Characteristics Of Conjugation Transfer Of Multidrug-resistant Plasmids In Livestock Farmsf And Its Influence Mechanism Of Composting Temperature

In recent years,antibiotics have been widely used in intensive animal farming,and even abused.Due to the long-term irrational use of antibiotics on farms,various animal sourced pathogens have developed antibiotic resistance.Antibiotic resistance has not only caused serious food safety problems,but has also become an important factor restricting the development of livestock and poultry farm.The emergence of multidrug-resistant bacteria and the spread of antibiotic resistance genes(ARGs)caused by the horizontal transfer of antibiotic-resistant plasmids have seriously threatened the ecological environment.In response to the above problems,this study carried out an evaluation of the mobility of multidrug-resistant bacteria carrying antibiotic-resistant plasmids in farms;analyzed the genome of conjugative multidrugresistant plasmids and the characteristics of ARGs carried by them;the effect of composting at different high temperature time on the reduction of ARGs carried by exogenous plasmids and the shifts of bacterial community were studied.The main conclusions are as follows:Firstly,the conjugative transfer experiment of 78 multidrug-resistant strains was carried out,and the multidrug-resistant strains with the ability of conjugative transfer of plasmids were screened.Then,the multi-drug resistant transconjugants were captured.The results showed that the proportion of multi-drug resistant plasmids capable of conjugative transfer in farms was low,and mainly existed in manure of food animals,such as pigs,beef cow and broiler chickens.ARGs on multidrug-resistant plasmids in food animals have a higher risk of horizontal transfer than that in functional animals.Secondly,the genome-sequencing results of the multi-drug resistance plasmids showed that the size of all the multi-drug resistance plasmids were above 130 kb.The S13A1-P and T5A4-P plasmids isolated from beef cow and broiler chicken both had gene island structures derived from horizontal transfer.Both G3A2-P and T5A4-P plasmids had prophage structures.The predicted results of ARGs based on the CARD database show that aminoglycoside resistance genes are the most common in conjugative plasmids,followed by sulfonamide resistance genes and β-lactam resistance genes.All the sequenced multidrug-resistant plasmids in our samples capable of conjugation transfer carried a variety of virulence factors,indicating that significant co-transfer of ARGs and virulence factors existed in multidrug-resistant bacteria from farms.Finally,the multidrug-resistant strains were added to the compost environment,and the conjugative multidrug-resistant plasmids and ARGs carried on them were used as exogenous pollutants to investigate the effects of the composting process and high temperature time on the conjugative drug-resistant plasmids and their horizontal transfer.In the experimental group,prolonged high temperature time was adopted to study the effect of high temperature duration on the reduction rule of ARGs,and the corresponding changes of compost flora were also analyzed.The results showed that prolonged high temperature can reduce the total number of mesophilic bacteria in compost products and effectively inhibit the activity of multi-drug resistant bacteria.The culturable multidrug-resistant bacteria were sequenced,and the results showed that the dominant multidrug-resistant bacteria in the control group before and after composting changed from E.The absolute abundance of ARGs in the whole composting process was measured by dd PCR and the relative abundance value was calculated.In the absolute abundance,the reduction effect of Enterobacter 16 S r RNA gene and MOBP gene was good,resistance genes APH(3)-Ib,resistance genes The abundance of gene Sul2 and integrase gene int I1 increased.In the fitting curve,Enterobacter 16 S r RNA gene and MOBP gene fit well.Statistical analysis of the alpha diversity index showed that composting could significantly reduce the uniformity of the community and increase the concentration of the bacterial community,and prolonging the high temperature time of composting could reduce the bacterial diversity of the final compost product.In the comprehensive analysis of community species composition and environmental factors,among the top 20 genera,most genera had strong correlation with environmental factors,and the temperature-affected genera in the experimental group were more than those in the control group.In the correlation analysis of community composition and ARGs abundance,it was found that the increase of ARGs abundance may be related to the enrichment of some genera,among which Bifidobacterium thermophilus was the dominant species in the experimental group.The characteristics of conjugation efficiency and conjugative transfer system of multidrugresistant plasmids on livestock farms were studied,and the removal of ARGs on conjugative multidrug-resistant plasmids with different composting high temperature times and the shifts of bacterial community were analyzed.The results provide a theoretical basis for the subsequent research on the control and removal of animal-sourced conjugative multidrug-resistant plasmids.