How heavy metal stress promotes dissemination of antibiotic resistance genes in the activated sludge process

Heavy metals have been recently revealed as promoters to antibiotic resistance gene (ARG) dissemination in water environment, but their influence on ARG transfer in the activated sludge process has not been clear. In this study, a set of sequencing batch reactors (SBRs) and micro-scale microfluidic chips were established to quantify the impacts of heavy metals (0.5 mM of Pb, 0.1 mM of As, and 0.005 mM of Hg) on the ARG spreading in the activated sludge consortium. Under heavy metal stress, transfer frequencies were 1.7-3.6 folds increase compared to the control. Gram-negative bacteria increased significantly after heavy metal added, which were more prone to receiving resistant plasmid from donors. Meanwhile, the relative expression of genes related to conjugation changed in activated sludge, especially the expression of outer membrane protein and oxidative stress regulatory genes increased by 2.9-7.4 folds and 7.8-13.1 folds, respectively. Furthermore, using microfluidic chips, the dynamics of ARG transfer was observed at single cell level under heavy metal pressure. Heavy metals firstly promoted conjugation and then vertical gene transfer played an important part for ARG spreading. The results provided in-depth understanding of the influence of heavy metals on ARG behavior in the environment.