In vitro assessment of the bacterial stress response and resistance evolution during multidrug-resistant bacterial invasion of the Xenopus tropicalis intestinal tract under typical stresses.

The intestinal microbiome might be both a sink and source of resistance genes (RGs). To investigate the impact of environmental stress on the disturbance of exogenous multidrug-resistant bacteria (mARB) within the indigenous microbiome and proliferation of RGs, an intestinal conjugative system was established to simulate the invasion of mARB into the intestinal microbiota in vitro. Oxytetracycline (OTC) and heavy metals (Zn, Cu, Pb), commonly encountered in aquaculture, were selected as typical stresses for investigation. Adenosine 5'-triphosphate (ATP), hydroxyl radical (OH·-) and extracellular polymeric substance (EPS) were measured to investigate their influence on the acceptance of RGs by intestinal bacteria. The results showed that the transfer and diffusion of RGs under typical combined stressors were greater than those under a single stressor. Combined effect of OTC and heavy metals (Zn, Cu) significantly increased the activity and extracellular EPS content of bacteria in the intestinal conjugative system, increasing intI3 and RG abundance. OTC induced a notable inhibitory response in Citrobacter and exerted the proportion of Citrobacter and Carnobacterium in microbiota. The introduction of stressors stimulates the proliferation and dissemination of RGs within the intestinal environment. These results enhance our comprehension of the typical stresses effect on the RGs dispersal in the intestine.

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.