Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile

Clostridioides difficile may have a negative impact on gut microbiota composition in terms of diversity and abundance, thereby triggering functional changes supported by the differential presence of genes involved in significant metabolic pathways, such as short-chain fatty acids (SCFA). This work h...

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Autores:
Herrera, Giovanny
Castañeda, Sergio
Arboleda Rivera, Juan Camilo
Pérez-Jaramillo, Juan Esteban
Patarroyo, Manuel-Alfonso
Ramírez, Juan David
Muñoz, Marina
Tipo de recurso:
Article of investigation
Fecha de publicación:
2024
Institución:
Universidad de Ciencias Aplicadas y Ambientales U.D.C.A
Repositorio:
Repositorio Institucional UDCA
Idioma:
eng
OAI Identifier:
oai:repository.udca.edu.co:11158/6028
Acceso en línea:
https://repository.udca.edu.co/handle/11158/6028
https://doi.org/10.1016/j.micres.2024.127739
Palabra clave:
610 - Medicina y salud::616 - Enfermedades
Disbiosis
Metagenoma
Redes y Vías Metabólicas
Microbioma Gastrointestinal
Clostridioides difficile
Rights
openAccess
License
https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.es
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dc.title.eng.fl_str_mv Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
title Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
spellingShingle Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
610 - Medicina y salud::616 - Enfermedades
Disbiosis
Metagenoma
Redes y Vías Metabólicas
Microbioma Gastrointestinal
Clostridioides difficile
title_short Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
title_full Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
title_fullStr Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
title_full_unstemmed Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
title_sort Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile
dc.creator.fl_str_mv Herrera, Giovanny
Castañeda, Sergio
Arboleda Rivera, Juan Camilo
Pérez-Jaramillo, Juan Esteban
Patarroyo, Manuel-Alfonso
Ramírez, Juan David
Muñoz, Marina
dc.contributor.author.none.fl_str_mv Herrera, Giovanny
Castañeda, Sergio
Arboleda Rivera, Juan Camilo
Pérez-Jaramillo, Juan Esteban
Patarroyo, Manuel-Alfonso
Ramírez, Juan David
Muñoz, Marina
dc.subject.ddc.none.fl_str_mv 610 - Medicina y salud::616 - Enfermedades
topic 610 - Medicina y salud::616 - Enfermedades
Disbiosis
Metagenoma
Redes y Vías Metabólicas
Microbioma Gastrointestinal
Clostridioides difficile
dc.subject.decs.none.fl_str_mv Disbiosis
Metagenoma
Redes y Vías Metabólicas
Microbioma Gastrointestinal
dc.subject.proposal.eng.fl_str_mv Clostridioides difficile
description Clostridioides difficile may have a negative impact on gut microbiota composition in terms of diversity and abundance, thereby triggering functional changes supported by the differential presence of genes involved in significant metabolic pathways, such as short-chain fatty acids (SCFA). This work has evaluated shotgun metagenomics data regarding 48 samples from four groups classified according to diarrhea acquisition site (community- and healthcare facility-onset) and positive or negative Clostridioides difficile infection (CDI) result. The metagenomic-assembled genomes (MAGs) obtained from each sample were taxonomically assigned for preliminary comparative analysis concerning differences in composition among groups. The predicted genes involved in metabolism, transport, and signaling remained constant in microbiota members; characteristic patterns were observed in MAGs and genes involved in SCFA butyrate and acetate metabolic pathways for each study group. A decrease in genera and species, as well as relative MAG abundance with the presence of the acetate metabolism-related gene, was evident in the HCFO/- group. Increased antibiotic resistance markers (ARM) were observed in MAGs along with the genes involved in acetate metabolism. The results highlight the need to explore the role of acetate in greater depth as a potential protector of the imbalances produced by CDI, as occurs in other inflammatory intestinal diseases
publishDate 2024
dc.date.accessioned.none.fl_str_mv 2024-08-20T20:55:52Z
dc.date.available.none.fl_str_mv 2024-08-20T20:55:52Z
dc.date.issued.none.fl_str_mv 2024
dc.type.none.fl_str_mv Artículo de revista
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dc.identifier.citation.none.fl_str_mv Herrera, G., Castañeda, S., Arboleda, J. C., Pérez-Jaramillo, J. E., Patarroyo, M. A., Ramírez, J. D., & Muñoz, M. (2024). Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile. Microbiological Research, 127739. https://doi.org/10.1016/j.micres.2024.127739
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identifier_str_mv Herrera, G., Castañeda, S., Arboleda, J. C., Pérez-Jaramillo, J. E., Patarroyo, M. A., Ramírez, J. D., & Muñoz, M. (2024). Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile. Microbiological Research, 127739. https://doi.org/10.1016/j.micres.2024.127739
09445013
url https://repository.udca.edu.co/handle/11158/6028
https://doi.org/10.1016/j.micres.2024.127739
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dc.relation.indexed.none.fl_str_mv LaReferencia
dc.relation.citationedition.none.fl_str_mv (Ago., 2024) Artículo número 127739
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dc.relation.ispartofjournal.none.fl_str_mv Microbiological Research
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spelling Herrera, Giovannyvirtual::111-1Castañeda, Sergiovirtual::112-1Arboleda Rivera, Juan Camilovirtual::113-1Pérez-Jaramillo, Juan Estebanvirtual::114-1Patarroyo, Manuel-Alfonsovirtual::115-1Ramírez, Juan Davidvirtual::116-1Muñoz, Marinavirtual::117-12024-08-20T20:55:52Z2024-08-20T20:55:52Z2024Herrera, G., Castañeda, S., Arboleda, J. C., Pérez-Jaramillo, J. E., Patarroyo, M. A., Ramírez, J. D., & Muñoz, M. (2024). Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile. Microbiological Research, 127739. https://doi.org/10.1016/j.micres.2024.127739https://repository.udca.edu.co/handle/11158/6028https://doi.org/10.1016/j.micres.2024.12773909445013Clostridioides difficile may have a negative impact on gut microbiota composition in terms of diversity and abundance, thereby triggering functional changes supported by the differential presence of genes involved in significant metabolic pathways, such as short-chain fatty acids (SCFA). This work has evaluated shotgun metagenomics data regarding 48 samples from four groups classified according to diarrhea acquisition site (community- and healthcare facility-onset) and positive or negative Clostridioides difficile infection (CDI) result. The metagenomic-assembled genomes (MAGs) obtained from each sample were taxonomically assigned for preliminary comparative analysis concerning differences in composition among groups. The predicted genes involved in metabolism, transport, and signaling remained constant in microbiota members; characteristic patterns were observed in MAGs and genes involved in SCFA butyrate and acetate metabolic pathways for each study group. A decrease in genera and species, as well as relative MAG abundance with the presence of the acetate metabolism-related gene, was evident in the HCFO/- group. Increased antibiotic resistance markers (ARM) were observed in MAGs along with the genes involved in acetate metabolism. The results highlight the need to explore the role of acetate in greater depth as a potential protector of the imbalances produced by CDI, as occurs in other inflammatory intestinal diseasesIncluye referencias bibliográficasapplication/pdfenghttps://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.eshttps://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_abf2https://www.sciencedirect.com/science/article/pii/S094450132400140Xeys0005610 - Medicina y salud::616 - EnfermedadesDisbiosisMetagenomaRedes y Vías MetabólicasMicrobioma GastrointestinalClostridioides difficileMetagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficileArtículo de 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jecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.</li>
      <li>
        Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:
        <ol type="i">
          <li>Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.</li>
          <li>Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, los consagrados por la SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.</li>
        </ol>
      </li>
      <li>Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, ACINPRO), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.</li>
    </ol>
  </li>
  <br/>
  <li>
    Representaciones, Garantías y Limitaciones de Responsabilidad.
    <p>A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.</p>
  </li>
  <br/>
  <li>
    Limitación de responsabilidad.
    <p>A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.</p>
  </li>
  <br/>
  <li>
    Término.
    <ol type="a">
      <li>Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.</li>
      <li>Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.</li>
    </ol>
  </li>
  <br/>
  <li>
    Varios.
    <ol type="a">
      <li>Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.</li>
      <li>Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.</li>
      <li>Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.</li>
      <li>Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.</li>
    </ol>
  </li>
  <br/>
</ol>
