Influência de agentes com alvos distintos na geração de células persistentes de Streptococcus mutans e caracterização dos biofilmes derivados das células persistentes
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Data
2022-02-10
Autores
Lopes, Ana Carolina Urbano de Araujo
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Universidade Estadual Paulista (Unesp)
Resumo
Cárie dentária é uma doença crônica multifatorial, biofilme-açúcar- dependente. Biofilme dental é uma comunidade microbiana, estruturada e envolta pela matriz extracelular. A recorrência e severidade de lesões cariosas podem estar relacionadas com a presença de células persistentes no biofilme. O objetivo deste trabalho foi investigar a influência de diferentes agentes de tratamento sobre a geração de células persistentes após sucessivos ciclos de tratamentos e caracterizar os biofilmes formados a partir das células persistentes. Para isso, utilizou-se a cepa Streptococcus mutans UA159 e como agentes de tratamento, com seus respectivos alvos principais: composto 1771 (metabolismo de ácidos lipoteicóicos), hidroxichalcona 4’ (síntese de exopolissacarídeos), miricetina (síntese de exopolissacarídeos), tt-farnesol (membrana citoplasmática), fluoreto de sódio (enolase da glicólise), digluconato de clorexidina (antimicrobiano; controle positivo) e veículo (diluente dos agentes). Inicialmente, os agentes foram testados em cultura planctônica, em duas fases de crescimento: meio da fase exponencial (mid-log) e fase estacionária, determinando o período mínimo necessário para matar as células (curva de sobrevivência). Após, células persistentes em biofilme foram geradas a partir da exposição sucessiva a cada agente, durante 10 ciclos de tratamentos consecutivos (após a adesão em modelo em fundo de placa de poliestireno; meio de cultura com sacarose). Ao final, as persistentes recuperadas de cada grupo de tratamento foram utilizadas para a formação de biofilmes sobre discos de hidroxiapatita com película salivar (meio de cultura contendo saliva e sacarose alternado com combinação de saliva, sacarose e amido). O pH do meio foi aferido em diversas fases de desenvolvimento do biofilme. Após 45h de incubação, os biofilmes foram processados para análise de expressão gênica (RT-qPCR); após 67h, para análises bioquímicas (peso seco, proteínas e componentes da matriz extracelular: eDNA, ácidos lipoteicóicos, exopolissacarídeos solúveis e insolúveis), microbiológica (população microbiana) e da arquitetura (microscopia confocal, com quantificação de exopolissacarídeos e bactéria). Os dados foram submetidos à análise estatística descritiva e inferencial (α=0,05). Os diferentes agentes de tratamento apresentaram comportamento distinto em ambas as curvas de sobrevivência. A geração de células persistentes foi distinta para cada agente testado. Os biofilmes formados por células persistentes apresentaram-se diferentes, em que os biofilmes formados pós 1771, hidroxichalcona 4’ e miricetina são mais jovens em relação aos demais, por apresentarem maior expressão dos genes 16S rRNA, gtfB (exopolissacarídeos insolúveis) e eno (glicólise), além de menor de peso seco e exopolissacarídeos insolúveis na matriz. Esses achados são confirmados pela diferença na distribuição (área de cobertura) e menor quantidade dos componentes bactéria e/ou exopolissacarídeos (biovolume) nas imagens de microscopia. Portanto, agentes com diferentes alvos originam células persistentes com capacidade de formação de biofilme alterada, o que é importante no desenvolvimento de formulações para controle de biofilme cariogênico.
Dental caries is a multifactorial biofilm/sugar disease. Biofilm is a microbial community structured and surrounded by the extracellular matrix. The recurrence and severity of carious lesions may be related to persisters cells in the biofilm. This study aimed to investigate the influence of different treatment agents on the generation of persisters cells after successive treatment cycles and to characterize the biofilms formed by these persisters. For this, strain Streptococcus mutans UA159 was used, and as treatment agents (targets) were: compound 1771 (lipoteichoic acids metabolism), hydroxychalcone 4’ (exopolysaccharides synthesis), tt-farnesol (cytoplasmatic membrane), sodium fluoride (enolase; glycolysis), chlorhexidine digluconate (antimicrobial, positive control) and vehicle (diluent of agents). First, the agents were tested in planktonic culture in two growth phases: mid-exponential (mid-log) and stationary phases, determining the minimum period necessary to kill the cells (killing curves). After, persisters in the biofilm were generated from successive exposure to each agent during 10 cycles of consecutive treatments (after adhesion in a model on a polystyrene plate bottom; culture medium with sucrose). Next, the persisters recovered from each group were used to grow biofilms on saliva-coated hydroxyapatite discs (culture medium with saliva and sucrose alternated with a combination of saliva, sucrose, and starch). The pH of the medium was measured at different stages of biofilm development. After 45h, biofilms were processed for gene expression analysis (RT-qPCR); after 67h, for biochemical analysis (dry weight, proteins, and extracellular matrix components: eDNA, lipoteichoic acids, soluble and insoluble exopolysaccharides), microbiological (microbial population), and architecture (confocal microscopy, with quantification of exopolysaccharides and bacteria). Data were subjected to descriptive and inferential statistical analysis (α=0,05). The agents showed different behavior in both killing curves. The persisters generation was different for each agent tested. Biofilms formed by persisters showed some specific differences, in which biofilms formed after compound 1771, 4’ hydroxychalcone, and myricetin were younger than the others, as they present greater expression of 16S rRNA, gtfB, and eno genes, in addition to lower dry weight and insoluble exopolysaccharides in the matrix. Those findings were confirmed by the difference in distribution (coverage area) and the smaller amount of bacterial and/or exopolysaccharide components (biovolume) in the microscopy images. Therefore, agents with different targets originate persisters with distinct biofilm formation capacity, which is important in formulations development for cariogenic biofilm control.
Dental caries is a multifactorial biofilm/sugar disease. Biofilm is a microbial community structured and surrounded by the extracellular matrix. The recurrence and severity of carious lesions may be related to persisters cells in the biofilm. This study aimed to investigate the influence of different treatment agents on the generation of persisters cells after successive treatment cycles and to characterize the biofilms formed by these persisters. For this, strain Streptococcus mutans UA159 was used, and as treatment agents (targets) were: compound 1771 (lipoteichoic acids metabolism), hydroxychalcone 4’ (exopolysaccharides synthesis), tt-farnesol (cytoplasmatic membrane), sodium fluoride (enolase; glycolysis), chlorhexidine digluconate (antimicrobial, positive control) and vehicle (diluent of agents). First, the agents were tested in planktonic culture in two growth phases: mid-exponential (mid-log) and stationary phases, determining the minimum period necessary to kill the cells (killing curves). After, persisters in the biofilm were generated from successive exposure to each agent during 10 cycles of consecutive treatments (after adhesion in a model on a polystyrene plate bottom; culture medium with sucrose). Next, the persisters recovered from each group were used to grow biofilms on saliva-coated hydroxyapatite discs (culture medium with saliva and sucrose alternated with a combination of saliva, sucrose, and starch). The pH of the medium was measured at different stages of biofilm development. After 45h, biofilms were processed for gene expression analysis (RT-qPCR); after 67h, for biochemical analysis (dry weight, proteins, and extracellular matrix components: eDNA, lipoteichoic acids, soluble and insoluble exopolysaccharides), microbiological (microbial population), and architecture (confocal microscopy, with quantification of exopolysaccharides and bacteria). Data were subjected to descriptive and inferential statistical analysis (α=0,05). The agents showed different behavior in both killing curves. The persisters generation was different for each agent tested. Biofilms formed by persisters showed some specific differences, in which biofilms formed after compound 1771, 4’ hydroxychalcone, and myricetin were younger than the others, as they present greater expression of 16S rRNA, gtfB, and eno genes, in addition to lower dry weight and insoluble exopolysaccharides in the matrix. Those findings were confirmed by the difference in distribution (coverage area) and the smaller amount of bacterial and/or exopolysaccharide components (biovolume) in the microscopy images. Therefore, agents with different targets originate persisters with distinct biofilm formation capacity, which is important in formulations development for cariogenic biofilm control.
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Palavras-chave
Biofilmes., Streptococcus mutans., Matriz Extracelular., Biofilms., Streptococcus mutans., Extracellular Matrix.