Removal of tetracycline resistance genes in water by ultrasound/hydrogen peroxide

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.