Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens

Serratia marcescens is a Gram-negative, motile, free-living, ubiquitous bacillus belonging to the family Yersiniaceae. In recent decades, S. marcescens has been recognized by the World Health Organisation as a priority antibiotic resistant pathogen. The emergence of S. marcescens in the in-hospital...

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Autor principal: Gambino, Anahí Samanta
Otros Autores: Quiroga, María Paula
Formato: Tesis doctoral acceptedVersion
Lenguaje:Español
Publicado: Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica 2023
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Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7826
https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7826.dir/7826.PDF
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Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_7826
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Español
orig_language_str_mv spa
topic Ciencias de la vida
spellingShingle Ciencias de la vida
Gambino, Anahí Samanta
Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens
topic_facet Ciencias de la vida
description Serratia marcescens is a Gram-negative, motile, free-living, ubiquitous bacillus belonging to the family Yersiniaceae. In recent decades, S. marcescens has been recognized by the World Health Organisation as a priority antibiotic resistant pathogen. The emergence of S. marcescens in the in-hospital niche combined with its intrinsic resistance to antibiotics (ATBs) could lead to the emergence of lineages with limited therapeutic options. Today, given the existence of multiple evidences on the accelerated acquisition of antibiotic resistance in this species, we hypothesize that the in-depth study of its resistome and mobilome will provide valuable information on the molecular basis associated with adaptation to the in-hospital niche. First, S. marcescens strain SCH909, a multidrug-resistant (MDR) isolate that has been used as a biological model in our laboratory, was selected and sequenced, which was the starting point for the genomic analyses of this thesis. Subsequently, the genomes of a collection of clinical strains of S. marcescens (n=152), obtained from different health entities located in four provinces of Argentina (14 hospitals, 1997-2018), were sequenced. As a result of the latter, a local database of genomes from our region was constructed. To understand the components that nourish the resistome, this database was compared with the genomes of other strains from different niches, including an environmental strain called S. marcescens He2Co2 isolated from the pavement of the Autonomous City of Buenos Aires. Finally, we evaluated the ability of S. marcescens to capture, maintain and disseminate plasmids carrying ?-lactamases both intraspecies and interspecies, evidencing the possibility of this species to microevolve towards extreme (XDR) and pandrug resistance (PDR). Two already sequenced S. marcescens isolates, one from the in-hospital niche (S. marcescens SM39) and one from the environmental niche (S. marcescens Db11), were selected; comparative genomics studies were performed with S. marcescens SCH909. The study of the mobilome of these three strains revealed that their genomes contain a wide variety of mobile genetic elements (MGEs). A total of 13 prophages, 39 insertion sequences (ISs), five distinct integrons, one Group II intron, one genomic island (GI), three plasmids and a total of 30 antibiotic resistance genes (ARGs) were identified in the three selected genomes. At the same time, when analyzing the resistome of the three strains, we found that S. marcescens SCH909 is the major carrier of ARGs (n=18), of which 61.11% (11/18) are inserted in the chromosome. Most of these ARGs were located within a novel resistance GI that we named SmaR, whose structure is similar to the characteristic GIs of A. baumannii. Likewise, within the resistome of S. marcescens strains circulating in Argentina, we also located other chromosomal resistance GIs that had not been previously described. One was named SmaR-2, which carries the blaCTX-M-2 gene disseminating in several clusters. In turn, within the vertically inherited resistome, we analyzed other components such as intrinsic ARGs, efflux pumps and antibiotic resistance proto-genes, observing their abundance and diversity. We identified all acquired ARGs contributing to the resistome (n=593), most of them spreading across different genetic platforms. Among them, the presence of the mcr9.1 gene, conferring resistance to colistin is highlighted. This ATB, to which S. marcescens is intrinsically resistant, is used as last resort for infections caused by XDR pathogens. Phylogenetic analysis of the strains revealed lineages exhibiting resistomes with high clinical impact including polyclonal dissemination of the blaCTX-M-2 and blaKPC-2 genes as well as the presence of plasmids carrying the carbapenemases blaNDM-1 and blaIMP-8 genes. We also found a cluster grouping three strains from different hospitals containing a novel allele of the carbapenemase blaSPR-1. To understand the role of S. marcescens in the dissemination of antibiotic resistance, we studied the activity of plasmids associated with the dissemination of ?-lactam resistance in our strain´s collection. Four Plasmids and two plasmid pseudo molecules were molecularly characterized. The conjugative capacity of these plasmids was evaluated, both interspecies (E. coli J53) and intraspecies (S. marcescens SCH909). In vivo assays resulted in pDCASG1-CTX (carrying the blaCTX-M-2 gene) and pDCASG6-NDM (carrying blaNDM-1 gene) were conjugative. When evaluating plasmid maintenance in the transconjugant strains, the results showed that S. marcescens SCH909 was able to maintain the pDCASG6-NDM plasmid longer than E. coli J53 (97,78% versus 20% at ten days, respectively). In conclusion, this work revealed the heterogeneity of the antibiotic resistance patterns that compose the resistome of S. marcescens species. Its ability to maintain and disseminate MGEs associated with MDR genetic platforms could serve as a "Trojan horse", silently contributing to the dissemination of clinically relevant acquired ARGs in the in-hospital niche over time. In turn, the carriage of a wide variety of genetic platforms allows for greater adaptability and survival in different habitats including both environmental and clinical niches, making this species a likely nexus between different bacterial groups.
author2 Quiroga, María Paula
author_facet Quiroga, María Paula
Gambino, Anahí Samanta
format Tesis doctoral
Tesis doctoral
acceptedVersion
author Gambino, Anahí Samanta
author_sort Gambino, Anahí Samanta
title Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens
title_short Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens
title_full Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens
title_fullStr Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens
title_full_unstemmed Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens
title_sort mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno serratia marcescens
publisher Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
publishDate 2023
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7826
https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7826.dir/7826.PDF
work_keys_str_mv AT gambinoanahisamanta mecanismosinvolucradosenlaevolucionhacialaextremadrogaresistenciadelpatogenoserratiamarcescens
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spelling I28-R145-HWA_78262025-08-01 Serratia marcescens is a Gram-negative, motile, free-living, ubiquitous bacillus belonging to the family Yersiniaceae. In recent decades, S. marcescens has been recognized by the World Health Organisation as a priority antibiotic resistant pathogen. The emergence of S. marcescens in the in-hospital niche combined with its intrinsic resistance to antibiotics (ATBs) could lead to the emergence of lineages with limited therapeutic options. Today, given the existence of multiple evidences on the accelerated acquisition of antibiotic resistance in this species, we hypothesize that the in-depth study of its resistome and mobilome will provide valuable information on the molecular basis associated with adaptation to the in-hospital niche. First, S. marcescens strain SCH909, a multidrug-resistant (MDR) isolate that has been used as a biological model in our laboratory, was selected and sequenced, which was the starting point for the genomic analyses of this thesis. Subsequently, the genomes of a collection of clinical strains of S. marcescens (n=152), obtained from different health entities located in four provinces of Argentina (14 hospitals, 1997-2018), were sequenced. As a result of the latter, a local database of genomes from our region was constructed. To understand the components that nourish the resistome, this database was compared with the genomes of other strains from different niches, including an environmental strain called S. marcescens He2Co2 isolated from the pavement of the Autonomous City of Buenos Aires. Finally, we evaluated the ability of S. marcescens to capture, maintain and disseminate plasmids carrying ?-lactamases both intraspecies and interspecies, evidencing the possibility of this species to microevolve towards extreme (XDR) and pandrug resistance (PDR). Two already sequenced S. marcescens isolates, one from the in-hospital niche (S. marcescens SM39) and one from the environmental niche (S. marcescens Db11), were selected; comparative genomics studies were performed with S. marcescens SCH909. The study of the mobilome of these three strains revealed that their genomes contain a wide variety of mobile genetic elements (MGEs). A total of 13 prophages, 39 insertion sequences (ISs), five distinct integrons, one Group II intron, one genomic island (GI), three plasmids and a total of 30 antibiotic resistance genes (ARGs) were identified in the three selected genomes. At the same time, when analyzing the resistome of the three strains, we found that S. marcescens SCH909 is the major carrier of ARGs (n=18), of which 61.11% (11/18) are inserted in the chromosome. Most of these ARGs were located within a novel resistance GI that we named SmaR, whose structure is similar to the characteristic GIs of A. baumannii. Likewise, within the resistome of S. marcescens strains circulating in Argentina, we also located other chromosomal resistance GIs that had not been previously described. One was named SmaR-2, which carries the blaCTX-M-2 gene disseminating in several clusters. In turn, within the vertically inherited resistome, we analyzed other components such as intrinsic ARGs, efflux pumps and antibiotic resistance proto-genes, observing their abundance and diversity. We identified all acquired ARGs contributing to the resistome (n=593), most of them spreading across different genetic platforms. Among them, the presence of the mcr9.1 gene, conferring resistance to colistin is highlighted. This ATB, to which S. marcescens is intrinsically resistant, is used as last resort for infections caused by XDR pathogens. Phylogenetic analysis of the strains revealed lineages exhibiting resistomes with high clinical impact including polyclonal dissemination of the blaCTX-M-2 and blaKPC-2 genes as well as the presence of plasmids carrying the carbapenemases blaNDM-1 and blaIMP-8 genes. We also found a cluster grouping three strains from different hospitals containing a novel allele of the carbapenemase blaSPR-1. To understand the role of S. marcescens in the dissemination of antibiotic resistance, we studied the activity of plasmids associated with the dissemination of ?-lactam resistance in our strain´s collection. Four Plasmids and two plasmid pseudo molecules were molecularly characterized. The conjugative capacity of these plasmids was evaluated, both interspecies (E. coli J53) and intraspecies (S. marcescens SCH909). In vivo assays resulted in pDCASG1-CTX (carrying the blaCTX-M-2 gene) and pDCASG6-NDM (carrying blaNDM-1 gene) were conjugative. When evaluating plasmid maintenance in the transconjugant strains, the results showed that S. marcescens SCH909 was able to maintain the pDCASG6-NDM plasmid longer than E. coli J53 (97,78% versus 20% at ten days, respectively). In conclusion, this work revealed the heterogeneity of the antibiotic resistance patterns that compose the resistome of S. marcescens species. Its ability to maintain and disseminate MGEs associated with MDR genetic platforms could serve as a "Trojan horse", silently contributing to the dissemination of clinically relevant acquired ARGs in the in-hospital niche over time. In turn, the carriage of a wide variety of genetic platforms allows for greater adaptability and survival in different habitats including both environmental and clinical niches, making this species a likely nexus between different bacterial groups. Fil: Gambino, Anahí Samanta. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Quiroga, María Paula Di Conza, José Centrón, Daniela Gambino, Anahí Samanta 2023-03-17 Serratia marcescens es un bacilo Gram-negativo, móvil, de vida libre y ubicuo, perteneciente a la familia Yersiniaceae. En las últimas décadas, S. marcescens ha sido reconocida por la Organización Mundial de la Salud como patógeno prioritario resistente a antibióticos (RAM). La emergencia de esta especie en el nicho intrahospitalario combinada con la resistencia intrínseca a los antibióticos (ATBs) que la caracteriza, podría derivar en la aparición de linajes con opción terapéutica limitada. Hoy en día, dada la existencia de múltiples evidencias sobre la adquisición acelerada de la RAM en S. marcescens nos permiten hipotetizar que el estudio profundo de su resistoma y moviloma proporcionará información sobre la base molecular asociada a la adaptación al nicho intrahospitalario. Primeramente, se seleccionó y secuenció la cepa S. marcescens SCH909, que es un aislamiento multirresistente (MDR) que en nuestro laboratorio se ha utilizado como modelo biológico, lo cual fue el punto de partida para los análisis genómicos de esta tesis. Posteriormente, se secuenciaron los genomas de una colección de cepas clínicas de S. marcescens (n=152), obtenida de distintas entidades de salud ubicadas en cuatro provincias de Argentina (14 hospitales, 1997-2018). Como resultado de esto último, se construyó una base de datos local de genomas de nuestra región. Para comprender los componentes que nutren el resistoma, dicha base de datos fue comparada con los genomas de otras cepas procedentes de distintos hábitats, incluida una cepa ambiental denominada S. marcescens He2Co2 aislada de una vereda de la Ciudad Autónoma de Buenos Aires. Finalmente, evaluamos la capacidad de S. marcescens de captar, mantener y diseminar plásmidos portadores de ?-lactamasas tanto intraespecie como interespecie evidenciando la posibilidad de esta especie de microevolucionar hacia la extrema (XDR) y pandroga resistencia (PDR). Se seleccionaron dos aislamientos de S. marcescens ya secuenciados, uno del nicho intrahospitalario (S. marcescens SM39) y otro del nicho ambiental (S. marcescens Db11) y se realizaron los estudios de genómica comparativa con el genoma de S. marcescens SCH909. El estudio del moviloma de estas tres cepas reveló que sus genomas contienen una amplia variedad de elementos genéticos móviles (EGMs). Se identificaron en los tres genomas seleccionados un total de 13 profagos, 39 secuencias de inserción (ISs), cinco integrones distintos, un intrón de Grupo II, una isla genómica (IG), tres plásmidos y un total de 30 genes de resistencia a ATBs (GRAs). Paralelamente, al analizar el resistoma de las tres cepas evidenciamos que S. marcescens SCH909 es la portadora mayoritaria de GRAs (n=18), de los cuales el 61,11% (11/18) se encuentran insertos dentro del cromosoma. En su mayoría, dichos GRAs se localizaron dentro de una nueva IG de resistencia que denominamos SmaR, la cual posee una estructura en forma de mosaico que conserva similitud con la región MARR descripta dentro de las IGs del tipo AbaR y AbGRI2, características de los cromosomas de los clones globales 1 y 2 de A. baumannii, respectivamente. Asimismo, dentro del resistoma de las 152 cepas de nuestro estudio, localizamos otras IGs de resistencia cromosómicas que tampoco habían sido descriptas previamente. Una de estas fue denominada SmaR-2, la cual porta el gen blaCTX-M-2 y gracias a los análisis filogenéticos detectamos que se encuentra diseminando en diversos clusters. A su vez, dentro del resistoma heredado verticalmente, analizamos otros componentes como los GRAs intrínsecos, bombas de eflujo y proto-genes de RAM observando la abundancia y diversidad de los mismos. Se identificaron la totalidad de GRAs adquiridos que contribuyen al resistoma (n=593), diseminando en distintas plataformas genéticas. Entre ellos se destaca la presencia del gen mcr9.1 que otorga resistencia al ATB colistín. Este ATB, para el cual S. marcescens es intrínsecamente resistente, es utilizado como ATB de último recurso para infecciones ocasionadas por patógenos XDR. El análisis filogenético de las cepas reveló linajes que exhiben resistomas con alto impacto clínico, entre los que se encuentra la diseminación policlonal del gen blaCTX-M-2 y de blaKPC-2, así como la presencia de plásmidos portadores de las carbapenemasas blaNDM-1 y de blaIMP-8; además se identificó un cluster que agrupa tres cepas procedentes de hospitales distintos que contienen un nuevo alelo de blaSPR-1. Para conocer el rol de S. marcescens en la diseminación de la RAM, se estudió la actividad de plásmidos relacionados con la diseminación de resistencia a los ?-lactámicos en nuestra colección de cepas. Se caracterizaron molecularmente 4 plásmidos y 2 pseudo moléculas plasmídicas. Se evaluó la capacidad conjugativa de dichos plásmidos, tanto interespecie (E. coli J53) como intraespecie (S. marcescens SCH909). Los ensayos in vivo dieron como resultado que pDCASG1-CTX (portador del gen blaCTX-M-2) y pDCASG6-NDM (portador de blaNDM-1) son conjugativos. Al evaluar el mantenimiento de los plásmidos en las cepas transconjugantes, los resultados mostraron que S. marcescens SCH909 fue capaz de mantener por más tiempo el plásmido pDCASG6-NDM por encima de lo observado para E. coli J53 (97,78% versus 20% a los diez días, respectivamente). Como conclusión, este trabajo reveló la heterogeneidad de los patrones de RAM que componen el resistoma de la especie S. marcescens. Su capacidad para mantener y diseminar EGMs asociados a plataformas genéticas MDR podría servir como un ?caballo de Troya?, contribuyendo silenciosamente a la diseminación de GRAs adquiridos de relevancia clínica en el nicho intrahospitalario a lo largo del tiempo. A su vez, la portación de una gran variedad de plataformas genéticas le permiten mayor adaptabilidad y supervivencia en diferentes hábitats incluyendo tanto nichos ambientales como clínicos, constituyendo así esta especie un nexo probable entre diferentes grupos bacterianos. application/pdf Rodriguez, Hernan Soria, Marcelo Viale, Alejandro spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Doctora de la Universidad de Buenos Aires en Ciencias Biológicas Mecanismos involucrados en la evolución hacia la extrema droga resistencia del patógeno Serratia marcescens info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7826 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7826.dir/7826.PDF

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