A Chip-Based Colony Fusion Recombinase Polymerase
Amplification Assay for Monitoring of Antimicrobial Resistance Genes
and Their Carrying Species in Surface Water
posted on 2021-02-10, 16:11authored byKatharina Sollweck, Philipp Streich, Martin Elsner, Michael Seidel
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.