Otimização da seleção de cepas potencialmente probióticas para terapia Personalizada
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2021-02-18
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Universidade Estadual Paulista (Unesp)
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Introdução: A ingestão de probióticos, prebióticos, o transplante de microbiota fecal e o transplante de consórcio bacteriano são as principais estratégias utilizadas para modular a microbiota intestinal (MI) humana e prevenir ou tratar diferentes doenças intestinais. Entretanto, os efeitos de tais tratamentos são inconsistentes, provavelmente devido à resiliência desse complexo ecossistema e às características genéticas do hospedeiro. Objetivo: Otimizar a seleção e caracterização de cepas potencialmente probióticas, a partir de amostras fecais de crianças saudáveis e a construção de um “banco de microbiota” individualizado para terapia personalizada. Metodologia: As cepas bacterianas comensais foram isoladas das fezes de crianças (n=5), com idades entre dois e seis anos, por plaqueamento em meios seletivos. Posteriormente, foi realizada a caracterização das cepas isoladas pela técnica de amplificação aleatória de DNA polimórfico (RAPD-PCR), coloração de Gram, teste de catalase, sequenciamento do gene 16S rRNA, teste de resistência a condições gastrointestinais simuladas e suscetibilidade a antibióticos. Resultados: Foram isoladas cepas pertencentes aos gêneros Enterococcus 45% (E. faecium, E. faecalis, E. avium, E. Hirae e E. galinarrium), Lactobacillus 31,7 % (L. paracasei e L. rhamnosus), Leuconostoc 12,7% (L. lactis), Weissella 7,9% (W.confusa) e Streptococcus 3,2 % (S. salivarius). Todas as cepas se mostraram altamente resistentes às condições gastrointestinais simuladas, com redução máxima de 1,4 log10 UFC / mL e população final entre 9 e 8 log10 UFC / mL e foram sensíveis a pelo menos três antibióticos (ampicilina, cloranfenicol e ciprofloxacina). Após identificar e garantir o atendimento aos critérios mínimos para cepas potencialmente probióticas, as mesmas foram armazenadas em bancos individuais de células microbianas, por criopreservação a -80 °C. Conclusão: A criação de bancos de células microbianas potencialmente probióticas individualizados, a partir de amostras de fezes humanas, se mostrou viável para a população estudada e representa uma opção promissora para o tratamento personalizado de doenças relacionadas à disbiose.
Background: The intake of probiotics, prebiotics, fecal microbiota transplantation and bacterial consortium transplantation are the main strategies used to modulate the human intestinal microbiota (IM) and prevent or treat different intestinal diseases. However, the effects of such treatments are inconsistent, probably due to the resilience of this complex ecosystem and the host's genetic characteristics. Objective: To optimize the selection and characterization of potentially probiotic strains, from fecal samples from healthy children and the construction of an individualized bacterial cell bank for personalized therapy. Methodology: Commensal bacterial strains were isolated from the feces of children (n = 5), aged between two and six years, by plating in selective media. Subsequently, isolated strains were characterized by random amplification of polymorphic DNA (RAPD-PCR), Gram stain, catalase test,16S rRNA gene sequencing, resistance to simulated gastrointestinal conditions and susceptibility to antibiotics. Results: It was isolate potentially probiotic strains of the genera Enterococcus 45% (E. faecium, E. faecalis, E. avium, E. hirae, E. galinarrium), Lactobacillus 31.7% (L. paracasei and L. rhamnosus) , Leuconostoc 12.7% (L. lactis), Weissella 7.9% (W.confusa) and Streptococcus 3.2% (S. salivarius). All strains proved to be highly resistant to in vitro simulated gastrointestinal conditions, with a maximum reduction of 1.4 log10 CFU / mL and final population between 9 and 8 log10 CFU / mL and were sensitive to at least three antibiotics (ampicillin, chloramphenicol and ciprofloxacin). After identifying and ensuring compliance with the minimum criteria for potentially probiotic strains, they were stored in individual microbial cell banks by cryopreservation at -80 ° C. Conclusion: The creation of potentially individualized probiotic microbial cell banks, based on human feces samples, proved to be viable for the population studied and represents a promising option for the personalized treatment of diseases related to dysbiosis.
Background: The intake of probiotics, prebiotics, fecal microbiota transplantation and bacterial consortium transplantation are the main strategies used to modulate the human intestinal microbiota (IM) and prevent or treat different intestinal diseases. However, the effects of such treatments are inconsistent, probably due to the resilience of this complex ecosystem and the host's genetic characteristics. Objective: To optimize the selection and characterization of potentially probiotic strains, from fecal samples from healthy children and the construction of an individualized bacterial cell bank for personalized therapy. Methodology: Commensal bacterial strains were isolated from the feces of children (n = 5), aged between two and six years, by plating in selective media. Subsequently, isolated strains were characterized by random amplification of polymorphic DNA (RAPD-PCR), Gram stain, catalase test,16S rRNA gene sequencing, resistance to simulated gastrointestinal conditions and susceptibility to antibiotics. Results: It was isolate potentially probiotic strains of the genera Enterococcus 45% (E. faecium, E. faecalis, E. avium, E. hirae, E. galinarrium), Lactobacillus 31.7% (L. paracasei and L. rhamnosus) , Leuconostoc 12.7% (L. lactis), Weissella 7.9% (W.confusa) and Streptococcus 3.2% (S. salivarius). All strains proved to be highly resistant to in vitro simulated gastrointestinal conditions, with a maximum reduction of 1.4 log10 CFU / mL and final population between 9 and 8 log10 CFU / mL and were sensitive to at least three antibiotics (ampicillin, chloramphenicol and ciprofloxacin). After identifying and ensuring compliance with the minimum criteria for potentially probiotic strains, they were stored in individual microbial cell banks by cryopreservation at -80 ° C. Conclusion: The creation of potentially individualized probiotic microbial cell banks, based on human feces samples, proved to be viable for the population studied and represents a promising option for the personalized treatment of diseases related to dysbiosis.
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probiótico, personalizado, microbiota, crianças, banco de células, cell bank