Changes In Multidrug Resistant Bacteria In Animal Manure After The Ban Of Antibiotic Growth Promoters And Its Control During Composting

Due to the frequent use of antibiotics,multidrug-resistant bacteria(MRB)in farm manure can pose ecological risks to the surrounding environment.In order to understand the change characteristics of MRB from farms after the ban of antibiotic growth promoters(AGPs),this study collected livestock and poultry manure samples from farms before and after the ban of AGPs,counted,isolated and identified MRB,and used 16 S r RNA gene amplicon sequencing to compare and analyze the cultureable MRB.The resistance characteristics of MRB before and after AGPs were analyzed by susceptibility tests.The effects of aeration rate on the removal of MRB and antibiotic resistance genes(ARGs)and the changes of bacterial community structure during composting were studied.The main conclusions are as follows:Firstly,the analysis of the change characteristics of MRB and their community structure before and after the ban of AGPs showed that the average multidrug-resistant rate of pig and chicken manure decreased by 64.55% and 11.59%,and the average number of MRB decreased by 37.61% and 13.51%,respectively.The results indicated that AGPs prohibition had a positive effect on the pollution reduction of MRB in animal manure.The diversity and evenness of cultivated MRB decreased after the ban.β-diversity analysis showed that there was a significant difference in the composition of MRB community in manure before and after the ban,and the concentration of oxytetracycline in manure significantly affected the distribution of MRB.The MRB were mainly distributed in Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes at the phylum level.On the genus level,Escherichia was the absolute dominant bacterium,while the relative abundance of multidrug-resistant genus such as Ignatzschineria,Corynebacterium and Enterococcus decreased after AGPs was banned.Secondly,after further isolation,264 strains of MRB were obtained.The antibiotic susceptibility testing showed that the ban on AGPs could reduce the MRB ’s resistance rate of some AGPs,such as tetracyclines,chloramphenicols and macroliactones,in pig and chicken manure,but also increase the MRB ’s resistance rate of antibiotics to therapeutic use such as aminoglycosides,cephalosporin and rifampicin.There was little difference in the dominant MRB between pig and chicken manure,but there were significant differences in the composition of MRB,indicating that in addition to the influence of AGPs,host animal species also affected the composition of MRB in manure.Finally,Kurthia gibsonii XT5A4 carrying multiple ARGs were added to manure as exogenous pollutants before composting.At the same time,different aeration rates of 0.01L/min·L,0.05 L/min·L and 0.1 L/min·L were set up to study the changes of ARGs and the effects of different aeration rates on the ARGs reduction rate during composting.The results showed that aerobic compost significantly reduced the total bacterial population and MRB,and the reduction rates were 70% and 100%,respectively.The0.05 L/min·L ventilation group has the highest reduction rate.Quantitative PCR analysis showed that compost treatment had a good effect on the removal of the conjungative plasmid(MOBP gene)and MRB(Kurthia 16 S r RNA gene),with the reduction rates of 99.98% and 93.22%,respectively.However,the remove efficiency of absolute abundance of APH(3)-Ib and int I1 in compost was limited.It shows the trend of decline in high temperature period and rebound in low temperature period.These results indicate that compost plays an important role in the elimination of MRB and their drug-resistant genes in livestock manure.Alpha diversity analysis of bactieral community in composts showed that both Sobs index and Shannon index decreased after composting,indicating that composting can effectively reduce species diversity and evenness in manure.After composting,Enterobacter and other high-temperature sesitive bacteria were killed,and Thermobifida and other thermophilic bacteria became the dominant bacteria.Combining the environmental factors with the OTU data of the bacterial community,it was found that more than 30% of the top 30 bacterial genera in the abundance of the compost samples were positively correlated with organic matter,and more than 50% were negatively correlated with potassium,and the bacterial community structure of 0.05 L/min·L ventilation group was more stable.In this paper,the change characteristics of MRB and ARGs from farms were analyzed before and after the ban of AGPs.The changes of ARGs during composting and the effect of different ventilation on the removal of MRB and carried ARGs were studied,which provided basic data for monitoring,prevention and control of farm-sourced antibiotic pollution.