| Background and objectivesAntibiotics are widely used in medical and health and agricultural breeding,in order to treat clinical infectious diseases,prevent and control the animal epidemics.Due to the unreasonable use of antibiotics,the acceleration of bacterial resistance has become an important cause of treatment failure in patients,prolonged hospital stays,increased medical costs and increased mortality.It is estimated that by 2050,bacterial resistance is expected to kill more than 10 million people worldwide,even more than cancer.If control actions are not taken in time,the threat of infectious diseases will increase,and the impact on social and economic development,human health will be incalculable.China is one of the world's largest producers and consumers of antibiotics.Research showed that approximately 162,000 tons of antibiotic were consumed in 2013 in China.Antibiotics can be harmful to human health,inducing the development of bacterial resistance,increasing the risk of clinical cross-infection,and causing adverse drug reactions,such as aminoglycoside antibiotics causing deafness and kidney damage.Antibiotics cannot be completely metabolized in humans or animals,about 30%to 90%of antibiotics are excreted into the environment through the urine and feces of humans or animals as parent compounds or active metabolites.Thus,the ecological risks and the human health impacts caused by antibiotics in the environment need to be properly evaluated.The environment is not only a repository of antibiotic residues,but also a reservoir of antibiotic-resistant bacteria(ARB)/antibiotic-resistance genes(ARGs).The environment is a central part of the "animal-environment-human"(One-Health concept)and plays a vital role in the transmission of ARB.Therefore,curbing the transmission of ARB is not only a challenge faced by the clinical and aquaculture industries,but also the environmental field.However,the current researches mainly focus ARB on single environmental media rather than multi-media transmission in the environment.The cross-media transmission mechanism and driving forces have not yet been clarified.Especially in the rural areas where the amount of livestock breeding is increasing year by year,due to the lack of special facilities to deal with livestock waste,leading to the direct discharge of breeding waste into the surrounding environment.Therefore,the cross-media transmission and driving forces of ARB in rural animal breeding environments need to be revealed.Therefore,the cross-media transmission and driving forces of ARB in the livestock breeding environment have become the focus of attention,which is the theoretical basis for the control of antibiotics/ARB pollution in the breeding areas.Extended-spectrum ?-lactamases(ESBLs)are plasmid-mediated enzymes,which can hydrolyze the b-lactam ring of ?-lactamase antibiotics,detected frequently in Escherichia coli and Klebsiella pneumoniae.CTX-M genes have become the predominant ESBL type,which was widely identified worldwide.CTX-M-producing E.coli has been reported frequently in the environment,and can be used as a suitable indicator of ARB in the environment.Its high detection rate provides an opportunity to study the cross-media transmission of ESBL in the environment.Therefore,this study assessed the ecological risks and human health risks of antibiotic resistance development by detecting the antibiotic residues in the livestock breeding environment.At the same time,the prevalence and characteristics of CTX-M-producing E.coli in the environment were explored to clarify the transmission routes and driving factors of ARB.Methods1.Quantitative analysis of antibiotic residues in environmental samples by high performance liquid chromatography-tandem mass spectrometry.The risk quotient(RQ)of the measured environmental concentration to the predicted no-effect concentration is used to assess the ecological risk of antibiotic resistance development.The ratio of the measured concentration of antibiotics in drinking water to the predicted no-effect concentration,the daily intake of antibiotics in vegetables and the daily allowable intake was used to assess antibiotic residues Health risks to the human body.2.CTX-M-producing E.coli was obtained through isolatio,screening,and identification of bacteria.Antibiotic-resistant phenotypes of strains were determined by antibiotic sensitivity tests.Pulsed-field gel electrophoresis(PFGE)and multi-locus sequence typing(MLST)were conducted for homology analysis and molecular typing.Results1.The samples from the livestock breeding environment were found to contain metronidazole,sulfapyridine,norfloxacin,levofloxacin,ciprofloxacin,enrofloxacin,doxycycline,sulfamethoxazole,florfenicol and chloramphenicol.Antibiotics with high residual levels in river water,wastewater,drinking water,river sediments,pig faeces,outlet sediments,soil,and vegetables are florfenicol(3.9 ng/L),levofloxacin(12.5 ng/L),ciprofloxacin(21.4 ng/L),chloramphenicol(1.12 ?g/kg),doxycycline(1.91 ?g/kg),levofloxacin(11.68 ?g/kg),chloramphenicol(2.5 ?g/kg)and chloramphenicol(27.2 ?g/kg),respectively.2.The RQs for resistance development were>1 for enrofloxacin,levofloxacin,and ranged between 0.1and 1 for ciprofloxacin.The RQs values were<1 from exposure to antibiotics through drinking water for both adults and children.The RQs values were<0.1 from exposure to antibiotics through vegetable intake.3.88 CTX-M-producing E.coli were isolated from 231 environmental samples,with the following frequencies in the different types of samples:pig feces,73%;river water,64%;river sediments,52%;wastewater,31%;drinking water,23%;outlet sediments,21%;soil,17%;and vegetables,4.4%.All strains were resistant to cefotaxime,and the majority of these isolates were also resistant to tetracycline,trimethoprim-sulfamethoxazole,and florfenicol.4.A total of 57 STs were detected,including 2 new STs,and the most prevalent STs were ST10(n=10),ST48(n=7),ST3489(n=4),ST34(n=3),and ST1638(n=3),ST206(n=2),ST226(n=2),ST542(n=2),ST602(n=2),ST1011(n=2),ST2526(n=2),ST2614(n=2)and ST2973(n=2).The PFGE analysis revealed a high clonal diversity among these isolates.Twenty-three groups of related isolates were identified,with 18 of the groups containing isolates originating from different environmental sources.Conclusions1.Antibiotic pollutants were ubiquitous in the livestock breeding environment in the study area.Residues of enrofloxacin,levofloxacin and ciprofloxacin in wastewater may pose an ecological risk for the development of antibiotic resistance.Exposure to antibiotics through drinking water and vegetables does not pose a direct health risk to humans,but there may be a risk of developing antibiotic resistance.2.Putative transmission routes were hypothesized by analyzing the genetic relatedness of isolates:from pig wastes to aquatic environment,among aquatic environment,from aquatic environment to soil,routes associated with wild birds.Human activities,meteorological events and birds have affected the transmission of ARB across the environmental matrices.Fertilization and irrigation are conducive to the transmission of ARB from animal faeces to the environment,drought as a protective factor impedes ARB spreading over long distances along rivers,wild birds can be used as a carrier of ARB to promote long-distance diffusion. |
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