Effects of free antibiotic resistance genes in the environment on intestinal microecology of mice

The rapid spread of antibiotic resistance genes (ARGs) is a great challenge to the ecological safety and human health. The intestine of humans and animals is an important site for the increase and spread of ARGs due to the great diversity and abundance of microorganisms in the intestinal microecology. ARGs, including the intracellular (iARGs) and the extracellular (eARGs) ARGs, are usually introduced into the intestinal tract through the diet, and the iARGs are colonized and spread in the intestinal microbiota with the help of the host bacteria. However, whether the eARGs can enter the intestinal microorganisms in the absence of host bacteria is not known. Here, we show the transformation and the diffusion of the ampramycin resistance gene (Ap) carried by the free plasmid RK2 in the intestinal microbiota of mice. After two days of consecutive gavage with free RK2, the intracellular Ap gene increases from days 0-8 in the feces of mice, and has remained constant. Bacterial transformation happens in the small intestine, including proximal and distal jejuna and proximal and distal ilea, at the early stage (first two days), and the intracellular RK2 is diffused into the intestinal microbiota of mice by conjugation on days 2-8 day, which is based on the distribution of eARG and iARG and the mRNA expression levels of trbBp, trfAp, korA, korB, and trbA. The characteristics of ARGs susceptible microbiota for transformation are analyzed using 16s rRNA gene sequencing, transmission electron microscopy, and flow cytometric. The ingestion of RK2 affects the composition of intestinal microbiota especially for Proteobacteria, and the antibiotic residue promotes the increase in Escherichia coli. These findings are important to assess the risk of ARGs, especially the eARGs in the intestinal microecology.