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
ABSTRACT Cefotaximase-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 wit...
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2023-02-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03316-22 |
| _version_ | 1811164387483844608 |
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| author | Owen Higgins Alexandra Chueiri Louise O'Connor Sinéad Lahiff Liam Burke Dearbhaile Morris Nicola Maria Pfeifer Belén González Santamarina Christian Berens Christian Menge Manuela Caniça Vera Manageiro Veljo Kisand Marwa M. Hassan Brian Gardner Arnoud H. M. van Vliet Roberto M. La Ragione Bruno Gonzalez-Zorn Terry J. Smith |
| author_facet | Owen Higgins Alexandra Chueiri Louise O'Connor Sinéad Lahiff Liam Burke Dearbhaile Morris Nicola Maria Pfeifer Belén González Santamarina Christian Berens Christian Menge Manuela Caniça Vera Manageiro Veljo Kisand Marwa M. Hassan Brian Gardner Arnoud H. M. van Vliet Roberto M. La Ragione Bruno Gonzalez-Zorn Terry J. Smith |
| author_sort | Owen Higgins |
| collection | DOAJ |
| description | ABSTRACT Cefotaximase-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. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance. |
| first_indexed | 2024-04-10T15:20:43Z |
| format | Article |
| id | doaj.art-a7b4d5be508d49fa98f657adbf9bddd6 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-10T15:20:43Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-a7b4d5be508d49fa98f657adbf9bddd62023-02-14T14:15:50ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-02-0111110.1128/spectrum.03316-22Portable 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 AmplificationOwen Higgins0Alexandra Chueiri1Louise O'Connor2Sinéad Lahiff3Liam Burke4Dearbhaile Morris5Nicola Maria Pfeifer6Belén González Santamarina7Christian Berens8Christian Menge9Manuela Caniça10Vera Manageiro11Veljo Kisand12Marwa M. Hassan13Brian Gardner14Arnoud H. M. van Vliet15Roberto M. La Ragione16Bruno Gonzalez-Zorn17Terry J. Smith18Molecular Diagnostics Research Group, School of Biological and Chemical Sciences, University of Galway, Galway, IrelandAntimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Galway, IrelandAntimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Galway, IrelandMolecular Diagnostics Research Group, School of Biological and Chemical Sciences, University of Galway, Galway, IrelandAntimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Galway, IrelandAntimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Galway, IrelandFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, GermanyFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, GermanyFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, GermanyFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, GermanyNational Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, PortugalNational Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, PortugalInstitute of Technology, University of Tartu, Tartu, EstoniaDepartment of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United KingdomDepartment of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United KingdomDepartment of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United KingdomDepartment of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United KingdomAntimicrobial Resistance Unit, Veterinary School and VISAVET, Complutense University of Madrid, SpainMolecular Diagnostics Research Group, School of Biological and Chemical Sciences, University of Galway, Galway, IrelandABSTRACT Cefotaximase-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. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.https://journals.asm.org/doi/10.1128/spectrum.03316-22AMRCTX-MESBLEnterobacteriaceaeLAMP |
| spellingShingle | Owen Higgins Alexandra Chueiri Louise O'Connor Sinéad Lahiff Liam Burke Dearbhaile Morris Nicola Maria Pfeifer Belén González Santamarina Christian Berens Christian Menge Manuela Caniça Vera Manageiro Veljo Kisand Marwa M. Hassan Brian Gardner Arnoud H. M. van Vliet Roberto M. La Ragione Bruno Gonzalez-Zorn Terry J. Smith 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 Microbiology Spectrum AMR CTX-M ESBL Enterobacteriaceae LAMP |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | AMR CTX-M ESBL Enterobacteriaceae LAMP |
| url | https://journals.asm.org/doi/10.1128/spectrum.03316-22 |
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