Research Unit of Innovative Diagnosis of Antimicrobial Resistance, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Suita, Japan.
PLoS One. 2021 Mar 15;16(3):e0248536. doi: 10.1371/journal.pone.0248536. eCollection 2021.
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.
产超广谱β-内酰胺酶（ESBL）大肠埃希菌的出现和传播是一个全球性的健康问题。包括猪在内的食用动物是抗生素耐药性（AMR）的重要储存库，这些耐药性可以传播给人类。因此，需要快速检测 ESBL 以有效进行流行病学控制和治疗。在这项研究中，建立了多重重组酶聚合扩增（RPA）与单链标签杂交色谱打印阵列条（STH-PAS）相结合的侧向流动条检测（LFA），用于在单个反应中快速同时检测多个 bla 基因。通过肉眼观察，在 10 分钟内可以观察到存在 blaCTX-M、blaSHV 和 blaOXA 基因的可见蓝色线条。所有三个基因的检测限均为 2.5 ng/25 μL，与七种共生需氧菌没有交叉反应。从猪肉和粪便样本中分离出的大肠埃希菌中，分别有 93.9%（92/98）和 96%（48/50）表达 ESBL 产生表型。PCR 扩增子的核苷酸测序显示，blaCTX-M 是最普遍的类型（91.3-95.83%），其中主要形式是 blaCTX-M-55。RPA-LFA 的灵敏度和特异性分别为 99.2%和 100%，与 PCR 测序结果几乎完全一致（κ=0.949-1.000）。因此，RPA-LFA 是一种快速、无需设备的 ESBL 检测的有前途的工具，可促进人类和兽医医学中的临床诊断，以及 AMR 监测和监测。
