Spectrochemical identification of kanamycin resistance genes in artificial microbial communities using Clover-assay

Persistent abuse and overuse of antibiotics induces a widespread bloom of antibiotic resistance genes (ARGs) and the emergence of superbugs. A method designed to rapidly quantify ARGs in real-world scenarios is urgently needed. Here, we present an orthogonal test of heavy water and kanamycin exposure, namely, a "clover-assay", to reveal the capability of state-of-the-art Raman microspectroscopy to identify ARGs within microbial communities. This assay successfully recognizes the discriminating spectral alterations from two genetically identical strains that differ only in terms of the expression of one kanamycin resistance gene. In addition to the previously reported Raman shift at carbon-deuterium vibration bands (2,040-2,300 cm^-1), we identify two new peak shifts (970-990 cm^-1) and (1,110-1,130 cm^-1) associated with deuterium labelling. Notably, the spectral alterations from 1,110-1,130 cm^-1 strongly correlate with kanamycin exposure. By introducing dispersion index (DI) and clover assay index (CAI) as indicators, this assay is able to quantify the abundance of kanamycin resistance genes within artificial microbiotas. Based on our results, the biospectral clover assay is a powerful tool for the in situ interrogation of the occurrence of ARGs within microbial communities, which displays great potential to eliminate the need for culture protocols in the future. Due to the non-destructive and non-intrusive features, this approach may therefore potentially be able to diagnose horizontal gene transfer (HGT) in real time.