Mechanisms Affecting The Fate Of Antibiotic Resistance Genes During Anaerobic Digestions Of Swine Manure At Different Temperatures

As an adverse result of the abuse of antibiotics,antibiotic resistance has become a growing and major threat to human health.It is critically important to controlthe emergence and spread of antibiotic-resistant pathogens.Animal manure is considered to be a significant source of antibiotic resistance due to the overuse of antibiotics in food animals.Antibiotic resistance genes(ARGs)are the genetic determinants of antibiotic resistance.ARGs can spread among bacteria via horizontal gene transfer.Without a proper treatment,the large amount of ARGs carried by animal manure can pollute the environment and eventually accelerate the emergence of antibiotic-resistant pathogens.Anaerobic digestion(AD)is an effective treatment method commonly used for manure management and the removal of ARGs.The objectives of this project were to determine the AD factors affecting the removal of ARGs and to understand the mechanisms underlying the removal.Since AD temperature plays a major role in AD process,its effect on the removal of ARGs was investigated.The ADs with different chemical oxygen demand(COD)concentrations(0.4,1.2,2,5 g/L)of swine manure were performed at psychrophilic(22°C),mesophilic(37°C),and thermophilic(55°C)AD,and their respective removal efficiencies of ARGs were compared using metagenomic approach.The fates and the relationship among microorganisms,plasmids,and ARGs were also examined through nexworking analyses.In addition,the fates of four commonly funded ARGs in swine manure were also evaluated using quantitative polymerase chain reaction(qPCR)assays.The results of qPCR and those of metagenomics were compared for the cons and pros of the two approaches.The major results and conclusions are summaried as follow:The most abundant ARGs types in swine manure appeared to be tetracycline resistance genes,accounting for 58% of the total ARGs detected,followed by aminoglycoside(15%),MLS(macrolides-lincosamids-streptogramins)(15%),and sulfonamide(10%)resistance genes.These four types of ARGs accounted for about 98% of the total ARGs.This finding was in line with the industry practice of using the antibiotics for the treatment and prevention of diseases in pig,and for promoting the pig's growth.Among the ARGs subtypes,tetW was the most abundant,followed by tetW(10.0%),tetQ(9.1%),sul2(8.7%)and tetX(6.0%).The thermophilic AD showed the highest overall removal efficiency of ARGs.Our data suggested that under lower COD concentrations(0.4 and 1.2 g/L),the AD was more effective to remove ARGs at 55°C than at 22°C or 37°C.However,with the COD concentration at 2 g/L,the mesophilic AD had the highest removal efficiency,indicating that COD was an affecting factor under certain conditions.Additionally,it was found that the fermentation duration might be one of the factors affecting the removal efficiency,at least for the thermophilic AD with COD at 2 g/L,since the abandances of tetW,sul2,tet44,and tetL reached to the lowest point at day 12 and then bounced to a higher level at day 16.This study found that most ARGs decreased over time during all the AD processes and that their decreases became obvious by day 4.Similarly,microorganism abundances changed dramatically by day 4.The correlation suggested that microorganism population was an important factor affecting the ARG abundances in ADs.The results of network analyses suggested that the potential hosts of the dominant ARGs were mainly bacteria of Bacteroides propionicifaciens,Pseudomonas caeni,Lactobacillus spp,and Bifidobacterium pseudolongum.Furthermore,among the ARGs subtypes,tetW and tet36 appeared to have the most diverse hosts.During the ADs,plasmids were found to be associated with the removal of ARGs.Our data further suggested that plasmids carried by bacteria of from Proteobacteria were more likely to carry an ARG,being the major source of ARGs in pig manure.The abundances of the four ARGs measured by the qPCR assays were not consistent with those detected by metagenomics.This conclusion was devidenced by that tetW was the most abundant ARG determined by the metagenomic approach,while sul2 was the most abundant one indicated by the qPCR assays.However,the two approaches showed a similar dynamic trend for all the four ARGs in the study.Since the PCR amplication can be biased,the results indicated that the qPCR approaches might not be effective to measure the abundances of the ARGs,but might be used to analyze the dynamic trend of ARG.In summary,swine manure was rich in ARGs and the thermophilic AD was more effective in removing the ARGs,compared with psychrophilic and mesophilic AD.While AD termperature,COD,and fermentation duration appeared to be the factors affecting the removal,termperature was the most important one.All those affecting factors might have affected the dynamics of the microbial community and their ARG-bearing plasmids and thus affecting the ARG removals.Our results can be an informative reference for the optimization of the imputs and outputs of pig-manure ADs.This study has enhanced our understanding of the mechnisms underlying the removal of ARGs during pig-manure ADs,which can have a positive impact on the study and application of other types of ADs.