High-Throughput Single-Cell Technology Reveals the Contribution of Horizontal Gene Transfer to Typical Antibiotic Resistance Gene Dissemination in Wastewater Treatment Plants

The spread of antibiotic resistance genes (ARGs) has gained much attention worldwide, while the contribution of vertical gene transfer (VGT) and horizontal gene transfer (HGT) is still elusive. Here, we improved an emerging high-throughput single-cell-based technology, emulsion, paired isolation, and concatenation polymerase chain reaction (epicPCR), by lengthening the sequence of ARG in the fused ARG-16S rRNA fragments to cover the variance of both ARG and its hosts. The improved epicPCR was applied to track the hosts of a widely detected ARG, sul1 gene, in five urban wastewater treatment plants (UWTPs) during two seasons. The sul1 host bacteria were highly diverse and mostly classified as Proteobacteria and Bacteroidetes. Clear seasonal divergence of α-diversity and interaction networks were present in the host community. The consensus phylogenetic trees of the sul1 gene and their host demonstrated incorrespondence on the whole and regularity on abundant groups, suggesting the important role of both HGT and VGT, respectively. The relative importance of these two ways was further measured; HGT (54%) generally played an equal or even more important role as VGT (46%) in UWTPs. The application of the improved epicPCR technology provides a feasible approach to quantify the relative contributions of VGT and HGT in environmental dissemination of ARGs.