Variaciones del perfil genómico de virulencia en aislamientos clínicos de Klebsiella pneumoniae productores de carbapenemasas mediante secuenciación de genoma completo (WGS)

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dc.contributor.advisorBarreto Hernandez, Emiliano
dc.contributor.advisorLeal Castro, Aura Lucía
dc.contributor.authorMelo Ortiz, Derly Dallana
dc.contributor.researchgroupBioinformática
dc.coverage.countryColombia
dc.date.accessioned2025-08-26T15:33:02Z
dc.date.available2025-08-26T15:33:02Z
dc.date.issued2025
dc.descriptionilustraciones a color, diagramas, fotografías, mapasspa
dc.description.abstractKlebsiella pneumoniae, es un patógeno oportunista asociado a infecciones nosocomiales y comunitarias, representa un desafío clínico debido a su alta virulencia y resistencia antimicrobiana. Este estudio evaluó las variaciones genómicas de 70 aislamientos clínicos de K. pneumoniae productoras de carbapenemasas recolectadas entre 2020 y 2021 en un hospital colombiano, empleando secuenciación de genoma completo (WGS). Los aislamientos fueron obtenidos de diversas fuentes clínicas, predominando sangre (37,14%), orina (30%) y secreciones orotraqueales (14,28%) (Figura 8-1). Las características epidemiológicas revelaron que el 54% de los aislamientos provenían de infecciones intrahospitalarias, con una alta prevalencia en pacientes mayores de 60 años (57%) y una tasa de mortalidad del 40% (Tabla 8-1). El análisis bioinformático identificó 5518 genes en los cromosomas bacterianos, destacando los genes de adherencia, sistemas de secreción (T6SS), mecanismos antifagocíticos (genes del operón capsular), bombas de eflujo y sideróforos básicos como la enterobactina. Asimismo, se detectaron 679 genes en plásmidos, principalmente relacionados con la producción de sideróforos de alta afinidad como la aerobactina y yersiniabactina, así como toxinas genotóxicas como la colibactina. Las divergencias filogenéticas observadas reflejan adaptaciones genéticas que favorecen la colonización en ambientes hospitalarios (Figuras 8-6). La tipificación molecular (MLST) reveló la prevalencia de diversos tipos de secuencia (ST), siendo el ST1082 el más frecuente (43%), seguido por ST236 (11%) y ST258 (10%), este último vinculado a brotes epidémicos y resistencia a carbapenémicos. Además, se identificó un caso del linaje hipervirulento ST23, recientemente alertado por la OMS debido a su capacidad para causar infecciones graves en personas sanas (Figura 8-7). El análisis filogenético mediante ANI permitió identificar cinco clústeres clonales, destacando el clúster 1 como un clado genéticamente distinto, posiblemente adaptado a nichos específicos. Este hallazgo refuerza la necesidad de monitorear linajes emergentes y desarrollar estrategias efectivas de control (Figura 8-8). En conclusión, los resultados subrayan la relevancia de integrar herramientas genómicas para caracterizar factores de virulencia y rastrear la diseminación de cepas de K. pneumoniae. La combinación de resistencia antimicrobiana e hipervirulencia enfatiza la urgencia de enfoques terapéuticos integrales para mitigar el impacto clínico de este patógeno emergente. (Texto tomado de la fuente).spa
dc.description.abstractKlebsiella pneumoniae is an opportunistic pathogen associated with both nosocomial and community-acquired infections, posing a clinical challenge due to its high virulence and antimicrobial resistance. This study evaluated the genomic variations of 70 carbapenemaseproducing K. pneumoniae clinical isolates collected between 2020 and 2021 in a Colombian hospital, using whole-genome sequencing (WGS). The isolates were obtained from various clinical sources, primarily blood cultures (37.14%), urine cultures (30%), and orotracheal secretions (14.28%) (Figure 8-1). Epidemiological characteristics revealed that 54% of the isolates were from nosocomial infections, with a high prevalence among patients over 60 years of age (57%) and a mortality rate of 40% (Table 8-1). Bioinformatic analysis identified 5,518 genes in bacterial chromosomes, highlighting genes involved in adherence, secretion systems (T6SS), anti-phagocytic mechanisms (capsular operon genes), efflux pumps, and basic siderophores such as enterobactin. Additionally, 679 genes were detected in plasmids, mainly associated with high-affinity siderophores like aerobactin and yersiniabactin, as well as genotoxic toxins such as colibactin. The observed phylogenetic divergences reflect genetic adaptations favoring colonization in hospital environments (Figures 8-6). Molecular typing (MLST) revealed the prevalence of various sequence types (ST), with ST1082 being the most frequent (43%), followed by ST236 (11%) and ST258 (10%), the latter associated with epidemic outbreaks and carbapenem resistance. Furthermore, one case of the hypervirulent ST23 lineage was identified, recently highlighted by the WHO due to its ability to cause severe infections in healthy individuals (Figure 8-7). Phylogenetic analysis using ANI identified five clonal clusters, with cluster 1 representing a genetically distinct clade, possibly adapted to specific ecological niches. This finding reinforces the need to monitor emerging lineages and develop effective control strategies (Figure 8-8). In conclusion, the results emphasize the importance of integrating genomic tools to characterize virulence factors and track the dissemination of K. pneumoniae strains. The combination of antimicrobial resistance and hypervirulence underscores the urgency of comprehensive therapeutic approaches to mitigate the clinical impact of this emerging pathogen.eng
dc.description.degreelevelMaestría
dc.description.degreenameMagíster en Ciencias – Microbiología
dc.description.methodsSe incluyeron 70 aislamientos clínicos de Klebsiella pneumoniae con perfil de resistencia a carbapenémicos (CRKP), recolectados en un hospital de alta complejidad de Colombia entre el 2 de enero de 2020 y el 10 de julio de 2021. Los aislamientos se obtuvieron de pacientes con infecciones asociadas a la atención en salud, quienes otorgaron su consentimiento informado bajo aprobación del comité de ética institucional. Identificación y caracterización fenotípica La identificación microbiológica inicial se realizó mediante el sistema automatizado Phoenix™ 100 (BD), empleando paneles NID y NMIC-101. Se confirmaron resultados con pruebas bioquímicas convencionales y espectrometría de masas MALDI-TOF (MicroScan WalkAway 96 Plus, Beckman Coulter). Extracción y control de calidad del ADN El ADN genómico fue extraído con los kits PureLink® Genomic DNA Mini Kit (Invitrogen) y DNeasy Blood & Tissue (Qiagen), siguiendo las instrucciones del fabricante. La calidad del ADN fue evaluada por espectrofotometría (NanoDrop ND-2000C) y electroforesis en gel de agarosa al 1%. La cuantificación se realizó mediante fluorometría con Qubit 4.0 (Invitrogen). Preparación de librerías y secuenciación Las librerías para secuenciación por Illumina se prepararon con el kit Illumina DNA Prep. Para muestras con cobertura <50X, se complementó con secuenciación por Oxford Nanopore Technologies (ONT) usando el kit SQK-LSK109 y el sistema de barcoding EXP-NBD196. La calidad de las librerías se evaluó con el sistema Qseq100 Fragment Analyzer. Las secuencias se generaron en plataformas Illumina MiSeq (2×300 bp) y ONT MinION (48 h). Análisis bioinformático Las lecturas crudas fueron procesadas según un pipeline desarrollado por la Universidad Nacional de Colombia (https://github.com/dctopro/Pipe_Plasmid). La calidad de lecturas Illumina se verificó con FastQC v0.11.5 y Trimmomatic v0.36, y las de ONT con Guppy v6.2.7. El ensamblaje de novo se realizó con SPAdes v3.10.1; los ensamblajes híbridos con Unicycler v0.4.8. La calidad se evaluó con QUAST v5.0.2 y se utilizó como referencia K. pneumoniae HS11286 (NC_016845). Anotación y análisis genómico Los genomas ensamblados fueron anotados con Prokka v1.14.6. La confirmación taxonómica se realizó mediante redes neuronales recurrentes (RNN). La tipificación por multilocus sequence typing (MLST) se ejecutó con MLST v2.22.0. El análisis filogenético empleó Snippy-core para identificación de SNPs y RAxML v8.2.4 para construcción del árbol filogenético, visualizado con iTOL. Identificación de factores de virulencia La identificación de genes de virulencia se realizó utilizando la base de datos VFDB y la herramienta VFanalyzer. Esta permitió detectar sistemáticamente genes ortólogos relacionados con factores de virulencia conocidos y potenciales, refinando los resultados por contexto genómico, sin intervención manual.
dc.description.researchareaBiología Molecular de Agentes Infecciosos
dc.format.extent124 páginas
dc.format.mimetypeapplication/pdf
dc.identifier.instnameUniversidad Nacional de Colombiaspa
dc.identifier.reponameRepositorio Institucional Universidad Nacional de Colombiaspa
dc.identifier.repourlhttps://repositorio.unal.edu.co/spa
dc.identifier.urihttps://repositorio.unal.edu.co/handle/unal/88469
dc.language.isospa
dc.publisherUniverisidad Nacional de Colombia
dc.publisher.branchUniversidad Nacional de Colombia - Sede Bogotá
dc.publisher.facultyFacultad de Ciencias
dc.publisher.placeBogotá, Colombia
dc.publisher.programBogotá - Ciencias - Maestría en Ciencias - Microbiología
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dc.rights.accessrightsinfo:eu-repo/semantics/openAccess
dc.rights.licenseAtribución-NoComercial-SinDerivadas 4.0 Internacional
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc570 - Biología::572 - Bioquímica
dc.subject.ddc570 - Biología::579 - Historia natural microorganismos, hongos, algas
dc.subject.ddc620 - Ingeniería y operaciones afines::628 - Ingeniería sanitaria
dc.subject.lembGenómicaspa
dc.subject.lembGenomicseng
dc.subject.lembBIOINFORMATICAspa
dc.subject.lembBioinformaticseng
dc.subject.lembGENOMICA MICROBIANAspa
dc.subject.lembMicrobial genomicseng
dc.subject.lembEPIDEMIOLOGIAspa
dc.subject.lembEpidemiologyeng
dc.subject.lembMICROBIOLOGIAspa
dc.subject.lembMicrobiologyeng
dc.subject.lembGENOMAS VIRALESspa
dc.subject.lembViral genomeseng
dc.subject.proposalKlebsiella pneumoniaespa
dc.subject.proposalFactores de virulenciaspa
dc.subject.proposalSecuenciación de genoma completospa
dc.subject.proposalVirulence factorseng
dc.subject.proposalWhole-genome sequencingeng
dc.titleVariaciones del perfil genómico de virulencia en aislamientos clínicos de Klebsiella pneumoniae productores de carbapenemasas mediante secuenciación de genoma completo (WGS)spa
dc.title.translatedVariations in the genomic virulence profile of carbapenemase-producing Klebsiella pneumoniae clinical isolates by whole genome sequencing (WGS)eng
dc.typeTrabajo de grado - Maestría
dc.type.coarhttp://purl.org/coar/resource_type/c_bdcc
dc.type.coarversionhttp://purl.org/coar/version/c_ab4af688f83e57aa
dc.type.contentDataPaper
dc.type.contentText
dc.type.driverinfo:eu-repo/semantics/masterThesis
dc.type.redcolhttp://purl.org/redcol/resource_type/TM
dc.type.versioninfo:eu-repo/semantics/acceptedVersion
dcterms.audience.professionaldevelopmentEstudiantes
dcterms.audience.professionaldevelopmentMaestros
dcterms.audience.professionaldevelopmentInvestigadores
dcterms.audience.professionaldevelopmentPúblico general
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