Eficácia de compostos com alvos distintos no controle de biofilmes de Streptococcus mutans e Candida albicans
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2022-02-24
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Universidade Estadual Paulista (Unesp)
Resumo
A doença cárie é um problema de saúde pública mundial, causada por biofilme patogênico modulado por uma dieta rica em açúcares (principalmente, sacarose). A associação de Candida albicans e Streptococcus mutans tem sido encontrada em biofilmes de lesões clínicas de cárie da infância. O biofilme misto dessas duas espécies é mais tolerante à tratamentos tópicos com agentes que controlam e diminuiem a virulência de biofilme simples de S. mutans. Portanto, avaliou-se as atividades antimicrobiana, antibiofilme e citotóxica de seis compostos com possíveis alvos em S. mutans e C. albicans: terpenóide (tt-farnesol ou Far), flavonoide (mirecetina ou Mir), três hidroxichalconas (2’ hidroxichalconas: AGN e R815; 4’ hidroxichalcona: C135) e um agente modulador da síntese de ácido lipoteicóico (composto 1771). Assim, determinou-se o potencial antimicrobiano (antibacteriano e antifúngico) e antibiofilme (modelos em placas de poliestireno: biofilmes iniciais de 24h e pré-formados de 48h) dos compostos isolados contra os microrganismos isolados. Após, combinou-se os compostos e concentrações promissoras entre si e com e sem flúoreto de sódio (F) para avaliação antibiofilme contra biofilmes mistos (iniciais e pré-formados). Posteriormente, avaliou-se a eficácia da aplicação tópica (5 min) dos compostos promissores contra biofilmes mistos formados em discos de hidroxiapatita com película salivar em 28h (expressão gênica) e 43h [população microbiana, biomassa, proteinas na porção insolúvel, componentes da matriz extracelular (exopolissacarídeos solúveis em água e em álcali, eDNA e proteinas), estrutura 3D e microscopia eletrônica de varredura (MEV) do biofilme]. Ainda, determinou-se o efeito citotóxico dos tratamentos usados para a aplicação tópica sobre monocamanda de queratinócitos orais. Os testes estatísticos consideraram α=0,05. Dentre os agentes avaliados, C135, Far, Mir e 1771 foram promissores para as atividades antimicrobiana e antibiofilme sobre biofilme simples de cada espécie (24 e 48h). Esses compostos combinados com e sem flúor foram eficazes contra as duas espécies microbianas (biofilme misto inicial); entretanto C135+Far+Mir, C135+Far+F e todos compostos combinados foram eficazes contra biofilmes mistos pré-formados. Os biofilmes mistos tratados topicamente com C135+Far+F apresentaram menor expressão gênica para genes de C. albicans associados à síntese (BGL2, FKS1) e remodelagem de -glucanos (XOG1, PHR1, PHR2) (componentes da matriz extracelular), estresse oxidativo (SOD1), efluxo de fármacos (CDR1, ERG11) e maior expressão do gene de S. mutans nox1 (tolerância à estresses oxidativo e ácido); já C135+Far resultou em menor quantidade de exopolissacarídeos insolúveis (quantificação bioquímica), biomassa e proteínas na porção insolúvel dos biofilmes, e redução de expressão de BGL2, ERG11, SOD1 e PHR2. A quantificação dos microrganismos e expopolissacarídeos a partir da microscopia confocal mostrou menor biovolume e porcentagem de cobertura da estrutura 3D para C135+F, C135+Far+F e C135; e nas imagens de MEV, C135+Far e C135+Far+F mostraram C. albicans apenas na forma de levedura. A exposição aos tratamentos por 30 min, mostrou citotoxicidade moderada para C135+F e C135 enquanto todos tratamentos contendo Far e a clorexidina (0,12%) foram altamente citotóxicos. Portanto, as formulações avaliadas afetaram alvos distintos dos biofilmes simples e misto de S. mutans e C. albicans; com efeito mais pronunciado contra o fungo em biofilmes mistos.
Dental caries is a worldwide public health problem caused by a pathogenic biofilm modulated by a diet rich in sugars (mainly sucrose). Candida albicans has been found in association with Streptococcus mutans in biofilms of clinical childhood dental caries lesions. Dual-species biofilms of these two species are more tolerant to topical treatments with agents that can control and decrease the virulence of single-species S. mutans biofilm. Therefore, the antimicrobial, antibiofilm, and cytotoxic activity of six compounds with possible targets in S. mutans and C. albicans were evaluated: a terpenoid (tt-farnesol or Far), a flavonoid (myricetin or Mir), three hydroxychalcones (2'-hydroxychalcones: AGN and R815; 4'-hydroxychalcone: C135) and a lipoteichoic acid synthesis modulating agent (compound 1771). Thus, the antimicrobial (antibacterial and antifungal) and antibiofilm (polystyrene model plates: initial 24h and 48h pre-formed biofilms) potential of the isolated compounds against isolated microorganisms were determined. Afterward, the promising compounds and concentrations were combined with each other and with or without sodium fluoride (F) for antibiofilm evaluation against dual-species biofilms (initial and pre-formed). After, the effectiveness of topical application (5 min) of the promising compounds with and without F against dual-species biofilms formed on hydroxyapatite discs with salivary pellicle was evaluated via different analyzes at 28h (gene expression) and 43h [microbial population, biomass, proteins of the insoluble portion, extracellular matrix components (water- and alkali-soluble exopolysaccharides, eDNA, and proteins), 3D structure and scanning electron microscopy (SEM) of the biofilm]. Also, the cytotoxicity of treatments used for topical application on oral keratinocytes (monolayer) was determined. Statistical tests followed α=0.05. Among the agents evaluated, C135, Far, Mir, and 1771 were promising for antimicrobial and antibiofilm activities on single-species biofilms of both species (24 and 48h). Combined compounds with and without fluoride were effective against both microbial species (initial dual-species biofilm); C135+Far+Mir, C135+Far+F and all compounds combined were effective against pre-formed dual-species biofilms. The topical treatments on dual-species biofilms with C135+Far+F showed lower gene expression for C. albicans genes associated with synthesis (BGL2, FKS1) and remodeling of b-glucans (XOG1, PHR1, PHR2) (extracellular matrix components), oxidative stress (SOD1), drug efflux (CDR1, ERG11) and increased expression of the S. mutans gene nox1 (tolerance to oxidative and acidic stress). C135+Far resulted in a lower amount of insoluble exopolysaccharides (biochemical quantification), biomass, and proteins in the insoluble portion of the biofilms and reduced the expression of BGL2, ERG11, SOD1, and PHR2. However, the quantification of microorganisms and exopolysaccharides from confocal microscopy showed lower biovolume and percentage of coverage of the 3D structure for C135+F, C135+Far+F, and C135. For SEM, biofilms treated with C135+Far e C135+Far+F presented C. albicans only as yeast form. The exposure to treatments (30 min) demonstrated moderate cytotoxicity for C135+F and C135, while treatments containing Far or chlorhexidine (0.12%) were highly cytotoxic. Therefore, the evaluated formulations affected different targets in single- and dual-species biofilm of S. mutans and C. albicans, with a more pronounced effect against C. albicans cells in dual-species biofilms
Dental caries is a worldwide public health problem caused by a pathogenic biofilm modulated by a diet rich in sugars (mainly sucrose). Candida albicans has been found in association with Streptococcus mutans in biofilms of clinical childhood dental caries lesions. Dual-species biofilms of these two species are more tolerant to topical treatments with agents that can control and decrease the virulence of single-species S. mutans biofilm. Therefore, the antimicrobial, antibiofilm, and cytotoxic activity of six compounds with possible targets in S. mutans and C. albicans were evaluated: a terpenoid (tt-farnesol or Far), a flavonoid (myricetin or Mir), three hydroxychalcones (2'-hydroxychalcones: AGN and R815; 4'-hydroxychalcone: C135) and a lipoteichoic acid synthesis modulating agent (compound 1771). Thus, the antimicrobial (antibacterial and antifungal) and antibiofilm (polystyrene model plates: initial 24h and 48h pre-formed biofilms) potential of the isolated compounds against isolated microorganisms were determined. Afterward, the promising compounds and concentrations were combined with each other and with or without sodium fluoride (F) for antibiofilm evaluation against dual-species biofilms (initial and pre-formed). After, the effectiveness of topical application (5 min) of the promising compounds with and without F against dual-species biofilms formed on hydroxyapatite discs with salivary pellicle was evaluated via different analyzes at 28h (gene expression) and 43h [microbial population, biomass, proteins of the insoluble portion, extracellular matrix components (water- and alkali-soluble exopolysaccharides, eDNA, and proteins), 3D structure and scanning electron microscopy (SEM) of the biofilm]. Also, the cytotoxicity of treatments used for topical application on oral keratinocytes (monolayer) was determined. Statistical tests followed α=0.05. Among the agents evaluated, C135, Far, Mir, and 1771 were promising for antimicrobial and antibiofilm activities on single-species biofilms of both species (24 and 48h). Combined compounds with and without fluoride were effective against both microbial species (initial dual-species biofilm); C135+Far+Mir, C135+Far+F and all compounds combined were effective against pre-formed dual-species biofilms. The topical treatments on dual-species biofilms with C135+Far+F showed lower gene expression for C. albicans genes associated with synthesis (BGL2, FKS1) and remodeling of b-glucans (XOG1, PHR1, PHR2) (extracellular matrix components), oxidative stress (SOD1), drug efflux (CDR1, ERG11) and increased expression of the S. mutans gene nox1 (tolerance to oxidative and acidic stress). C135+Far resulted in a lower amount of insoluble exopolysaccharides (biochemical quantification), biomass, and proteins in the insoluble portion of the biofilms and reduced the expression of BGL2, ERG11, SOD1, and PHR2. However, the quantification of microorganisms and exopolysaccharides from confocal microscopy showed lower biovolume and percentage of coverage of the 3D structure for C135+F, C135+Far+F, and C135. For SEM, biofilms treated with C135+Far e C135+Far+F presented C. albicans only as yeast form. The exposure to treatments (30 min) demonstrated moderate cytotoxicity for C135+F and C135, while treatments containing Far or chlorhexidine (0.12%) were highly cytotoxic. Therefore, the evaluated formulations affected different targets in single- and dual-species biofilm of S. mutans and C. albicans, with a more pronounced effect against C. albicans cells in dual-species biofilms
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Biofilmes, Streptococcus mutans, Candida albicans, Biofilms, Streptococcus mutans, Candida albicans