The Influence Of Fishery Oxidants And Bacillus Subtilis On Sulphonamide Resistance Sul1 Genes In The Aquaculture Pond Water

In China,antibiotics are widely used in the treatment of human and animal diseases.Agriculture and animal husbandry also use antibiotics as growth promoters.This makes antibiotics remain in various environments such as river water bodies and sediments,soil and sewage treatment plants.The large amount of antibiotic residues in the environment makes it enter the breeding environment in the form of aquaculture water source and leads to its accumulation in aquatic products,which affects the microecological balance in the aquatic animals and directly induces antibiotic resistant bacteria（ARB）and antibiotic resistance genes（ARGs）.ARGs spread horizontally in the aquaculture environment and transfer to pathogenic bacteria in aquatic animals,which may eventually pose a threat to the health of farmed animals and even humans.Although the scientific use of antibiotics contributes to the healthy development of the aquaculture industry and has created good benefits,they have received widespread attention in recent years as the problem of antibiotics and their ARGs residues has become increasingly serious.Many current studies have shown that different types of ARGs have spread in aquaculture environment.Both fishery oxidants and bacillus are commonly used additives in aquaculture and are widely used in aquaculture production,but their impacts on ARGs in the aquaculture environment are rarely reported and thus worth studying.In this study,the commonly used fishery oxidants in aquaculture,i.e.,sodium hypochlorite,dibromohydantoin and potassium permanganate and the commonly used probiotic,i.e.,Bacillus subtilis TY7210 were selected,and Escherichia coli SR1 and sulfonamide resistance gene sul1 carried by p MD18-T were respectively used as the model ARB and ARG.In order to explore the feasibility of using fishery oxidants to remove ARGs in the aquaculture environment and control their spread,the SR1 and sul1inactivation/removal efficiencies of fishery oxidants at three concentrations（0.5 mg/L,5mg/L and 15 mg/L）were firstly studied in a clean water matrix[phosphate-buffered saline（PBS）]（25℃,p H=7）,and the kinetics of the inactivation and removal of ARB and ARGs were studied based on the Chick-Watson model to explain disinfection abilities of oxidants,respectively.Then,the experiment was conducted in pond water to futher explore the modeled ARB and ARG disinfection effects of fishery oxidants in the aquaculture environment.The kinetics models were verified and influence of aquaculture water on ARGs removal was studied.Finally,fishery oxidation methods were applied to aquaculture pond water matrix to study indigenous ARB and ARG disinfection efficiencies.In order to study the effect of Bacillus subtilis on sul1 in aquaculture water environment,Bacillus subtilis was selected as a model probiotic.In the pond water containing plasmid p MD18-T（carrying sul1）and pond water containing E.coli SR1（the selected pond water contained native extracellular sul1）,its effects on sul1 concentrations and whether it could obtain exogenous sul1 during the experiment were studied.Research indicates:1.The kinetics models for the ARB/ARG inactivation/removal processes of three fishery oxidants（sodium hypochlorite,dibromohydantoin and potassium permanganate）were determined in the PBS matrix,and the kinetics data could be partially tested in the pond water matrix to predict the disinfection efficiencies of ARB/ARGs.Among the three fishery oxidants,chlorine had the strongest inactivation effect on E.coli SR1(k=6.36×10⁴ M^-1·s^-1),followed by bromine(k=1.16×10⁴ M^-1·s^-1)and potassium permanganate(k=2.63×10² M^-1·s^-1).The k-values for e ARGs（extracellular antibiotic resistant genes）removal of active chlorine and active bromine were 1.42×10² M^-1·s^-1 and1.68×10² M^-1·s^-1,respectively.The k-values for iARGs（intracellular antibiotic resistant genes）removal of active chlorine and active bromine were 1.54-1.89×10² M^-1·s^-1 and1.71-1.76×10² M^-1·s^-1,respectively.The abilities of active chlorine and active bromine to remove ARGs were in moderate level(10-1000 M^-1·s^-1).The k-value showed that the removal of ARGs lagged behind the inactivation of ARB,and the removal process of iARGs is close to that of e ARGs（p=0.7）.The kinetics models provided a basis for scientific evaluation of the ARB/ARGs disinfection ability of oxidants,and can be partially verified in the pond water environment to predict the ARB/ARGs inactivation/removal effect.The fishery oxidants at 5 mg/L could completely inactivate SR1.The fishery oxidants at 15 mg/L could effectively remove iARGs,and the active chlorine and potassium permanganate at 15 mg/L could effectively remove e ARGs.2.In the pond water environment,the results of study on indigenous ARB/ARGs disinfection showed that the concentration of iARGs is much higher than that of e ARGs,and the ARB inactivation in the pond water environment influenced the ARG removal.The removal effects of chlorine and bromine on iARGs significantly exceeded that on e ARGs.Among the three fishery oxidants,15 mg/L of chlorine could efficiently remove ARGs in the pond water matrix（1.0-log）.The scientific use of chlorine-containing oxidants can effectively remove ARGs in the pond water environment to control their spread.3.In the pond water environment,Bacillus subtilis had no significant effect on the concentration of sul1,and it had not acquired sul1 during the experimental process.It preliminarily shows that the application of Bacillus subtilis in aquaculture does not promote the sul1 spread,which is conducive to the promotion of antibiotic-free breeding.