The Fate Of Antibiotic Resistance Genes Anddiversity Of Microflora Under Antimicrobial Pressureinsoil-manure-earthworms Model

Antibiotic resistance genes(ARGs),as a new environmental pollutants,has aroused great concern in society in recent years.In livestock and poultry breeding industry,due to long-term and extensive use of antibiotics to prevent disease and promote animal growth,antibiotics remained in fecal greatly induced ARGs andspread them in the farmland,soil and rivers,which not only affects the diversity of environmental indigenous microorganisms but also enter the surrounding water vegetableand fish,posing a great threat to public health.In order to evaluate the behavior of ARGs in soil environment after the application of feces.By constructing fecal-soil-antibiotic indoor soil model,we want to explore the effect of the application of antimicrobial agents on the elimination of antibiotic resistance genes and the diversity of bacteria in fecal soil.At the same time,another experimental group swere also designed,tostudy the effect of earthworms' presence and sterilized soil.The untreated soil was set as control,and the antibiotics was set in three concentrations,0 mg/kg(pure manure);0.5 mg/kg(low antibiotic);5 mg/kg(high antibiotic).In the other two groups,the earthworms Eisenia fetida were added and the experimental soil was autoclaved.The abunda nce of various antibiotic resistance genes was detected by real-time fluorescence quantification at the 1th,7th,15 th,30th and 60 th day after the exposure of the antimicrobial agents.The Illumina 454 sequencing platform was used to study the 16 S r DNA diversity.The results showed that the resistant genes(tetracycline-resistant genes: tet B,tet W and tet M,sulfonamides-resistant genes: sul1 and sul2,plasmid-mediated fluoroquinolone-resistant gene,aac(6')-Ib-cr,oqx A and qnrS)were significantly increased(P ?0.05).During the experiment time,the logarithmic regression model(N = mlnt + b)was used to study the reduction of drug-resistant genes.The reduction rate of drug resistance gene is assessed by m-value reaction(the greater the m value,the slower the reduction rate).S urprised that the addition of earthworms significantly accelerated the reduction of all resistance genes(except for the oqx A in low).Under the antimicrobial pressure,the antibiotic resistance gene was slower than the control group,and the reduction rate was related to the concentration.The reduction rate of resistance genes in the original soil group,earthworm group and sterilized soil group showed that the tetracycline resistance gene was significantly higher than that of tet B in all the groups,and the reduction rate was reverse correlated with the concentration.The sulfonamide-resistant gene sul1 and sul2 had no obvious trend under different antibiotic concentration(P>0.05).The PMQR genes showedthat environmental change had no obvious effect on their reduction,but oqx A was significantly higher than the other two PMQ R genes.In this experiment,16 S r DNA sequencing only came from the first day,the 30 th day and the 60 th day including the earthworms group and untreated group.The diversity results showed that it has a greater volatility in the original group.With the addition of earthworms,the abundance and the stability of bacteria maintained all the time.At the same time,the classification of O TU showed that the addition of earthworms did not significantly change the species and abundance of the various types of bacteria at the phlym level,Actinobacteria and Firmicutes occupied the main position at the first day,while in the 30 th and 60 th day,Bacteroidetes dominated.However,at the level of genus classification,the presence of earthworms significantly affected the abundance of species.The results shown showed no difference at the first day,and Corynebacterium was the dominant genus.At the 60 th day,the dominant genus in the original soil group were Lysobacter,while the earthworm group was Luteimonason the 30 th day and Rhodanobacter on the 60 th day.From the experimental results of this paper,we can acquire that the activity of earthworms affects the structure of bacteria in the soil.In conclusion,the differences in the reduction effiency of antibiotic resistance genes in soils were discussed by different treatments(steeding,antimicrobial stress,adding earthworms and soil sterilization).Steeding and antibiotics stress were significantly increased the ARGs abundance.Manure and earthworm added increase the reduction rate,while the antimicrobial and sterilization slowed down the reduction.The earthworm group diversity increased,and species have also changed.If the combination of specific bacteria and the elimination efficiency of specific drug resistance genes,we can found a way to improve the elimination of drug resistance genes.