Rapid detection of extended spectrum β-lactamase producing Escherichia coli isolated from fresh pork meat and pig cecum samples using multiplex recombinase polymerase amplification and lateral flow strip analysis.

Sitthichai Kanokudom,Nuntaree Chaichanawongsaroj,Rungtip Chuanchuen,Thachaporn Assawakongkarat,Yukihiro Akeda,Panan Ratthawongjirakul,Iddya Karunasagar

doi:10.1371/journal.pone.0248536

Sitthichai Kanokudom, Nuntaree Chaichanawongsaroj + Show 5 more

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https://doi.org/10.1371/journal.pone.0248536

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Abstract

The emergence and dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a global health issue. Food-producing animals, including pigs, are significant reservoirs of antimicrobial resistance (AMR), which can be transmitted to humans. Thus, the rapid detection of ESBLs is required for efficient epidemiological control and treatment. In this study, multiplex recombinase polymerase amplification (RPA) combined with a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS), as a lateral flow strip assay (LFA), was established for the rapid and simultaneous detection of multiple bla genes in a single reaction. Visible blue lines, indicating the presence of the blaCTX-M, blaSHV, and blaOXA genes, were observed within 10 min by the naked eye. The limit of detection of all three genes was 2.5 ng/25 μL, and no cross-reactivity with seven commensal aerobic bacteria was observed. A total of 93.9% (92/98) and 96% (48/50) of the E. coli isolates from pork meat and fecal samples, respectively, expressed an ESBL-producing phenotype. Nucleotide sequencing of the PCR amplicons showed that blaCTX-M was the most prevalent type (91.3-95.83%), of which the main form was blaCTX-M-55. The sensitivity and specificity of the RPA-LFA were 99.2% and 100%, respectively, and were in almost perfect agreement (κ = 0.949-1.000) with the results from PCR sequencing. Thus, the RPA-LFA is a promising tool for rapid and equipment-free ESBL detection and may facilitate clinical diagnosis in human and veterinary medicine, as well as AMR monitoring and surveillance.

Highlights

The rising level of antimicrobial resistance (AMR) worldwide has a significant impact on humans, animals, and the environment
From the 100 pork samples individually collected from 100 retail markets, a single E. coli isolate was selected from 68 positive samples
Of these screened E. coli isolates, 93.9% (92/98) were ESBL producers, and three isolates were resistant to carbapenem (Fig 2A)

Summary

Introduction

The rising level of antimicrobial resistance (AMR) worldwide has a significant impact on humans, animals, and the environment. Extended spectrum β-lactamase (ESBL)-producing E. coli have been categorized by the World Health Organization (WHO) as being the most critical AMR pathogen to human health and a major public health concern. Broad-spectrum β-lactamase enzymes hydrolyze penicillins, cephalosporins (first-, second- and third-generation), and aztreonam but not carbapenems. These enzymes are inhibited by clavulanate [10]. Up to 86% of bacterial isolates (97.8% E. coli) from food animals, including pigs, cattle, chicken, and sheep, produce CTX-M group 1 [2]. Up to 92% of CTX-M groups have been detected among the multidrug-resistant (MDR) E. coli isolates from healthy finisher and breeder pigs and boot swabs from pig farms [12]

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