Temperature Effect On The Removal Of Antibiotic Resistance Gene OptrA In Enterococci During Anaerobic Digestion Treating Livestock Manure Wastes

Anaerobic digestion could reduce pathogenic microorganisms in solid wastes.Enterococci is an important zoonotic opportunistic pathogen,which exists widely in human,animal feces and domestic sewage.Vancomycin-resistant enterococci is important antibiotic resistant bacteria in humans.Linezolid is an effective antibiotic treating vancomycin-resistant enterococci infection.The extensive use of florfenicol in animal industry leads to the emergence of optrA,a gene confers cross-resistance to linezolid and florfenicol,posing potential threats for clinical treatment.Therefore,it is of vital importance to control the dissemination of enterococci and optrA gene during anaerobic digestion.In the present study,optrA and enterococci were studied on their distributions in both full-sacle(operation temperature at 26℃)and lab-scale(operation temperature at25℃,37℃,55℃ and 70℃,respectively)anaerobic digestion systems treating animal manure wastes.Characteristics of molecular genetic optrA in anaerobic digestion at different temperatures were further revealed.The main results are as follows:(1)The characterization of enterococci and optrA in full-scale(26℃)and lab-scale(25℃)anaerobic digestion systems were investigated,using isolation,culture,and PCR methods,respectively.It was found that enterococci and gene optrA could be persistant in effluents of different anaerobic digestion system.A total of 254 enterococci strains were isolated from full-scale anaerobic digestion system,and the detection rates of opr A in influent and effluent were 73.5% and 38.9%,respectively.A total of 140 enterococci strains were isolated from lab-scale anaerobic digestion system,and the detection rates of optrA in influent and effluent were 49.2% and 43.8%,respectively.Most strains harbored more than two resistance genes optrA/fex A and were resistant to florfenicol and linezolid.(2)The removal of enterococci and optrA was studied in anaerobic digestion system treating animal manure wastes at different temperatures.Hyperthermophic anaerobic digestion could effectively reduce enterococci and optrA.The detection rates of optrA in enterococci from influent,mesophilic effluent(37℃),thermophilic effluent(55℃),and hyperthermophilic effluent(70℃)were 42.6%,72.0%,56.7% and 6.9%,respectively.Compared with the influent,the detection rate of optrA increased by 29.4%and 14.1% in mesophilic and thermophilic effluents,respectively,while the detection rate of optrA in hyperthermophilic effluent decreased by 35.9%.(3)The evolution and relationship of optrA-positive enterococci clone strains in anaerobic digestion systems at different temperatures were investigated by whole-genome sequencing.A total of 16 different sequence types(STs)were obtained from anaerobic digestion systems at different temperatures,among which ST631 and ST368 were the dominant clones that harbored optrA/fex A or optrA/fex A/cat.Enterococci strains ST631(CHH8 and CHH78)in hyperthermophilic effluent showed high levels of mutations.(4)The genetic environment of optrA harbored enterococci in anaerobic digestion system at different temperatures was further analyzed.Two genetic elements of optrA-positive enterococci,IS1216E-erm(A)-optrA-fex A and Tn554-erm(A)-ant(9)-Ifex A-optrA were found from influent,mesophilic effluent and thermophilic effluent.In contrast,only Tn554-erm(A)-ant(9)-I-fex A-optrA was found in hyperthermophilic effluent,,indicating effective removal of IS1216E-erm(A)-optrA-fex A in hyperthermophilic(70℃)anaerobic digestion.Figures,33;Tables,39;References,147.