The Occurrence And Distribution Of Antibiotics,Antibiotic Resistance Genes And Their Relationships Among Physicochemical Factors In Landfill Sites Of Chinese Mainland

Antibiotics are a kind of an effective antimicrobial agent,which have been extensively and effectively used in veterinaey and human medicines.However,most of antibiotics are released into environments in its original form since antibiotics are poorly absorbed in humans and animals.Thereby,most of antibiotics residues can be detected in environments.Meanwhile,the increased occurrence of antibiotics in environments leads to the dissemination and proliferation of antibiotic resistance genes（ARGs）.ARGs have been regarded as a new type of persistent environmental pollutants,thus,posed a threat to environmental ecology.China is the world’s largest producer and consumer of antibiotics,and due to improper disposal of unused or expird durgs,which are gathered in the municipal solide waste（MSW）landfill eventually.MSW has thereby become an potential hotspot of antibiotics and ARGs.Besides,the usage of antibiotics in various areas existed difference,which lead to different distributions of antibiotics and antibiotic resistance genes in landfills at different areas,where have diverse environments.Therefore,it will be meaningful to study the occurrence and regional distribution of antibiotics,ARGs and their relationships among physicochemical factors in landfill sites.In this study,the leachates are sampled from seven MSW landfills（Xi’an,Guiyang,Nanjing,Suzhou,Shanghai,Hangzhou,Shenzhen）with different geographical locations,and the refuses are sampled from Xi’an and Guiyang landfill according to various landfill time.The target of study is to reveal the regional characteristics of antibiotics and ARGs as well as the relationships among antibiotics,ARGs and physicochemcal factors（heavy metals carbons,nutrients levels）in landfill across Chinese Mainland.The results are as followed:（1）The residues of five classes of antibiotics,including sulfonamides（SAs）,tetracyclines（TCs）,quinolones（FQs）,macrolides（MLs）,β-lactams（BLs）in landfill leachates and refuses were quantified by Ultra Performance Liquid Chromatograph-tandem Mass Spectrometry（UPLC-3Q-MS）.In landfill leachates,the results showed that the concentration of total target antibiotics in Shanghai was the highest 52,223.6±877.89 ng/L,and the content of antibiotics in Xi’an and Guiyang-gaoyan landfill were also reached at 10,000 ng/L level（22,683.23±570.45 and 10,732.52±147.25 ng/L respectively）.The risk quotient method（RQ）assessed that the RQ value of Sulfadiazine（SD）,Sulfamethoxazole（SMX）,Sulfamethazine（SMZ）,Erythromycin-H₂O（ETM-H₂O）,Roxithromycin（RTM）were high risk（RQ>1）.There was a connection with economic development level,population scale and landfill volume in antibiotics.The further analysis indicated that FQs showed a positive relation with population scales（p<0.01）,while SAs negatively correlated with per capita GDP level（p<0.01）,while MLs correlated with landfill volume（p<0.05）.In refuses,the concentration of FQs was the highest（268.56μg/kg）,followed by that of TCs（68.66μg/kg）.The RQ method showed that only TCs（TC,OTC）reached to high risk（RQ>1）,while the further analysis revealed that there was a difference between young refuses and other lanfill time refuses（p<0.01）in both targeted landfill sites,indicating that the differece landfilling time do have an effect on the distribution of antibiotics.（2）The target ARGs,including sulfonamides resistance genes（sul1,sul2）,tetracyclines resistance genes（tetM,tetQ）,macrolides resistance genes（ermB,mefA）,β-lactams resistance genes(bla_CTX-M),multiple resistance gene（mexF）and Class I integron（intl1）were quantified by quantitative real-time PCR（qPCR）.In landfill leachates,the abundance of ARGs and intl1 in Suzhou landfill were the highest,was 56.54±1.81 and 9.61±0.89 lg copies/ng DNA respectively,while the contents of those in Shanghai landfill were the lowest,was 42.49±2.48 and 5.50±0.58lg copies/ng DNA respectively The further analysis indicated that there was a regional difference rather than a distinct geographical characteristic in ARGs.In refuses,the relative concentration of ARGs in Guiyang landfill was much higher than that of Xi’an（8.06±0.23 vs 7.71±0.43 lg copies/ng DNA）.The total concentration of ARGs in young refuse of Guiyang was the highest,which was8.27±0.42 lg copies/ng DNA,whereas the figure of ARGs in old refuse of Xi’an landfill was the lowest,which was 7.11±0.96 lg copies/ng DNA.The distribution of ARGs in landfill refuses was analyzed via cluster analysis,the reslut revealed that there were differences between various landfill time.（3）The physicochemical parameters and common heavy metals were measured by discrete auto analyzer（SmartChem 200）and Inductively Coupled Plasma-Atomic Emission Spectrometry（ICP-AES）respectively.The relationship between ARGs and antibiotics,physicochemical factors were analyzed by redundancy analysis（RDA）and bivariate correlation analysis.In landfill leachates,the total concentration of heavy metals in Shanghai was the highest,which was significantly different from others（p<0.05）.In target heavy metals,the figure of Fe was the highest,followed by Cr and Zn.Further analysis indicated that BLs,TCs,FQs antibiotics were correlated their related resistance genes repectively（p<0.05）.Besides,physicochemical parameters like COD,and NH₄⁺-N were the primary effect factors on ARGs,while heavy metals Pb,Fe,Cd,Cu,Zn and Cr are the main factors of ARGs.In refuses,the concentration of Fe was the highest both in Guiyang and Xi’an landfill refuses（20774.67-38233.33 mg/kg and 17038.00-26953.33 mg/kg respectively）.The further analysis revealed that TCs and FQs displayed a significant relation with their related genes.In addition,physicochemical indicators like C,N,P and heavy metals（Pb,Sb,Cu,Mn）both have great influence on the distribution of ARGs.（4）Clinically important FQs antibiotic resistance genes（qnrA,qnrB,qnrD,qnrS）,BLs antibiotic resistance genes(ampC,bla _CTX-M,bla _OXA10,bla_OXY,bla_PER,bla_SFO,bla_SHV,penA)and mobile gene elements（MGEs）（intl1,intl2,tnpA01,tnpA02,tnpA03）were quantified by high-throughput quantitative PCR（HT-qPCR）in landfill leachates.In landfill leachates,among the detected ARGs,qnrA and bla_OXA10 were the major subtypes,high contributed 99.99%and 83%of the total contents of FQs’and BLs’resistance genes.The relative abundance of qnrA was the highest in Nanjing landfill(7.57±3.79×10^-6（ARGs/16S）),while the figure of qnrS was 9.94×10^-7±5.53×10^-7（ARGs/16S）in Guiyang landfill.Additionally,the figure of bla_OXA10XA10 Suzhou landfill was the highest(1.54×10^-3±5.46×10^-4（ARGs/16S）),followed by Xi’an(5.24×10^-4±8.55×10^-5（ARGs/16S）)and Hangzhou(5.15×10^-4±2.33×10^-4（ARGs/16S）).The relative abundance of MGEs appeared to be significantly higher than that of ARGs（p<0.05）.The spatial distribution of FQs and BLs ARGs,MGEs in landfill leachates were analyzed by Principal Component Analysis（PCA）,which demontrated that there was no significant regional characteristic in FQs and BLs ARGs,while in which MGEs existing.The relationship among FQs ARGs,BLs ARGs,MGEs and anthropological activities were analyzed by correlation analysis,which revealed that the regional characteristic of ARGs and MGEs were mainly influenced by population scale.According to the results of RDA,FQs were positively correlated to relating gens qnr D and qnrS,BLs were negatively correlated to bla_CTX-M,bla_OXY,bla_SFO,while revealed the opposite correlated with bla_OXA10,penA,bla_SHV.FQs and BLs ARGs and MGEs were mainly effected by As,Cr,COD and NH₄⁺-N.