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

被引:8
作者
Higgins, Owen [1 ,3 ]
Chueiri, Alexandra [2 ,3 ]
O'Connor, Louise [1 ,2 ,3 ]
Lahiff, Sinead [3 ]
Burke, Liam [2 ,3 ]
Morris, Dearbhaile [2 ,3 ]
Pfeifer, Nicola Maria [4 ]
Santamarina, Belen Gonzalez [4 ]
Berens, Christian [4 ]
Menge, Christian [4 ]
Canica, Manuela [4 ,5 ]
Manageiro, Vera
Kisand, Veljo [6 ]
Hassan, Marwa M. M. [7 ]
Gardner, Brian [7 ]
van Vliet, Arnoud H. M. [7 ]
La Ragione, Roberto M. [7 ,8 ]
Gonzalez-Zorn, Bruno [9 ]
Smith, Terry J. J. [1 ,3 ]
机构
[1] Univ Galway, Sch Biol & Chem Sci, Mol Diagnost Res Grp, Galway, Ireland
[2] Univ Galway, Sch Med, Antimicrobial Resistance & Microbial Ecol Grp, Galway, Ireland
[3] Univ Galway, Ryan Inst, Ctr One Hlth, Galway, Ireland
[4] Friedrich Loeffler Inst, Fed Res Inst Anim Hlth, Inst Mol Pathogenesis, Jena, Germany
[5] Natl Inst Hlth Dr Ricardo Jorge, Dept Infect Dis, Natl Reference Lab Antibiot Resistances & Healthca, Lisbon, Portugal
[6] Univ Tartu, Inst Technol, Tartu, Estonia
[7] Univ Surrey, Fac Hlth & Med Sci, Sch Vet Med, Dept Comparat Biomed Sci, Guildford, England
[8] Univ Surrey, Fac Hlth & Med Sci, Sch Biosci & Med, Dept Microbial Sci, Guildford, England
[9] Univ Complutense Madrid, Vet Sch, Antimicrobial Resistance Unit, VISAVET, Madrid, Spain
来源
MICROBIOLOGY SPECTRUM | 2023年 / 11卷 / 01期
关键词
AMR; CTX-M; ESBL; Enterobacteriaceae; LAMP; SPECTRUM-BETA-LACTAMASE; REAL-TIME PCR; ANTIBIOTIC-RESISTANCE; COMMUNITY; EMERGENCE; CARRIAGE; SPREAD; CLONE;
D O I
10.1128/spectrum.03316-22
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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. 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 bla(CTX-M-1) and bla(CTX-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 bla(CTX-M-1) and bla(CTX-M-15). Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for bla(CTX-M-1) (n = 18), bla(CTX-M-15) (n = 35), and other closely related bla(CTX-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 bla(CTX-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 bla(CTX-M-1) and bla(CTX-M-15), respectively, and E. coli bla(CTX-M-1) was identified in all bla(CTX-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 bla(CTX-M-1) and bla(CTX-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.
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页数:12
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共 55 条
  • [1] Clonal Composition and Community Clustering of Drug-Susceptible and -Resistant Escherichia coli Isolates from Bloodstream Infections
    Adams-Sapper, Sheila
    Binh An Diep
    Perdreau-Remington, Francoise
    Riley, Lee W.
    [J]. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2013, 57 (01) : 490 - 497
  • [2] Anjum MF, 2018, ANTIMICROBIAL RESISTANCE IN BACTERIA FROM LIVESTOCK AND COMPANION ANIMALS, P33, DOI 10.1128/9781555819804.ch3
  • [3] Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype
    Bevan, Edward R.
    Jones, Annie M.
    Hawkey, Peter M.
    [J]. JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, 2017, 72 (08) : 2145 - 2155
  • [4] Prevalence of day-care centre children (France) with faecal CTX-M-producing Escherichia coli comprising O25b:H4 and O16:H5 ST131 strains
    Blanc, Veronique
    Leflon-Guibout, Veronique
    Blanco, Jorge
    Haenni, Marisa
    Madec, Jean-Yves
    Rafignon, Gwenaele
    Bruno, Pascale
    Mora, Azucena
    Lopez, Cecilia
    Dahbi, Ghizlane
    Dunais, Brigitte
    Anastay, Magali
    Branger, Catherine
    Moreau, Richard
    Pradier, Christian
    Nicolas-Chanoine, Marie-Helene
    [J]. JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, 2014, 69 (05) : 1231 - 1237
  • [5] Growing group of extended-spectrum β-lactamases:: The CTX-M enzymes
    Bonnet, R
    [J]. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2004, 48 (01) : 1 - 14
  • [6] Extended-spectrum β-lactamases in the 21st century:: Characterization, epidemiology, and detection of this important resistance threat
    Bradford, PA
    [J]. CLINICAL MICROBIOLOGY REVIEWS, 2001, 14 (04) : 933 - 951
  • [7] Past and Present Perspectives on β-Lactamases
    Bush, Karen
    [J]. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2018, 62 (10)
  • [8] β-Lactams and β-Lactamase Inhibitors: An Overview
    Bush, Karen
    Bradford, Patricia A.
    [J]. COLD SPRING HARBOR PERSPECTIVES IN MEDICINE, 2016, 6 (08):
  • [9] The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
    Bustin, Stephen A.
    Benes, Vladimir
    Garson, Jeremy A.
    Hellemans, Jan
    Huggett, Jim
    Kubista, Mikael
    Mueller, Reinhold
    Nolan, Tania
    Pfaffl, Michael W.
    Shipley, Gregory L.
    Vandesompele, Jo
    Wittwer, Carl T.
    [J]. CLINICAL CHEMISTRY, 2009, 55 (04) : 611 - 622
  • [10] Emerging Escherichia coli O25b/ST131 Clone Predicts Treatment Failure in Urinary Tract Infections
    Can, Fusun
    Azap, Ozlem Kurt
    Seref, Ceren
    Ispir, Pelin
    Arslan, Hande
    Ergonul, Onder
    [J]. CLINICAL INFECTIOUS DISEASES, 2015, 60 (04) : 523 - 527