Study On The Effect Of Chlortetracycline On The Transfer Of Resistance Genes During Swine Manure Composting

After long-term intake of trace heavy metals and antibiotics are used for prevention and treatment of diseases in livestock and poultry,the intestinal microorganisms are subjected to the co-selective pressure of antibiotics and heavy metals.The feces and urine contain antibiotic resistance bacteria,antibiotic resistance genes(ARG)and heavy metal resistance gene(HMRG).If they return to the field without treatment,the resistance genes carried by drug-resistant bacteria will spread in the environment through horizontal transfer.More resistant bacteria will increase the risk of drug resistance in the environment,thus threatening human and animal health.Chlortetracycline(CTC)is one of widely used tetracyclines in pig farms in northern China.There is lack of the study on the effect of CTC on the transmission mechanism of resistance genes.This study focused on the effects of different initial CTC residue on swine manure composting,to analysis of the changes in the abundance of ARG and HMRG during manure composting with different initial concentrations of CTC,and to explore the transmission mechanism of the class ? integron mediated resistance genes.Aimed at the reduction of CTC and other trace antibiotics in manure composting with low dose CTC residues,the changes of the abundance of ARGs such as tetracyclines,quinolones,sulfonamides and macrolides in the composting process,and the effect of CTC on microbial community in the composting process were studied at first.The removal of pollutants in swine manure composting process with two kinds of low concentration antibiotic residues was studied.Treatment CK does not add CTC,treatment T1 added 10 mg/kg CTC,treatment T2 added 20 mg/kg CTC.As a result,compost maturity meets the hygienic requirements for harmless feces(GB 7959-2012)and technical specification for harmless treatment of feces(GB 36195-2018).CTC,sulfamerazine,enrofloxacin and erythromycin were all degraded after composting.Composting can effectively reduce the level of ARG in the range of 0.26?3.35 logs.However,the absolute abundance of sulfonamides ARG(sul?,sul?,sul?),quinolones ARG(gyr A and par E),tetracyclines ARG(tet A and tet M)increased.Clostridium sensus tricot,Clostridium ? and Bacteroides are the dominant bacteria contributed to the ARG in the heating phase.Sporosarcina was the main genus in thermophilic phase,while Clostridium ?,Clostridium sensus tricot,Pseudoxanthomonas contributed on the maturation stage.In addition to antibiotic residues,the concentrations of Cu and Zn in pig manure were 32.52-42.27 mg/kg and 282.39-308.21 mg/kg,respectively.The transformation of heavy metal speciation,removal of HMRGs and transfer of mobile genetic elements(MGEs)during composting with high concentration of antibiotics(100 mg / kg)and low concentration of antibiotics(20 mg / kg)were investigated.The results showed that the content of bioavailable Cu were reduced by 14.5%,27.1% and 26.7% in treatment CK,P1 and P2,respectively.The content of bioavailable Zn decreased 15.31%,29.47% and 12.07% in treatment CK,P1 and P2,respectively.The absolute abundance of Cu resistance genes pco A,pco D and cus A were reduced by 1.24-4.1 logs,1.28-2.82 logs and 0.43-1.71 logs,respectively.The absolute abundance of Zn resistance gene znt A were reduced by 0.93-2.82 logs.Therefore,high concentration of antibiotics has improve the co-selection pressure of heavy metal and antibiotic on bacteria,the total removal ability of resistant genes decreased.Pearson's correlation analysis showed that Bacteroides and Escherichia/Shigella were the dominant bacteria play an important role in the transmission of pco A and pco D in CK treatment.Comamonas is the potential host of pco A and pco D,and Clostridium sensus tricto is the potential host of znt A in the compost with low CTC concentration.The class 1 integron was the potential MGE of cus A,and the CTC residual at a higher concentration can indirectly promote cus system-mediated Cu-resistance gene transfer.Based on the previous study,transmission mechanism of resistance genes in composting with with high concentration of antibiotics(100mg/kg)and low concentration of antibiotics(20mg/kg)were investigated.The results showed that the higher the concentration of CTC,the longer the degradation time.The higher CTC concentration of treatment P2 need more 10 days than that of treatment P1.Because the high concentration of antibiotic residues brings great co-selection pressure on ARG transfer.The abundance of sulfonamides,quinolones,macrolides resistance genes in treatment P1 and P2 was significantly higher than that in treatment CK in the beginning of composting,which indicated that the increase of single antibiotic concentration would enhance the transfer of other ARGs.The results of the distribution of resistant gene cassette mediated by the ? class integrons showed that: gene cassettes detected in pig manure and composting products were assigned to aminoglycosides(aad,aac),quaternary ammonium compounds(qac),trimethoprim(dfr,dhfr),?-lactamides(bla),and chloramphenicol resistance(cml).Trimethoprim resistance gene cassette resistant to CTC,which may be due to the effect of different concentrations of CTC on the transfer of resistance genes in manure composting process.Because of the style of gene cassettes detected in the thermophilic phase was less than that in the initial,it suggested that the thermophilic phase of compost could inhibit the horizontal gene transfer mediated by class ? integron.The number of CTC,SMZ and ERY resistant bacteria decreased at the end of the composting.Only the number of ENR thermophilic resistant bacteria has increased.Twenty-five strains of drug-resistant bacteria were screened from the composting samples,and the detection rate of multi-drug-resistant bacteria was 20%.Based on the study of the effects of composting environmental factors on the spread and diffusion of ARG,the partial least squares path model(PLS-PM)was used to obtain the results.CTC is the main factor affecting the horizontal transfer of tetracycline ARG,which exists through co-selection of sulfonamides and quinolone ARG.