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Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification

2022-12-13Journal article Open access
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dc.contributor.authorHiggins, Owen
dc.contributor.authorChueiri, Alexandra
dc.contributor.authorO'Connor, Louise
dc.contributor.authorLahiff, Sinéad
dc.contributor.authorBurke, Liam
dc.contributor.authorMorris, Dearbhaile
dc.contributor.authorPfeifer, Nicola Maria
dc.contributor.authorSantamarina, Belén González
dc.contributor.authorBerens, Christian
dc.contributor.authorMenge, Christian
dc.contributor.authorCaniça, Manuela
dc.contributor.authorManageiro, Vera
dc.contributor.authorKisand, Veljo
dc.contributor.authorHassan, Marwa M.
dc.contributor.authorGardner, Brian
dc.contributor.authorvan Vliet, Arnoud H.M.
dc.contributor.authorLa Ragione, Roberto M.
dc.contributor.authorGonzalez-Zorn, Bruno
dc.contributor.authorSmith, Terry J.
dc.date.accessioned2023-03-20T13:37:18Z
dc.date.available2023-03-20T13:37:18Z
dc.date.issued2022-12-13
dc.description.abstractCefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested.pt_PT
dc.description.sponsorshipThis research was funded by the European Union Horizon 2020 Research and Innovation Program under grant agreement No. 773830: One Health European Joint Program, JRP13-AMRSH5-WORLDCOM project.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationMicrobiol Spectr. 2023 Feb 14;11(1):e0331622. doi: 10.1128/spectrum.03316-22. Epub 2022 Dec 13pt_PT
dc.identifier.doi10.1128/spectrum.03316-22pt_PT
dc.identifier.issn2165-0497
dc.identifier.urihttp://hdl.handle.net/10400.18/8571
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherAmerican Society for Microbiologypt_PT
dc.relationPromoting One Health in Europe through joint actions on foodborne zoonoses, antimicrobial resistance and emerging microbiological hazards.
dc.relation.publisherversionhttps://journals.asm.org/doi/full/10.1128/spectrum.03316-22?rfr_dat=cr_pub++0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.orgpt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectAMRpt_PT
dc.subjectCTX-Mpt_PT
dc.subjectESBLpt_PT
dc.subjectEnterobacteriaceaept_PT
dc.subjectLAMPpt_PT
dc.subjectResistência aos Antimicrobianospt_PT
dc.titlePortable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplificationpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitlePromoting One Health in Europe through joint actions on foodborne zoonoses, antimicrobial resistance and emerging microbiological hazards.
oaire.awardURIinfo:eu-repo/grantAgreement/EC/H2020/773830/EU
oaire.citation.issue1pt_PT
oaire.citation.titleMicrobiology Spectrumpt_PT
oaire.citation.volume11pt_PT
oaire.fundingStreamH2020
project.funder.identifierhttp://doi.org/10.13039/501100008530
project.funder.nameEuropean Commission
rcaap.embargofctAcesso de acordo com política editorial da revista.pt_PT
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isProjectOfPublication94d118fb-33ce-49fa-b1ed-d5bddf63581d
relation.isProjectOfPublication.latestForDiscovery94d118fb-33ce-49fa-b1ed-d5bddf63581d

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