Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética
In 2014, the World Health Organization published the first global report on Surveillance of Antimicrobial resistance. It addressed the urgent need to come up with a coordinated set of strategies to fight the problem in a One Health approach, which contributes to protect the public health through the...
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Universidad de La Rioja (España)
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In 2014, the World Health Organization published the first global report on Surveillance of Antimicrobial resistance. It addressed the urgent need to come up with a coordinated set of strategies to fight the problem in a One Health approach, which contributes to protect the public health through the control of antibiotic resistance at the interface between humans, animals and the environment. According to the World Organisation for Animal Health, about 60% of human pathogens are of animal origin. However, antimicrobial resistance is not limited to bacteria causing infectious diseases, it also affects those non-pathogenic ones occupying diverse ecological niches. Thus, commensal bacteria are also a threat, since they can act as important reservoirs of resistance and virulence genes. This thesis aims at exploring the extent of antimicrobial resistant E. coli in wildlife, the major circulating clones, the mechanisms and genetic platforms involved in the flow of resistance genes between humans, animals, and the environment, and potential wild reservoirs of zoonotic E. coli pathotypes.
In the first and second chapters, population structure and antimicrobial resistance of E. coli from wildlife were investigated. A low to moderate frequency of antibiotic resistant strains was observed in mammals (11.1%), predominantly against old agents such as tetracyclines, penicillins and sulfonamides. The prevalence of ESBL/AmpC-producing E. coli was high among birds (16%), with SHV-12 as the main enzyme variant, followed by CTX-M-1, CTX-M- 14 and CMY-2. Eighty-two percent of the ESBLs from wildlife showed a multi-resistant genotype, often in relation to the carriage of class 1 integrons containing long arrays of gene cassettes. Although quinolone resistance was mainly due to chromosomal mutations in GyrA/ParC, a qnrS1 gene was identified in an E. coli from a barn owl. All the ESBL and plasmid-mediated AmpC encoding genes were transferable by conjugation, and we observed some associations like blaCTX-M-1/IncN, blaSHV-12/IncI1 or blaCMY-2/IncI1. Although some ESBL/AmpC-producing E. coli lineages were first described in wildlife (ST4564, ST4954, ST4996, ST7624, ST7629, ST7630, ST7631, ST7632), most of the isolates belonged to clones frequently detected among humans, domestic animals and food (ST131, ST10, ST155, ST224, ST38, ST57), which support the existence of successful lineages strongly associated with the bidirectional dissemination of ESBL/AmpC genes. Cryptic Escherichia clades were identified in various wild mammals, highlighting the detection of a clade V member carrying blaCTX-M-14 and different virulence factors.
The third chapter focused on the analysis of distinct genetic elements involved in the selection, persistence and spread of resistance determinants among E. coli from different ecosystems (humans, domestic animals, wildlife, food). The first paper provides insights into the molecular background of plasmids and additional genetic platforms associated with the dissemination of SHV-12 encoding gene. The horizontal transfer of blaSHV-12 was mainly driven by IncI1 plasmids, with the pST3 subtype prevailing in poultry and the pST26 (and other CC26 associated subtypes) being equally distributed among isolates from various origins. The complete sequencing of an IncI1 plasmid revealed the presence of a Tn21-blaSHV-12-ΔTn1721 resistance complex, containing the atypical intI1-estX-psp-aadA2-cmlA1-aadA1-qacI-IS440- sul3 integron and the tet(A) gene, which seems to play an important role in the spread of SHV- 12. Restriction analysis of IncK plasmids suggest the occurrence of horizontal blaSHV-12 events from local non-ST131 isolates to the pandemic ST131 clone. We identified an IncX3 plasmid, not typeable by PBRT, co-harbouring qnrS1 and blaSHV-12 genes, the latter one integrated in a composite transposon structure that could facilitate the en-bloc mobilization of the ESBL. The second paper aimed at gaining knowledge about the location and genetic organization of class 2 integrons in E. coli from different hosts and countries. Although a low diversity of gene cassettes was shown, many novel structures were identified due to the integration of IS at different sites. These IS elements might modulate the expression and movilization of adjacent genes, or even define the genomic location and dissemination capability of class 2 integrons. Most class 2 integrons were chromosomally inserted at the attTn7 site, adjacent to the essential glmS gene. Only a few were transferable by conjugation and the comobilization of BLEE genes was never involved in the process. Preliminary functional assays showed that class 1 integrase was capable of efficiently excising the aadA1 cassette found in the variable region of class 2 integrons.
In the fourth chapter, the epidemiological role of wildlife in the maintenance and dissemination of enteropathogenic (EPEC) and Shiga toxin-producing E. coli (STEC) was investigated. Among STEC recovered from wild animals, the stx2b/subAB2/ehxA virulence profile was the most common and 9 isolates belonged to seropathotypes frequently associated with hemolytic uremic syndrome (seropathotypes B -O145:[H28] - and C -O22:H8, O128:[H2] -) or diarrhea (seropathotype D -O110:H28, O146:H21, O146:[H28], ONT:H8-) in humans.We first reported a wild boar as carrier of a bfpA-positive O49:[H10] eae-κ strain of the same characteristics as tEPEC isolated from human diarrhea. Wild ruminants (deer and mouflon), as well as wild boar, act as important reservoirs of potentially pathogenic STEC and EPEC.
In the final chapter, we studied by WGS the accessory and core genome of 38 commensal E. coli strains from wildlife. We analyzed the CRISPR/Cas systems as well as the resistance and virulence modules, which show similar genetic structures and organizations than those reported in strains from the human setting. WGS enabled a more consistent characterization of the composition and diversity of the plasmidome, facilitating the study of small replicons, the differentiation of extra-chromosomal phages or phage-like elements and the detection of non-typeable plasmids. Our results suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems, rather than the occurrence of a parallel microevolution in the gut of wild animals. |
| author2 |
Torres Manrique, Carmen (null) |
| author_facet |
Torres Manrique, Carmen (null) Alonso Arribas, Carla Andrea |
| format |
text (thesis) |
| author |
Alonso Arribas, Carla Andrea |
| spellingShingle |
Alonso Arribas, Carla Andrea Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| author_sort |
Alonso Arribas, Carla Andrea |
| title |
Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| title_short |
Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| title_full |
Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| title_fullStr |
Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| title_full_unstemmed |
Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| title_sort |
epidemiología molecular en escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genética |
| publisher |
Universidad de La Rioja (España) |
| publishDate |
2019 |
| url |
https://dialnet.unirioja.es/servlet/oaites?codigo=221324 |
| work_keys_str_mv |
AT alonsoarribascarlaandrea epidemiologiamolecularenescherichiacoliprocedentedefaunasalvajeresistenciaantimicrobianavirulenciaydiversidadydiversidadgenetica |
| _version_ |
1718346677388050432 |
| spelling |
oai-TES00000228922019-05-15Epidemiología molecular en Escherichia coli procedente de fauna salvaje: resistencia antimicrobiana, virulencia y diversidad y diversidad genéticaAlonso Arribas, Carla AndreaIn 2014, the World Health Organization published the first global report on Surveillance of Antimicrobial resistance. It addressed the urgent need to come up with a coordinated set of strategies to fight the problem in a One Health approach, which contributes to protect the public health through the control of antibiotic resistance at the interface between humans, animals and the environment. According to the World Organisation for Animal Health, about 60% of human pathogens are of animal origin. However, antimicrobial resistance is not limited to bacteria causing infectious diseases, it also affects those non-pathogenic ones occupying diverse ecological niches. Thus, commensal bacteria are also a threat, since they can act as important reservoirs of resistance and virulence genes. This thesis aims at exploring the extent of antimicrobial resistant E. coli in wildlife, the major circulating clones, the mechanisms and genetic platforms involved in the flow of resistance genes between humans, animals, and the environment, and potential wild reservoirs of zoonotic E. coli pathotypes. In the first and second chapters, population structure and antimicrobial resistance of E. coli from wildlife were investigated. A low to moderate frequency of antibiotic resistant strains was observed in mammals (11.1%), predominantly against old agents such as tetracyclines, penicillins and sulfonamides. The prevalence of ESBL/AmpC-producing E. coli was high among birds (16%), with SHV-12 as the main enzyme variant, followed by CTX-M-1, CTX-M- 14 and CMY-2. Eighty-two percent of the ESBLs from wildlife showed a multi-resistant genotype, often in relation to the carriage of class 1 integrons containing long arrays of gene cassettes. Although quinolone resistance was mainly due to chromosomal mutations in GyrA/ParC, a qnrS1 gene was identified in an E. coli from a barn owl. All the ESBL and plasmid-mediated AmpC encoding genes were transferable by conjugation, and we observed some associations like blaCTX-M-1/IncN, blaSHV-12/IncI1 or blaCMY-2/IncI1. Although some ESBL/AmpC-producing E. coli lineages were first described in wildlife (ST4564, ST4954, ST4996, ST7624, ST7629, ST7630, ST7631, ST7632), most of the isolates belonged to clones frequently detected among humans, domestic animals and food (ST131, ST10, ST155, ST224, ST38, ST57), which support the existence of successful lineages strongly associated with the bidirectional dissemination of ESBL/AmpC genes. Cryptic Escherichia clades were identified in various wild mammals, highlighting the detection of a clade V member carrying blaCTX-M-14 and different virulence factors. The third chapter focused on the analysis of distinct genetic elements involved in the selection, persistence and spread of resistance determinants among E. coli from different ecosystems (humans, domestic animals, wildlife, food). The first paper provides insights into the molecular background of plasmids and additional genetic platforms associated with the dissemination of SHV-12 encoding gene. The horizontal transfer of blaSHV-12 was mainly driven by IncI1 plasmids, with the pST3 subtype prevailing in poultry and the pST26 (and other CC26 associated subtypes) being equally distributed among isolates from various origins. The complete sequencing of an IncI1 plasmid revealed the presence of a Tn21-blaSHV-12-ΔTn1721 resistance complex, containing the atypical intI1-estX-psp-aadA2-cmlA1-aadA1-qacI-IS440- sul3 integron and the tet(A) gene, which seems to play an important role in the spread of SHV- 12. Restriction analysis of IncK plasmids suggest the occurrence of horizontal blaSHV-12 events from local non-ST131 isolates to the pandemic ST131 clone. We identified an IncX3 plasmid, not typeable by PBRT, co-harbouring qnrS1 and blaSHV-12 genes, the latter one integrated in a composite transposon structure that could facilitate the en-bloc mobilization of the ESBL. The second paper aimed at gaining knowledge about the location and genetic organization of class 2 integrons in E. coli from different hosts and countries. Although a low diversity of gene cassettes was shown, many novel structures were identified due to the integration of IS at different sites. These IS elements might modulate the expression and movilization of adjacent genes, or even define the genomic location and dissemination capability of class 2 integrons. Most class 2 integrons were chromosomally inserted at the attTn7 site, adjacent to the essential glmS gene. Only a few were transferable by conjugation and the comobilization of BLEE genes was never involved in the process. Preliminary functional assays showed that class 1 integrase was capable of efficiently excising the aadA1 cassette found in the variable region of class 2 integrons. In the fourth chapter, the epidemiological role of wildlife in the maintenance and dissemination of enteropathogenic (EPEC) and Shiga toxin-producing E. coli (STEC) was investigated. Among STEC recovered from wild animals, the stx2b/subAB2/ehxA virulence profile was the most common and 9 isolates belonged to seropathotypes frequently associated with hemolytic uremic syndrome (seropathotypes B -O145:[H28] - and C -O22:H8, O128:[H2] -) or diarrhea (seropathotype D -O110:H28, O146:H21, O146:[H28], ONT:H8-) in humans.We first reported a wild boar as carrier of a bfpA-positive O49:[H10] eae-κ strain of the same characteristics as tEPEC isolated from human diarrhea. Wild ruminants (deer and mouflon), as well as wild boar, act as important reservoirs of potentially pathogenic STEC and EPEC. In the final chapter, we studied by WGS the accessory and core genome of 38 commensal E. coli strains from wildlife. We analyzed the CRISPR/Cas systems as well as the resistance and virulence modules, which show similar genetic structures and organizations than those reported in strains from the human setting. WGS enabled a more consistent characterization of the composition and diversity of the plasmidome, facilitating the study of small replicons, the differentiation of extra-chromosomal phages or phage-like elements and the detection of non-typeable plasmids. Our results suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems, rather than the occurrence of a parallel microevolution in the gut of wild animals.En 2014 la Organización Mundial de la Salud publicó el primer informe mundial sobre la situación de la resistencia a los antibióticos. En él destacaba la necesidad de un abordaje urgente del problema, con un enfoque One Health, que contribuyese a la protección de la Salud Pública por medio del control de la resistencia en la población humana, animal y el medio ambiente. Según la Organización Mundial de Sanidad Animal (OIE), cerca del 60% de los patógenos humanos son de origen animal. Pero la resistencia a antibióticos no se limita a las bacterias causantes de procesos infecciosos, también afecta a aquellas no patógenas presentes en diversos nichos ecológicos. Estas bacterias comensales representan igualmente una amenaza, pues pueden constituir un reservorio importante de genes de resistencia y virulencia. En esta tesis aportamos nuevos datos acerca del grado de diseminación de Escherichia coli resistente a los antibióticos en fauna silvestre, caracterizamos los clones circulantes y su potencial epidémico, analizamos los mecanismos y plataformas genéticas implicadas en el flujo humano-animal-medio ambiente e identificamos reservorios salvajes de cepas con potencial zoonótico. Por un lado, estudiamos la estructura poblacional y la caracterización molecular de la resistencia en E. coli de fauna salvaje. Observamos una moderada frecuencia de detección de cepas resistentes a los antibióticos en mamíferos (11,1%), predominando la resistencia a agentes clásicos como tetraciclinas, penicilinas y sulfonamidas. Destacó la alta prevalencia de E. coli BLEE/AmpC en aves (16%), siendo SHV-12 la principal variante implicada, seguida de CTX-M-1, CTX-M-14 y CMY-2. El 82% de las cepas BLEE aisladas de fauna silvestre mostraron un genotipo multirresistente, muchas veces en relación a la portación de integrones de clase 1 conteniendo complejos arreglos de genes cassettes. Aunque la resistencia a quinolonas estuvo mayormente mediada por mutaciones cromosómicas en GyrA/ParC, detectamos el gen qnrS1 en una cepa de lechuza. Los genes codificantes de BLEEs y AmpC plasmídicas fueron transferibles por conjugación, estableciéndose las asociaciones blaCTX-M-1/IncN, blaSHV-12/IncI1 y blaCMY-2/IncI1. Los resultados avalan el flujo bidireccional de la resistencia y la existencia de linajes exitosos en la diseminación de genotipos BLEE/AmpC ya que, si bien describimos nuevas secuencias tipo en fauna silvestre (ST4564, ST4954, ST4996, ST7624, ST7629, ST7630, ST7631 y ST7632), muchos de los clones son también frecuentes en humanos, animales domésticos y alimentos (ST131, ST10, ST155, ST224, ST38, ST57). Se detectaron clados crípticos de Escherichia en algunos mamíferos silvestres, con especial relevancia de una cepa perteneciente al clado V, portadora de blaCTX-M-14 y diversos factores de virulencia. Además, desde una perspectiva One Health, quisimos estudiar diversos elementos genéticos involucrados en la selección, persistencia y dispersión de la resistencia entre distintos hospedadores (humanos, animales domésticos, fauna silvestre). Respecto a la transferencia horizontal del gen blaSHV-12, destacó la frecuente implicación de plásmidos IncI1, prevaleciendo el subtipo pST3 en aves de corral y el pST26 (y derivados del CC26) en cepas de orígenes muy variados. Describimos el complejo de resistencia Tn21-blaSHV-12-∆Tn1721, conteniendo el integrón atípico IntI1-estX-psp-aadA2-cmlA1-aadA1-qacI-IS440-sul3 y el gen tet(A), y su implicación en la diseminación de SHV-12. Además,los ensayos de restricción revelaron que la adquisición de blaSHV-12 por parte del clon pandémico ST131 podría deberse a la transferencia lateral de plásmidos IncK desde cepas locales no-ST131. Identificamos un plásmido IncX, conteniendo los genes qnrS1 y blaSHV-12, este último integrado en una nueva estructura tipo transposón compuesto que podría facilitar su movilización en bloque. Por otro lado, estudiamos la localización y organización genética de los integrones de clase 2 en E. coli de distintos orígenes y áreas geográficas. Su estructura se mantenía altamente conservada, mostrando escasa diversidad de genes cassettes. Las variaciones se debían fundamentalmente a la integración de ISs a diferentes niveles, lo que podría modular la expresión y movilización de genes adyacentes o influir en su localización genómica y capacidad de diseminación. Los integrones de clase 2 se hallaron mayoritariamente localizados a nivel cromosómico en el sitio attTn7, adyacente al gen esencial glmS. Sólo unos pocos fueron transferibles por conjugación, y en ningún caso ésta implicó movilización de genes bla. Los ensayos funcionales preliminares mostraron que la integrasa de tipo 1 es capaz de escindir eficazmente el cassette aadA1 localizado en la región variable del integrón de clase 2. Asimismo, se investigó el papel epidemiológico de la fauna silvestre en el mantenimiento y diseminación de E. coli verotoxigénico (STEC) y enteropatógeno (EPEC). Entre las cepas STEC de animales salvajes destacó el genotipo stx2b/subAB2/ehxA y se identificaron hasta 9 seropatotipos asociados a síndrome urémico hemolítico (seropatotipos B -O145:H28- y C -O22:H8, O128:H2-) y diarrea (seropatotipo D -O110:H28, O146:H21, O146:H28, ONT:H8-) en humanos. Se detectó una cepa tEPEC en un jabalí, con un serotipo y perfil de virulencia (O49:H10 eae-κ) descrito en cepas aisladas de pacientes con diarrea. Los rumiantes silvestres (muflones y ciervos), así como los jabalíes, actúan como importantes reservorios de STEC y EPEC. Por último, se estudió el genoma accesorio y el genoma core de 38 cepas comensales de E. coli desde un abordaje genómico. Analizamos los sistemas CRISPR/Cas así como los módulos de resistencia y virulencia, los cuales generalmente conservaban estructuras altamente similares a las descritas en cepas del entorno humano. Los datos de WGS y la reconstrucción del genoma bacteriano por PLACNETw nos permitió estudiar la población de plásmidos pequeños y tipar todos los replicones, lo que nos brindó más información que las metologías convencionales. Nuestros resultados sugieren la existencia de un flujo continuo de bacterias entre los ecosistemas humano y natural, más que una microevolución paralela asociada al nicho u hospedador.Universidad de La Rioja (España)Torres Manrique, Carmen (null)2019text (thesis)application/pdfhttps://dialnet.unirioja.es/servlet/oaites?codigo=221324spaLICENCIA DE USO: Los documentos a texto completo incluidos en Dialnet son de acceso libre y propiedad de sus autores y/o editores. Por tanto, cualquier acto de reproducción, distribución, comunicación pública y/o transformación total o parcial requiere el consentimiento expreso y escrito de aquéllos. Cualquier enlace al texto completo de estos documentos deberá hacerse a través de la URL oficial de éstos en Dialnet. 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