A Chip-Based Colony Fusion Recombinase Polymerase Amplification Assay for Monitoring of Antimicrobial Resistance Genes and Their Carrying Species in Surface Water

This work presents a new approach to the rapid characterization of antibiotic resistance in environmental settings. For monitoring purposes and risk assessment, it is important to link the detection of resistance genes to the actual carrying species and their potential pathogenicity for humans. Starting from a bacterial culture plate, this proof of principle study developed a colony-based fusion recombinase polymerase amplification (RPA) reaction that detects species and resistance genes simultaneously within 1.5 h. A first step generates the fusion product by homogeneous RPA, while detection occurs in a second step via heterogeneous asymmetric RPA (haRPA) on a flow-based microarray chip. The assay system successfully discriminated between Escherichia coli colonies carrying blaCTX-M cluster 1 resistance genes and E. coli carrying blaCTX-M genes of other clusters as well as other bacterial species carrying blaCTX-M resistance genes. A threshold value of 17% was determined for the differentiation between positive and negative samples. Analysis of water from the river Lech demonstrated the possibility of addressing real environmental samples. The potential for multiplexing was demonstrated by successful formation of fusion products by homogeneous RPA also in Klebsiella pneumoniae. This study uses RPA for the first time to create molecular fusion products providing a promising tool for future multiplex analyses of antibiotic resistance in the environment.