Abstract:
Background Tetracycline resistance genes are the most representative new environmentallypolluting antibiotic resistance genes (ARGs), mostly in the form of antibiotic resistant bacteria (ARB), and are widely distributed in the water environment, posing potential harms to human health and ecological environment stability.
Objective This study explores a new and effective method for removing tetracycline resistance genes in the water environment, and analyzes the factors affecting the removal, in order to provide reference for the removal of ARGs in the environment.
Methods Escherichia coli (E.coli) containing tetracycline resistance genes tetA and tetR was constructed and inoculated in sterile water (D600=1.0). The two resistance genes were removed by ultrasound, H2O2, and ultrasound/H2O2, respectively. Three groups of ultrasonic treatment were set: 20 times (C20), 40 times (C40), and 80 times (C80), taking 0 times (C0) as the control group. Three groups of H2O2 treatment were set: 10, 20, and 60 mg·L-1, with 0 mg·L-1 H2O2 as the control group. Ultrasound/H2O2 combined treatment was divided into nine combinations of the two separate treatments. For the H2O2 treatment groups, samples were collected at five time points: 30min (T30), 60 min (T60), 120 min (T120), 240 min (T240), and 360 min (T360), and 0 min (T0) was used as the control group. The sample size of each treatment was 3. The removal effect of each treatment was evaluated by the reduction of the magnitude order of target tetA and tetR before and after treatmentlogarithm base 10 of the ratio of the concentration after (ρ) to before (ρ0).
Results When resistant E.coli was treated by ultrasound alone, it was found that with increasing number of repetitions, more tetA and tetR decreased, but the overall decrease was not significant: When ultrasound was administered 80 times, tetA and tetR decreased by 0.62 and 0.17 orders of magnitude respectively, and the removal efficiency of ultrasound on tetA was significantly higher than that on tetR (P < 0.05). Under electron microscope, the surface of resistant bacteria showed spherical protuberance and some bacterial structures were broken after the ultrasound treatment. When H2O2 acted on resistant E.coli alone, there was a significant interaction between H2O2 dose and treatment time (P < 0.05), and a significant difference among different H2O2 doses (P < 0.05). The removal effect of lowconcentration H2O2 on tetA and tetR in resistant E.coli was better than that of high-concentration H2O2. H2O2 removed tetA over time by up to 0.51 orders of magnitude (10 mg·L-1 H2O2, 360 min), but increased tetR by up to 0.45 orders of magnitude (60 mg·L-1 H2O2, 240 min). When the resistant E.coli was treated with ultrasound and H2O2 combination, there was a significant interaction between ultrasound treatment repetition and H2O2 dose (P < 0.001). Higher ultrasound repetitions and lower H2O2 doses were associated with better removal effects: tetA (C80, 10 mg·L-1 H2O2, 30 min) and tetR (C40, 10 mg·L-1 H2O2, 120 min) decreased by 1.15 and 0.56 orders of magnitude, respectively. After the combined treatment for 30 min, the changes of tetA and tetR were not significant over time (P > 0.05). However, different doses of H2O2 showed significant differences in the removal effect on the two resistance genes (P < 0.001).
Conclusion Ultrasonic treatment can cause mechanical damage to E.coli, resulting in E.coli fragmentation and entocyte outflow. H2O2 has an obvious removal effect on tetracycline resistance genes tetA and tetR in sterile water, as well as an obvious removal effect on tetA and a reverse increasing effect on tetR in E.coli. Compared with single treatment, ultrasound combined with low-concentration H2O2 can effectively remove tetA and tetR in resistant E.coli with a higher efficiency.