Effect Of Composting On Antibiotic-resistant Integrons Abundance In Livestock And Poultry Manure

In recent years,with the increasing use of veterinary antibiotics in intensive livestock and poultry farming,antibiotic pollution has become more and more serious,and the emergence of antibiotic-resistant bacteria(ARB)and antibioticresistance genes(ARGs)has drawn increasing attention to this emerging pollution.In this study,multi-resistant bacteria(MRB)and antibioticresistance genes were used as research objects.The contamination of multi-resistant bacteria in large-scale livestock and poultry manure was investigated,and the integrons associated gene cassettes in typical MRB were analyzed.The removal of multi-resistant bacteria and integrons in cultured manure from high temperature aerobic composting were analyzed,which laid a theoretical foundation for further control of the horizontal transfer of ARGs and the safety evaluation of ARGs in organic fertilizer.The main conclusions are as follows:First,twelve typical farms and the manure compost plants were selected.Fresh manure and compost samples were collected.MRB were isolated by adding two sets antibiotics,respectively.The MRB communities of different animal manure wereanalyzed.Bacterial count results showed that:for tetracycline,enrofloxacin,sulfamethoxazole,and tylosin(class A),and for anti-flufenicol,ampicillin,ceftiofur,and colistin(class B),the pollution status(absolute number and relative number)of the two types of multi-drug-resistant bacteria were both chicken manure>pig manure>cow manure.The absolute and relative numbers of cultivable MRB in composts were decreased.It shows that composting is an effective method to kill MRB.Analysis of the bacterial community structure based on 16S rDNA sequence amplicon sequencing shows that the diversity of MRB groups with different antibiotics sets is different.On the alpha-diversity,the highest diversity of type A MBR is found in the layer chicken manure,while the highest diversity of type B MRB is found in swine manure.On the beta-diversity,the smallest community differences among samples of type A MRB are found in cattle manure.The community differences among the samples of B type MRB were minimal.The species richness and diversity of MRB in compost are both smaller than those in manure,which indicates that composting can effectively reduce the richness and diversity of MRB in both type A and B.Type A and B MRB are mainly distributed at the phylum level of Proteobacteria,Firmicutes,Actinobacteria,and Bacteroidetes.Unidentified-Enterobacteriaceae is the dominant genus in type A MRB,while Myroides is the dominant genus of MRB in type B.Then,126 multidrug-resistant bacteria were isolated and purified,we carried out molecular biological identification based on 16S rDNA sequence alignment to determine the species status of the isolated MRB strains,and analysis of drug resistance characteristics based on drug sensitivity tests.The types and structures of multidrug-resistant integrons were analyzed.The results show that theMRB in large-scale poultry and animal husbandry are mainly concentrated in the four phyla of Proteobacteria,Firmicutes,Actinobacteria,and Bacteroidetes.Among them,the dominant type for MRB of type A is Enterobacter;The dominant genus of MRB is Shigella.The susceptibility test results show that the type A MRB in manure are all resistant to common antibiotics such as chloramphenicol,fluoroquinolone,?-lactam,sulfonamide,tetracycline,and macrolide.They are all sensitivity to aminoglycosides and cephalosporin.While type B MRB has little isolates,they are almost resistant to common antibiotics.Through the amplification of class ? and ? integron gene cassettes,it was clarified that the type ?integron gene cassettes that are widely present in manure and compost are aadA2 and dfrA17,and the main type of type ? integron gene cassette is sat2-dfrA1.Finally,a typical MBR Shigella flexneri 5A5 was selected and its antibiotic resistant plasmid was sequenced.Specific primers for integrase gene(int1)and ARGs such as aadA,sul2,mcrl and oqxb were designed.The 5A5 were added to the composting to simulate MRB and ARGs contamination,and observe the abundance of MRB and the removal of multi-drug resistant bacteria and integrons during high-temperature composting.The results showed that no matter whether the MRB added or endogenously,the composting process had a significant removal effect on them.After 3 days of high-temperature period,the colony-forming units(CFU)of cultivatable multi-resistant E.coli was approaching 0,and for the total MRB,after a 10-day composting process,the CFU also decreased by about 4-6 orders of magnitude.At the same time,during composting,the absolute abundance of ARGs showed a slight increase and then continued to decrease,and the removal efficiency of ARGs was above 89%,and the efficiency of intI removal was as high as.80%.The relative abundance of mostARGs showed a trend of decreasing first and then rising slightly.Among them,the remval rate of relative abundance ARGs and integrons was above 86%.This shows that composting is an effective way to deal with MRB and ARGs in manure from livestock and poultry farming.