Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Repositório Institucional da FIOCRUZ (ARCA) |
Texto Completo: | https://www.arca.fiocruz.br/handle/icict/55241 |
Resumo: | Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pREFOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPRCas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria. |
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Walflor, Haniel Siqueira MortaguaLucena, Aline Rodrigues CastroTuon, Felipe FranciscoMedeiros, Lia Carolina SoaresFaoro, Helisson2022-10-21T18:52:47Z2022-10-21T18:52:47Z2022WALFLOR. Haniel Siqueira Mortagua et al. Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system. Int. J. Mol. Sci., v. 23, n. 9175, p. 1–10, 2022.1422-0067https://www.arca.fiocruz.br/handle/icict/55241porMDPIMetaloenzima FosA3CRISPR–Cas9Escherichia coliFosfomycinDrug Resistance, MicrobialEnzymesCRISPR-Associated Protein 9FosfomicinaFarmacorresistencia MicrobianaProteína 9 Asociada a CRISPRFosfomycineRésistance microbienne aux médicamentsEnzymesProtéine-9 associée à CRISPRFosfomicinaResistência Microbiana a MedicamentosEnzimasProteína 9 Associada à CRISPRResensitization of fosfomycin-resistant Escherichia coli using the CRISPR systeminfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleAntimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pREFOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPRCas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZORIGINALijms-23-09175ok.pdfijms-23-09175ok.pdfapplication/pdf2959004https://www.arca.fiocruz.br/bitstream/icict/55241/2/ijms-23-09175ok.pdf6e81316286181dd352a5246eb6cd7161MD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/55241/1/license.txt5a560609d32a3863062d77ff32785d58MD51icict/552412022-10-21 15:52:48.374oai:www.arca.fiocruz.br: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ório InstitucionalPUBhttps://www.arca.fiocruz.br/oai/requestrepositorio.arca@fiocruz.bropendoar:21352022-10-21T18:52:48Repositório Institucional da FIOCRUZ (ARCA) - Fundação Oswaldo Cruz (FIOCRUZ)false |
dc.title.en_US.fl_str_mv |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
title |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
spellingShingle |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system Walflor, Haniel Siqueira Mortagua Metaloenzima FosA3 CRISPR–Cas9 Escherichia coli Fosfomycin Drug Resistance, Microbial Enzymes CRISPR-Associated Protein 9 Fosfomicina Farmacorresistencia Microbiana Proteína 9 Asociada a CRISPR Fosfomycine Résistance microbienne aux médicaments Enzymes Protéine-9 associée à CRISPR Fosfomicina Resistência Microbiana a Medicamentos Enzimas Proteína 9 Associada à CRISPR |
title_short |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
title_full |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
title_fullStr |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
title_full_unstemmed |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
title_sort |
Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system |
author |
Walflor, Haniel Siqueira Mortagua |
author_facet |
Walflor, Haniel Siqueira Mortagua Lucena, Aline Rodrigues Castro Tuon, Felipe Francisco Medeiros, Lia Carolina Soares Faoro, Helisson |
author_role |
author |
author2 |
Lucena, Aline Rodrigues Castro Tuon, Felipe Francisco Medeiros, Lia Carolina Soares Faoro, Helisson |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Walflor, Haniel Siqueira Mortagua Lucena, Aline Rodrigues Castro Tuon, Felipe Francisco Medeiros, Lia Carolina Soares Faoro, Helisson |
dc.subject.other.en_US.fl_str_mv |
Metaloenzima FosA3 CRISPR–Cas9 Escherichia coli |
topic |
Metaloenzima FosA3 CRISPR–Cas9 Escherichia coli Fosfomycin Drug Resistance, Microbial Enzymes CRISPR-Associated Protein 9 Fosfomicina Farmacorresistencia Microbiana Proteína 9 Asociada a CRISPR Fosfomycine Résistance microbienne aux médicaments Enzymes Protéine-9 associée à CRISPR Fosfomicina Resistência Microbiana a Medicamentos Enzimas Proteína 9 Associada à CRISPR |
dc.subject.en.en_US.fl_str_mv |
Fosfomycin Drug Resistance, Microbial Enzymes CRISPR-Associated Protein 9 |
dc.subject.es.en_US.fl_str_mv |
Fosfomicina Farmacorresistencia Microbiana Proteína 9 Asociada a CRISPR |
dc.subject.fr.en_US.fl_str_mv |
Fosfomycine Résistance microbienne aux médicaments Enzymes Protéine-9 associée à CRISPR |
dc.subject.decs.en_US.fl_str_mv |
Fosfomicina Resistência Microbiana a Medicamentos Enzimas Proteína 9 Associada à CRISPR |
description |
Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pREFOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPRCas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria. |
publishDate |
2022 |
dc.date.accessioned.fl_str_mv |
2022-10-21T18:52:47Z |
dc.date.available.fl_str_mv |
2022-10-21T18:52:47Z |
dc.date.issued.fl_str_mv |
2022 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
WALFLOR. Haniel Siqueira Mortagua et al. Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system. Int. J. Mol. Sci., v. 23, n. 9175, p. 1–10, 2022. |
dc.identifier.uri.fl_str_mv |
https://www.arca.fiocruz.br/handle/icict/55241 |
dc.identifier.issn.en_US.fl_str_mv |
1422-0067 |
identifier_str_mv |
WALFLOR. Haniel Siqueira Mortagua et al. Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system. Int. J. Mol. Sci., v. 23, n. 9175, p. 1–10, 2022. 1422-0067 |
url |
https://www.arca.fiocruz.br/handle/icict/55241 |
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MDPI |
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