,001) a viabilidade de CaS do biofilme maduro e em formação. Na análise dos componenetes da matriz dos bofilmes, foi observada uma tendência da NAC 100 mg/mL (biofilmes maduros) e 50 mg/mL (biofilmes em formação) em diminuir o peso seco do precipitado, proteínas do sobrenadante, WSP, ASP e eDNA. Assim, a NAC apresentou efeito fungistático contra as CaS e CaR, com um efeito concentração-dependente. O efeito antibiofilme da NAC foi decorrente de sua ação fungistática, uma vez que somente as concentrações que inibiram a viabilidade fúngica reduziram a biomassa. Houve uma tendência da NAC em diminuir os componentes solúveis da matriz. Este foi um estudo experimental in vitro inicial, que visa futuramente a aplicabilidade clínica da NAC como um coadjuvante no controle de biofilme de Ca sobre a superfície de próteses dentárias.
Candida albicans is the main fungal pathogen in humans, responsable for local and systemic infections, such as oropharyngeal candidiasis and candidemia. These fungal infections are associated with biofilm formation. The abusive use of antifungals has led to the development of fungal resistance. The therapies towards the biofilms, mainly against the matrix, are relevant for the development of more effective new treatments. One of the agents that have demonstrated antibiofilm action is N-acetylcysteine (NAC). Therefore, the aim of this study was to evaluate the effect in vitro of NAC on biofilms of C. albicans susceptible (CaS) and resistant to fluconazole (CaR). CaS and CaR were submitted to the susceptibility test to NAC. The time-kill curves of NAC and its effect on biofilm formation and mature biofilms were also determined for both strains. Finally, the effect of NAC on the composition of the biofilms matrix was evaluated [total and pellets’s dry weight, water (WSP) and alkali soluble polysaccharides (ASP), supernatant and pellet’s proteins, and extracellular DNA (eDNA)]. The data were descriptively analyzed and submitted to the ANOVA/Welch and Tukey/Gomes-Howell or Kruskal-Wallis tests (= 0.05). The minimum inhibitory concentration of NAC with 90% of reduction (MIC90) was 25 mg/mL. Although no minimum fungicidal concentration (MFC) value was found, NAC reduced (p≤ 0,001) the fungal viability by 1.81 to 4.06 log10 for both strains. In the time-kill curves, only concentrations ≥ MIC reduced the fungal growth for 24 h, this fungistatic action was most evident between 8-10 h. Concentrations < MIC increased the fungal growth. For biofilm formation, concentrations > MIC (100 and 50 mg/mL) inhibited the viability (reduction of 1.41 to 2.77 log10) and the biomass (reduction of 39 to 88%) for both strains, and for CaS NAC at 25 and 12.5 mg/mL reduced the biomass in 77 and 34%, respectively. This inhibitory effect of NAC was also observed in differents stages of biofilm formation (24, 12, and 6h). For mature biofilms, only NAC at 100 mg/mL reduced the viability (1.12 to 2.30 log10) of both strains, and for CaR 50 and 25 mg/mL of NAC reduced 1.26 and 0.64 log10, respectively. NAC 100 mg/mL reduced the biomass in 69-72% for both strains, and for CaR NAC at 50 and 25 mg/mL also reduced the biomass in 64 and 48%, respectively. NAC 12.5 mg/mL also increased (p< 0.001) the CaS viability during biofilm formation and for mature biofilm. For the components of biofilms matrix, NAC at 100 mg/mL (mature biofilms) and 50 mg/mL (biofilm formation) seems to show a tendency in decreasing the pellet’s dry weight, supernatant’s proteins, WSP, ASP, and eDNA. Therefore, NAC showed a fungistatic effect against CaS and CaR, with a concentration-dependent effect. The antibiofilm effect was due to its fungistatic action, since only concentrations that inhibited fungal viability reduced the biofilms biomass. NAC seems to reduce the soluble components of the biofilms matrix. This is an initial in vitro study, which will drive the future clinical application of NAC as a coadjuvant in the control of Ca biofilm on the surface of dental prostheses.
Candida albicans is the main fungal pathogen in humans, responsable for local and systemic infections, such as oropharyngeal candidiasis and candidemia. These fungal infections are associated with biofilm formation. The abusive use of antifungals has led to the development of fungal resistance. The therapies towards the biofilms, mainly against the matrix, are relevant for the development of more effective new treatments. One of the agents that have demonstrated antibiofilm action is N-acetylcysteine (NAC). Therefore, the aim of this study was to evaluate the effect in vitro of NAC on biofilms of C. albicans susceptible (CaS) and resistant to fluconazole (CaR). CaS and CaR were submitted to the susceptibility test to NAC. The time-kill curves of NAC and its effect on biofilm formation and mature biofilms were also determined for both strains. Finally, the effect of NAC on the composition of the biofilms matrix was evaluated [total and pellets’s dry weight, water (WSP) and alkali soluble polysaccharides (ASP), supernatant and pellet’s proteins, and extracellular DNA (eDNA)]. The data were descriptively analyzed and submitted to the ANOVA/Welch and Tukey/Gomes-Howell or Kruskal-Wallis tests (= 0.05). The minimum inhibitory concentration of NAC with 90% of reduction (MIC90) was 25 mg/mL. Although no minimum fungicidal concentration (MFC) value was found, NAC reduced (p≤ 0,001) the fungal viability by 1.81 to 4.06 log10 for both strains. In the time-kill curves, only concentrations ≥ MIC reduced the fungal growth for 24 h, this fungistatic action was most evident between 8-10 h. Concentrations < MIC increased the fungal growth. For biofilm formation, concentrations > MIC (100 and 50 mg/mL) inhibited the viability (reduction of 1.41 to 2.77 log10) and the biomass (reduction of 39 to 88%) for both strains, and for CaS NAC at 25 and 12.5 mg/mL reduced the biomass in 77 and 34%, respectively. This inhibitory effect of NAC was also observed in differents stages of biofilm formation (24, 12, and 6h). For mature biofilms, only NAC at 100 mg/mL reduced the viability (1.12 to 2.30 log10) of both strains, and for CaR 50 and 25 mg/mL of NAC reduced 1.26 and 0.64 log10, respectively. NAC 100 mg/mL reduced the biomass in 69-72% for both strains, and for CaR NAC at 50 and 25 mg/mL also reduced the biomass in 64 and 48%, respectively. NAC 12.5 mg/mL also increased (p< 0.001) the CaS viability during biofilm formation and for mature biofilm. For the components of biofilms matrix, NAC at 100 mg/mL (mature biofilms) and 50 mg/mL (biofilm formation) seems to show a tendency in decreasing the pellet’s dry weight, supernatant’s proteins, WSP, ASP, and eDNA. Therefore, NAC showed a fungistatic effect against CaS and CaR, with a concentration-dependent effect. The antibiofilm effect was due to its fungistatic action, since only concentrations that inhibited fungal viability reduced the biofilms biomass. NAC seems to reduce the soluble components of the biofilms matrix. This is an initial in vitro study, which will drive the future clinical application of NAC as a coadjuvant in the control of Ca biofilm on the surface of dental prostheses.
Descrição
Palavras-chave
Acetilcisteína, Biofilmes, Matriz extracelular, Candida albicans, Farmacorresistência fúngica, Acetylcysteine, Biofilms, Extracellular matrix, Candida albicans, Fungal drug resistance