Evaluación in vitro del efecto de bactiófagos específicos para Fusobacterium nucleatum en biopelículas formadas sobre distintos tejidos del diente
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Fecha
2012
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
Fusobacterium nucleatum es un bacilo alargado, Gram negativo y anaerobio
estricto, que juega un importante rol en el desarrollo de la enfermedad periodontal. Debido
a las limitaciones del tratamiento de esta patología, una terapia antimicrobiana que está
tomando fuerza es la fagoterapia. Ésta consiste en utilizar bacteriófagos para erradicar una
infección bacteriana de forma efectiva y específica. En nuestro laboratorio, se han descrito
dos bacteriófagos específicos para F. nucleatum, denominados Fnp~02-14 y Fnp~ll, que
producen una disminución significativa del recuento microbiano en una biopelícula in vitro
sobre poliestireno. El objetivo de este trabajo fue determinar si los bacteriófagos generan el
mismo efecto en una biopelícula formada sobre distintas superficies del diente.
Para evaluar el efecto de los bacteriófagos sobre la biopelícula, inicialmente se
determinaron las condiciones para mejorar la formación de biopelícula (saliva, pH,
nutrientes, triptófano, tiempo de incubación). Con las condiciones definidas se evaluó la
formación de biopelícula de Fusobacterium nucleatum sobre tres tejidos diferentes:
esmalte, dentina de corona y dentina de raíz, a través de cuantificación con cristal violeta.
Finalmente, la biopelícula fue tratada con los bacteriófagos Fnp~02-14 y Fnp~ 11 y se
realizó un recuento final de unidades formadoras de colonia, tanto en una biopelícula
simple como una mixta.
Los resultados mostraron que la formación de biopelícula, luego de una incubación
de 48 h, se ve favorecida en una superficie cubierta con saliva concentrada, en un medio
tamponado a pH 7,4 con buffer fosfato y suplementado con triptófano. Se determinó que la
formación de biopelícula es significativamente mayor cuando se forma sobre dentina. La
acción de la mezcla de bacteriófagos fue capaz de eliminar por completo las bacterias
establecidas sobre los diferentes tejidos con un MOl 0,01. Utilizando el mismo MOl en una
biopelícula mixta la mezcla de bacteriófagos disminuye un 93% la población de
Fusobacterium nucleatum.
Nuestros resultados sugieren fuertemente que la fagoterapia sería efectiva como una
terapia complementaria al actual tratamiento para el biocontrol de Fusobacterium
nucleatum en la enfermedad periodontal.
Fusobacterium nucleatum is an anaerobic, Gram-negative and long bacillus that plays an important role in the development of periodontal disease. Due to the limitations of treatment of this disease, the antimicrobial therapy that is gaining ground is the phagetherapy. This correspond to the use of bacteriophages to eradicate a bacteria! infection in an effective and specific manner. In our laboratory, we have described two specific bacteriophages for Fusobacterium nucleatum, called Fnp~02-14 and Fnp~ 11, which cause in vitro a significant reduction of the microbial count in a biofilm over polystyrene. The aim of this study was to determine whether bacteriophages ha ve the same effect on a biofilm formed over different tooth surfaces. To evaluate the effect of bacteriophages on the biofilm were initially determined the conditions for improving the formation of this biofilm (saliva, pH, nutrients, tryptophan, and incubation time) and was evaluated on three different tissues: enamel, crown dentin and root dentin. With the conditions optimized it was evaluated the biofilm formation of Fusobacterium nucleatum on each tissue through quantification with crystal violet. Finally, the biofilm was treated with bacteriophages Fnp~02-14 and Fnp~ 11 and made a final count of colony forming units in a simple and mixed biofilm. The results showed that the biofilm formation after an incubation of 48 h was favored on a surface area covered with saliva, in a culture media buffered to pH 7.4 with phosphate buffer, supplemented with tryptophan. It was found that biofilm formation is significantly higher when formed on dentin. The action of the mixture of phages was able to completely eliminate the bacteria established on the different tissues with MOl 0,01. Using the same MOl in a mixed biofilm, the bacteriophages mixture decreased by 93% the population of Fusobacterium nucleatum. Our results strongly suggest that the phagetherapy could be effective as an adjunctive therapy to current treatment for biocontrol of Fusobacterium nucleatum in periodontal disease.
Fusobacterium nucleatum is an anaerobic, Gram-negative and long bacillus that plays an important role in the development of periodontal disease. Due to the limitations of treatment of this disease, the antimicrobial therapy that is gaining ground is the phagetherapy. This correspond to the use of bacteriophages to eradicate a bacteria! infection in an effective and specific manner. In our laboratory, we have described two specific bacteriophages for Fusobacterium nucleatum, called Fnp~02-14 and Fnp~ 11, which cause in vitro a significant reduction of the microbial count in a biofilm over polystyrene. The aim of this study was to determine whether bacteriophages ha ve the same effect on a biofilm formed over different tooth surfaces. To evaluate the effect of bacteriophages on the biofilm were initially determined the conditions for improving the formation of this biofilm (saliva, pH, nutrients, tryptophan, and incubation time) and was evaluated on three different tissues: enamel, crown dentin and root dentin. With the conditions optimized it was evaluated the biofilm formation of Fusobacterium nucleatum on each tissue through quantification with crystal violet. Finally, the biofilm was treated with bacteriophages Fnp~02-14 and Fnp~ 11 and made a final count of colony forming units in a simple and mixed biofilm. The results showed that the biofilm formation after an incubation of 48 h was favored on a surface area covered with saliva, in a culture media buffered to pH 7.4 with phosphate buffer, supplemented with tryptophan. It was found that biofilm formation is significantly higher when formed on dentin. The action of the mixture of phages was able to completely eliminate the bacteria established on the different tissues with MOl 0,01. Using the same MOl in a mixed biofilm, the bacteriophages mixture decreased by 93% the population of Fusobacterium nucleatum. Our results strongly suggest that the phagetherapy could be effective as an adjunctive therapy to current treatment for biocontrol of Fusobacterium nucleatum in periodontal disease.
Notas
Tesis (Magister en Biotecnología)
Palabras clave
Bacteriófagos, Enfermedades Periodontales