Vesículas extracelulares y Transferencia Horizontal Genética en la adaptación hacia la extrema droga resistencia de E. coli en el marco "Una Salud"
One of the major challenges in addressing multidrug resistance is understanding the true burden of AMR. This is especially true where surveillance is minimal and data is scarce, such as in environmental ecosystems. In the context of One Health, there is evidence that antimicrobial resistance genes (...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2024
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7866 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7866.dir/7866.PDF |
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| Sumario: | One of the major challenges in addressing multidrug resistance is understanding the true burden of AMR. This is especially true where surveillance is minimal and data is scarce, such as in environmental ecosystems. In the context of One Health, there is evidence that antimicrobial resistance genes (ARG) in environmental bacteria can be rapidly acquired by human- and animal-associated pathogens and vice versa. Therefore, the transmission mechanisms of ARG in the different known reservoirs need to be characterized in detail. One of these mechanisms is the Horizontal genetic Transfer (HGT), which is mediated by canonical processes such as conjugation, transformation and transduction, and by non-canonical processes such as the novel mechanism known as vesiduction, which is generated by the release of EV.\nThree E. coli isolates of different origin and epidemiologic behavior were used as biological models in this study. The first is an isolate of environmental origin, the E. coli 4IgSN1 strain, being the other two clinical isolates, one a sporadic clone represented by E. coli M19736 ST615 strain, and the second a clone with pandemic behavior represented by E. coli SM5 ST131 strain. We performed genomic analysis of our three models in search of determinants of AMR. The genome of the E. coli 4IgSN1 strain was sequenced and its chromosome contains a genetic platform with a deleted intI1 gene, encoding a type 1 integrase, followed by the dfrB2 gene cassette. Both genes are inserted into the xerD gene, a site-specific tyrosine recombinase. Genomic analysis of biological model E. coli M19736 revealed that this strain carries chromosomal AMR determinants including fosL1, tet(B) and mutations at the quinolone resistance determining region. The presence of 3 plasmids was also detected, two of which harbor ARG. The first plasmid pM19736-MCR-1, previously reported, harbors the mcr-1 gene, while the second plasmid pM19736-IncFIB_IncFII harbors aph(6)-Id, aph(3'')-Ib, blaTEM-1B, floR and sul2 genes. The genome of E. coli SM5 strain has been analyzed in previous studies from our laboratory. This strain harbors the plasmid pseudomolecule pDCAG1-CTX-M-15. Several GRAs were found such as: tet(B), catB3, dfrA8, sul2, blaCTX-M-15, blaTEM-1B, blaOXA-1, aac(6?)-Ib-cr, aph(6)-Id and aph(3??)-Ib.\nOn the other hand, we have confirmed the ability of the three biological models of E. coli to receive clinically crucial ARG carried by MDR plasmids by transformation or conjugation using as donors the plasmids pM19736-MCR-1 (>63230, Incl2) carrying the mcr-1 gene, pDCAG1-CTX-M-15 (>112,000 bp, IncFII) carrying the blaCTX-M-15 gene, pDCCK1-KPC (>77,218 bp, IncM1) which contains the blaKPC-2 gene, pDCVA3-NDM -5 (>534,520 bp, IncFII) which contains the blaCTX-M-15 and blaNDM-5 genes, pDCASG-NDM-1 (137,269 bp, IncC) harboring the blaNDM-1 gene, pDCPR3-VIM-2 (pDCPR3-VIM-2) harboring the blaVIM-2 gene, and non-mobilized pAO1Bc (5,877 bp, p15A) harboring the aadB gene cassette. PCR, MIC, antibiogram and phenotypic detection of ?-lactamases were used to verify the acquisition of ARG. The ability of each transconjugant or transformant to maintain ARG over time without antibiotic pressure was then evaluated. Environmental isolate E. coli 4IgSN1 and sporadic clone E. coli M19736 were able to receive and maintain plasmids pDCAG1-CTX-M-15, pDCVA3-NDM-5 and pDCASG-NDM-1 over time. On the other hand, the three biological models E. coli 4IgSN1, E. coli M19736 and E. coli SM5 were able to acquire the plasmids pDCCK1-KPC and pAO1Bc, but could not maintain them over time. In sequential experiments, the E. coli M19736 strain acquired multiple plasmids giving rise to strains that we refer as evolved strains MDR and XDR-E. coli M19736 strains. Evolved MDR-E. coli strain M19736 sequentially acquired the blaCTX-M-15 and blaNDM-1 genes. Over time, it maintained ARG of 41.1% and 91.1%, during 10 days without antibiotic pressure, respectively. When this strain sequentially acquired the aadB gene (XDR-E. coli M19736 strain), the maintenance pattern changed, to 100% loss for blaCTX-M-15 and aadB genes, 98.9% maintenance of blaNDM-1 gene, and 100% maintenance of mcr-1 gene. Interestingly, evolved MDR and XDR-E. coli M19736 simultaneously disseminated the acquired conjugative plasmids pDCASG-NDM-1 and pDCAG1-CTX-M-15 as well as the native pMCR1-M19736 with different combinations of co-infection, although the selection was ceftazidime in all cases.\nFinally, we characterized the OMV of the E. coli strains carrying the MDR plasmids used as donors (E. coli M19736, E. coli SM5, K. pneumoniae HA7pKpn, K. pneumoniae HA31, S. marcescens SM938 and P. aeruginosa PAE981) in our conjugation experiments. It was possible to detect by PCR the blaCTX-M-15 genes in OMV of E. coli SM5, K. pneumoniae HA31 and evolved XDR-E. coli M19736; the blaNDM-1 gene in OMV of S. marcescens SM938; the blaNDM-5 gene in OMV of K. pneumoniae HA31 and the mcr-1 gene in OMV of E. coli M19736. Interestingly, OMV from evolved XDR-E. coli M19736 harbored blaCTX-M-15 though the plasmid pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments along time. Furthermore, by LC/MS-MS we could detect MCR-1 protein in E. coli M19736 OMV; AAC (6'), ?-lactamases TEM and CTX-M-1 and MBL NDM in K. pneumoniae HA31 OMV; KPC carbapenemase in K. pneumoniae HA7pKpn OMV.\nTogether, these results evidenced the genetic plasticity of E. coli and emphasize the relevance of focusing studies on environmental and sporadic clones´ models to evaluate adaptation to extreme drug resistance. Based on this, environmental strains and sporadic clones could play a crucial role in a clinical context in the maintenance and transmission of AMR, either by adapting to different selection pressures and at the same time spreading plasmids to other bacteria, ensuring an important link in the chain of events that leads bacteria to resist in the nosocomial niche. Finally, we demonstrate how OMV are active reservoirs of AMR determinants, having enormous potential to disseminate them. |
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