Study On The Effect Of Ultrasound/H₂O₂ On The Removal Of Tetracycline Antibiotic Resistance Genes In Water

ObjectiveTo explore a new and effective method to remove antibiotic resistance genes in the environment,and analyze its influencing factors,in order to provide some reference for the effective methods of removing antibiotic resistance genes in the environment.MethodsEscherichia coli（E.coli）containing tet A and tet R was constructed and inoculated in sterile water(OD₆₀₀=1.0).The aqueous solution containing resistant E.coli was treated at different ultrasonic frequencies of C0（0 times）,C20（20 times）,C40（40 times）and C80（80 times）,respectively.After treatment,the 1m L solution was stored at-20℃.Add 10mg/L,20 mg/L,60 mg/L H₂O₂to treat,collect the bacterial liquid before and after ultrasonic treatment,the collection time is T30（30 min）,T60（60 min）,T120（120 min）,T240（240 min）,T360（min）,each take 1m L bacterial solution to store at-20℃,take untreated bacterial liquid 1m L（T0）,extract DNA,to do q PCR（real-time quantitative PCR detecting system,q PCR）,and do the same treatment to tet A and tet R in aqueous solution.At each time point,20μL samples were stored for q PCR analysis.The effect of ultrasound,hydrogen peroxide and their combination on the removal of tetracycline resistance genes in water environment was studied.ResultsWhen the resistant Escherichia coli was treated by ultrasound alone,it was found that with the increase of ultrasound times,the number of tet A and tet R decreased more,but the overall decrease was not significant（p<0.05）.Under electron microscope,the surface of resistant bacteria showed spherical protuberance and some bacterial structures were broken.When H₂O₂acted on resistant Escherichia coli alone,it was found that there was a significant interaction between H₂O₂dose and treatment time,and there was significant difference among different H₂O₂doses.Tet A and tet R in H₂O₂E.coli showed that the removal effect of tet A and tet R at low concentration was better than that at high concentration.When the resistant E.coli was treated with ultrasound and H₂O₂,it was found that there was a significant interaction between ultrasound treatment and H₂O₂dose（p<0.001）.The more ultrasound times and the lower H₂O₂dose,the better the removal effect.After the combined action of ultrasound and H₂O₂for 30 min,the cumulative change of tet A and tet R was not significant（p>0.001）.However,different doses of H₂O₂showed significant differences in the removal of the two resistance genes（p<0.001）.The number of tet A and tet R,exposed in aqueous solution increased slightly by ultrasound alone,but the increase did not change with the increase of ultrasound times（p>0.05）.When H₂O₂acted on resistant Escherichia coli alone,it was found that there was a significant interaction between H₂O₂dose and treatment time.The higher the concentration of H₂O₂,the longer the treatment time,the better the removal of resistance genes.Under the combined action of ultrasound and H₂O₂,it was found that there was a significant interaction between ultrasound treatment and H₂O₂dose（p<0.001）,tet A、tet R decreased steeply and rapidly,and the removal effect was very significant,which showed a significant promoting effect as a whole.ConclusionThe combined effect of ultrasound/H₂O₂is better than the single method in the removal of two tetracycline resistance genes in Escherichia coli,and the removal effect of tetracycline resistance genes in aqueous solution is very significant.