School of Environment, Tsinghua University, Beijing 100084, PR China; Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK.
Lancashire Teaching Hospitals NHS Trust, Fulwood, Preston PR2 9HT, UK.
J Pharm Biomed Anal. 2020 Mar 20;181:113108. doi: 10.1016/j.jpba.2020.113108. Epub 2020 Jan 15.
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), we identify two new peak shifts (970-990 cm) and (1,110-1,130 cm) associated with deuterium labelling. Notably, the spectral alterations from 1,110-1,130 cm 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.
持续滥用和过度使用抗生素会导致抗生素耐药基因（ARGs）广泛传播，并出现超级细菌。因此，我们迫切需要一种能够在实际情况下快速定量 ARGs 的方法。在这里，我们通过氘水和卡那霉素暴露的正交测试，即“三叶草分析”，来展示最先进的拉曼微光谱技术识别微生物群落中 ARGs 的能力。该分析成功地识别了两个遗传上相同的菌株之间的有区别的光谱变化，这两个菌株仅在表达一个卡那霉素耐药基因方面存在差异。除了先前报道的碳-氘振动带（2040-2300cm）的拉曼位移外，我们还确定了两个新的峰位移（970-990cm）和（1110-1130cm）与氘标记有关。值得注意的是，来自 1110-1130cm 的光谱变化与卡那霉素暴露强烈相关。通过引入色散指数（DI）和三叶草分析指数（CAI）作为指标，该分析能够定量人工微生物群中卡那霉素耐药基因的丰度。基于我们的结果，生物光谱三叶草分析是一种用于原位检测微生物群落中 ARGs 发生的强大工具，具有在未来消除培养方案需求的巨大潜力。由于其非破坏性和非侵入性的特点，该方法可能有潜力实时诊断水平基因转移（HGT）。
