Formación de células persistentes por daño en el DNA inducido por vancomicina y metronidazol en Clostridioides difficile
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Fecha
2024
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Clostridioides difficile es un bacilo Gram positivo, anaerobio estricto, formador de esporas el cual es
conocido como el causante de enfermedades nosocomiales como la diarrea asociada a antibióticos. Las
enfermedades causadas por la infección de C. difficile (ICD), que van desde leves a potencialmente mortales,
se propagan principalmente por la vía fecal-oral y son tratados principalmente con vancomicina y
metronidazol. Ambos corresponden a antibióticos bactericidas, en el caso de vancomicina actúa a nivel de
pared celular bacteriana mientras que el metronidazol afecta a nivel de ADN bacteriano, sin embargo,
estudios indican un mecanismo común de muerte para los antibióticos bactericidas, correspondería al estrés
oxidativo/nitrosativo, que incluye entre otros blancos el ADN. C. difficile, entre sus formas de respuesta al
estrés, tiene como mecanismo propuesto y estudiado, la formación de biopelículas. Estas estructuras
extracelulares se han observado en otras bacterias que contienen un subtipo celular el cual se ha
responsabilizado de la tolerancia al estrés. Este subtipo celular se denomina células persistentes, las cuales
son variantes transitorias de una población genéticamente homogénea y cuyo metabolismo se encuentra
disminuido al igual que su división celular, lo que le permite tolerar distintos mecanismos de estrés. A partir
de los expuesto, se plantea que el daño en el ADN producido por vancomicina y metronidazol inducen la
formación de células persistentes en Clostridioides difficile. Por consiguiente, se evaluó si los tratamientos
con vancomicina y metronidazol eran capaces de inducir la formación de células persistentes mediante un
enriquecimiento de este subcultivo con un tratamiento de lisis. Además, se determinó la viabilidad y el
metabolismo del RNA por medio de una doble tinción (Tioflavina-T e Ioduro de Propidio) donde se
confirmó la presencia de células vivas con metabolismo de RNA disminuido, lo que sugiere la presencia de
la subpoblación persistente. Por otro lado, se encontró que la cantidad de bacterias persistentesinducidas
con vancomicina son de un porcentaje cercano al 12% lo cual representa una amenaza clínica. Para evaluar
si la inducción de esta subpoblación estaba dada por el daño en el ADN, se realizó un ensayo cometa, el
cual permitió determinar que ambos antibióticos estarían provocando daño en el ADN bacteriano, sugiriendo
que este podría ser el mecanismo por el que se estarían induciendo la expresión de este fenómeno.
Clostridioides difficile is a Gram-positive bacillus, strictly anaerobic, and spore-forming, known as the causative agent of nosocomial diseases, such as antibiotic-associated diarrhea. Diseases caused by C. difficile infection (CDI), which range from mild to potentially life-threatening, are primarily spread via the fecal-oral route and are mainly treated with vancomycin and metronidazole. Both are bactericidal antibiotics; vancomycin acts on the bacterial cell wall, while metronidazole affects the bacterial DNA. However, studies indicate a common mechanism of death for bactericidal antibiotics, which corresponds to oxidative/nitrosative stress, including DNA among other targets. One of C. difficile's stress response mechanisms is the formation of biofilms. These extracellular structures have been observed in other bacteria containing a cellular subtype responsible for stress tolerance. This cellular subtype is known as persister cells, which are transient variants of a genetically homogeneous population with reduced metabolism and cell division, allowing them to tolerate various stress mechanisms. Based on this, it is hypothesized that DNA damage caused by vancomycin and metronidazole induces the formation of persister cells in Clostridioides difficile. Consequently, it was evaluated whether treatments with vancomycin and metronidazole were capable of inducing the formation of persister cells through enrichment of this subculture with a lysis treatment. Additionally, the viability and RNA metabolism were determined through a double staining (Thioflavin-T and Propidium Iodide), confirming the presence of live cells with reduced RNA metabolism, suggesting the presence of the persister subpopulation. Furthermore, it was found that the number of persister bacteria induced with vancomycin is close to 12%, representing a clinical threat. To assess whether the induction of this subpopulation was due to DNA damage, a comet assay was performed, determining that both antibiotics cause bacterial DNA damage, suggesting that this could be the mechanism inducing the expression of this phenomenon.
Clostridioides difficile is a Gram-positive bacillus, strictly anaerobic, and spore-forming, known as the causative agent of nosocomial diseases, such as antibiotic-associated diarrhea. Diseases caused by C. difficile infection (CDI), which range from mild to potentially life-threatening, are primarily spread via the fecal-oral route and are mainly treated with vancomycin and metronidazole. Both are bactericidal antibiotics; vancomycin acts on the bacterial cell wall, while metronidazole affects the bacterial DNA. However, studies indicate a common mechanism of death for bactericidal antibiotics, which corresponds to oxidative/nitrosative stress, including DNA among other targets. One of C. difficile's stress response mechanisms is the formation of biofilms. These extracellular structures have been observed in other bacteria containing a cellular subtype responsible for stress tolerance. This cellular subtype is known as persister cells, which are transient variants of a genetically homogeneous population with reduced metabolism and cell division, allowing them to tolerate various stress mechanisms. Based on this, it is hypothesized that DNA damage caused by vancomycin and metronidazole induces the formation of persister cells in Clostridioides difficile. Consequently, it was evaluated whether treatments with vancomycin and metronidazole were capable of inducing the formation of persister cells through enrichment of this subculture with a lysis treatment. Additionally, the viability and RNA metabolism were determined through a double staining (Thioflavin-T and Propidium Iodide), confirming the presence of live cells with reduced RNA metabolism, suggesting the presence of the persister subpopulation. Furthermore, it was found that the number of persister bacteria induced with vancomycin is close to 12%, representing a clinical threat. To assess whether the induction of this subpopulation was due to DNA damage, a comet assay was performed, determining that both antibiotics cause bacterial DNA damage, suggesting that this could be the mechanism inducing the expression of this phenomenon.
Notas
Memoria (Ingeniera en Biotecnología)
Palabras clave
Clostridium Difficile, Investigaciones, Resistencia a los Antibióticos