Evolution And Spread Of Resistance Genes In Simulated Fish-duck Intergrated Pond

Fish-duck polyculture pond is a typical breeding model in the Pearl River Delta,and it is a hot environment with resistance genes co-selection conditions.In order to promote the growth of livestock and poultry and prevent diseases of livestock and poultry,antibiotics are widely used as feed additives and veterinary drugs.Most of the drugs and their induced resistance genes are excreted with the excrement and urine of livestock and poultry,causing serious pollution of resistance genes in the surrounding soil,river and aquaculture environment.A large number of resistance genes in the environment and livestock and aquaculture animals may cause potential harm to human health through the food chain.In view of the increasing level of resistance gene pollution in the environment,it is also necessary to consider fecal pollution while studying the selection pressure of residual antibiotics.Therefore,it is very necessary to study the transmission and distribution of resistance gene in the integrated culture environment,which will provide basic data for comprehensive understanding of the environmental behavior,ecological risks and health hazards of resistance gene leaving the hot environment.In this study,duck manure,cefotaxime,and arsenic were used as stress factors.A smallscale simulated fish-duck polyculture pond system was established,including control tank,duck manure tank,duck manure + cefotaxime tank,duck manure + arsenic tank and duck manure + cefotaxime + arsenic tank.The experimental period lasted for 100 days.The diversity and relative abundance of resistance genes in water,biofilm and fish intestine samples were detected by qualitative PCR and real-time fluorescence quantitative q PCR,and comparatively analyze the effects of duck manure or the addition of cefotaxime and arsenic on resistance genes,so as to explore the transmission and distribution of resistance genes under stress.16 S r DNA high-throughput sequencing technology was used to detect the bacterial structure in fish tanks and analyze the bacterial effect under different stress conditions.The results of this study indicate that the fish-duck integrated culture model promotes the spread of resistance genes,and duck manure is a potential source of resistance genes in the aquaculture environment.The addition of duck manure resulted in an increase in the diversity and abundance of resistance genes in the fish tanks.Tetracycline resistance genes(tet A,tet C,tet G,tet M,tet O,tet X),sulfonamide resistance genes(sul1,sul2),cephalosporin resistance genes(MOX,FOX,DHA,CIT,TEM,EBC)and arsenic resistance genes(ars B,ars C)were detected in the fish tank,of which tetracycline resistance genes are dominant resistance genes.Its total relative abundance is 1 to 2 orders of magnitude higher than that of the other three types of resistance genes,and it is more sensitive to being induced to evolve.In addition,the total relative abundance of resistance genes in the tanks with duck manure and stress factors was 4 ? 5 orders of magnitude higher than that of the control tank and 1 ? 2 orders of magnitude higher than that of the duck manure tank.The induction effect of cefotaxime was greater than that of arsenic on the evolution of resistance genes,and the induction effect of cefotaxime and arsenic co stress on resistance genes was greater than that of single stress under low concentration.Resistance genes in duck manure preferred to spread from water to biofilm and then fish intestine,however,the addition of stress factors changed the spread of resistance genes,of which the cefotaxime and As co-stress most obviously triggered the spread of RGs to fish intestine.Stress factors also accelerated the accumulation of resistance genes in fish intestine,and the enrichment multiple is: duck manure + cefotaxime tank(0.35 ? 7.25)> duck manure + cefotaxime + arsenic tank(0.46 ? 5.78)> duck manure + arsenic tank(0.41 ? 5.30)> duck manure tank(0.05 ? 0.6)> control tank(0.02 ? 0.12).The analysis of bacterial structure showed that the proteobacteria,Firmicutes,and Fusobacteria were the dominant bacteria in the fish intestine of the control tank,and duck manure,cefotaxime,and arsenic changed the bacterial structure and increased the species richness in the fish tank system,significantly.The order of the OUTs in fish intestine of different tanks was: duck manure + cefotaxime + arsenic tank(372)> duck manure + cefotaxime tank(365)> duck manure + arsenic tank(351)> duck manure tank(328)> control tank(102).In addition,correlation analysis showed int I1 was significant correlated to sul1,tet A,tet M,tet X,ars B,MOX,TEM(P < 0.05 or P < 0.01),hinting that there was a potential transmission risk of resistance genes.