One Health EJP

Part of EU Open Research Repository
Published December 13, 2022 | Version v1
Journal article Open

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

Creators

  • 1. Molecular Diagnostics Research Group, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland; Centre for One Health, Ryan Institute, University of Galway, Galway, Ireland
  • 2. Antimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Galway, Ireland; Centre for One Health, Ryan Institute, University of Galway, Galway, Ireland
  • 3. Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany
  • 4. National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal
  • 5. Institute of Technology, University of Tartu, Tartu, Estonia
  • 6. Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom
  • 7. Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom; Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom
  • 8. Antimicrobial Resistance Unit, Veterinary School and VISAVET, Complutense University of Madrid, Spain
  • 9. Molecular Diagnostics Research Group, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland

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 ESBLproducing
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.

Files

spectrum.03316-22.pdf

Files (2.5 MB)

Name Size Download all
md5:f22da4f7853404d8e10d137288424f61
2.5 MB Preview Download

Additional details

Funding

European Commission
One Health EJP – Promoting One Health in Europe through joint actions on foodborne zoonoses, antimicrobial resistance and emerging microbiological hazards. 773830