Oxytetracycline and heavy metals promote the migration of resistance genes in the intestinal microbiome by plasmid transfer

Horizontal gene transfer (HGT) has been considered the most important pathway to introduce antibiotic resistance genes (ARGs), which seriously threatens human health and biological security. The presence of ARGs in the aquatic environment and their effect on the intestinal micro-ecosystem of aquatic animals can occur easily. To investigate the HGT potential and rule of exogenous ARGs in the intestinal flora, a visual conjugative model was developed, including the donor of dual-fluorescent bacterium and the recipient of Xenopus tropicalis intestinal microbiome. Some common pollutants of oxytetracycline (OTC) and three heavy metals (Zn, Cu and Pb) were selected as the stressor. The multi-techniques of flow cytometry (FCM), scanning electron microscopy (SEM), atomic force microscopy (AFM), single-cell Raman spectroscopy with sorting (SCRSS) and indicator analysis were used in this study. The results showed that ARG transfer could occur more easily under stressors. Moreover, the conjugation efficiency mainly depended on the viability of the intestinal bacteria. The mechanisms of OTC and heavy metal stressing conjugation included the upregulation of ompC, traJ, traG and the downregulation of korA gene. Moreover, the enzymatic activities of SOD, CAT, GSH-PX increased and the bacterial surface appearance also changed. The predominant recipient was identified as Citrobacter freundi by SCRSS, in which the abundance and quantity of ARG after conjugation were higher than those before. Therefore, since the diversity of potential recipients in the intestine are very high, the migration of invasive ARGs in the microbiome should be given more attention to prevent its potential risks to public health.