The Distribution Of Antibiotic Resistance Genes And Its Influencing Factors In Agricultural Soil Nearby A Smelting Plant

ObjectiveTo investigate the distribution of antibiotic resistance genes（ARGs）and mobile genetic elements（MGEs）in agricultural soil surrounding a smelting plant,and to study the effects of contents of antibiotics,heavy metals and structures of bacterial community on the distribution of ARGs.Methods1.Relative abundances of 310 antibiotics resistance genes（ARGs）,and 9 mobile genetic elements（MGEs）were investigated by high-throughput quantitative PCR.16S rDNA was used as an internal reference gene.The method ofΔC_T was used to calculate relative abundances of ARGs and MGEs.2.Ultra performance liquidchromatography-tandem mass spectrometry was used to detect the concentrations of sulfadiazine（SDZ）,norfloxacin（NOR）,roxithromycin（ROX）,tetracycline（TC）and chloramphenicol（CAP）.The contents of eight heavy metals and five physicochemical properties were measured.3.The soil cultivable bacterial community and total bacterial community were analysed by denaturing gel gradient electrophoresis（DGGE）and Illunima Miseq respectively.4.Excel 2010 was used to process the data and SPSS 21.0 was used to analyze the normality of the data respectively.Principal component analysis（PCA）and clustering analysis were conducted by SPSS.Redundancy analysis（RDA）was performed by Canoco 5.0 and R software.Network analysis was constructed using Gephi based on Spearson rank correlation with R²>0.65 and P<0.05.Pearson correlation and Spearman rank correlation were used to analyze relationships among contents of antibiotics,heavy metals,total numbers of culturable bacteria and relative abundances of ARGs,MGEs.Results1.The distribution of ARGs and MGEsTotal of 149 ARGs were detected in 7 soil samples.The positive rate of FCA resistance genes was the highest,which was 77.8%,followed by aminoglycoside resistance genes（61.1%）and sulfonamide resistance genes（60.0%）.Total of 90ARGs were detected in G7 sample,which were the most abundant in seven samples.The total relative abundances of ARGs were 1.09×10^-23.63×10^-1,which was the highest in G1 sample(3.63×10^-1).Total relative abundances of sulfonamide resistance genes were the highest which was 3.44×10^-1.Network analysis indicated that ARGs resistant to 13 vancomycins,14tetracyclines,3 sulfonamides,23 beta lactamases,22 aminoglycosides,22 MLSB,21FCA,30 other/efflux and 5 MGEs were significantly correlated（P<0.05）.2.The contents of antibiotics and heavy metalsSDZ,NOR,ROX,TC were found except CAP in 7 soil samples.The concentration of TC was 1738.534μg/kg in G7 sample,which was much higher than other antibiotics.Contents of six heavy metals（Cd,Pb,As,Hg,Cu and Zn）exceeded standards except Ni,Cr.G1 sample was polluted by heavy metals seriously.3.The results of bacterial communityTotal numbers of culturable bacteria ranged from 1.67×10⁵to 8.33×10⁵.Eleven dominant genera were found in cultivable bacterial community by DGGE,which belonged to Proteobacteria and Firmicutes.PCA and clustering analysis showed that cultivable bacterial community structures of G1 and G2 samples were similar,which were different from other samples.RDA indicated that the contents of TC and Cu significantly influenced the structure of cultivable bacterial community（P<0.05）.Relative abundances of Proteobacteria,Actinobacteria,Acidobacteria,Chloroflexi and Gemmatimonadetes were abundant in total bacterial community by Illumina.Relative abundances of Armatimonadetes,Elusimicrobia,Chlorobi and Bacteroidetes were significantly different（P values were all less than 0.05）.The heat map showed that dominant genera of G1 and G2 samples were similar that were different from other samples.RDA displayed that contents of TC,Cd,Pb,As,Hg,Cu and Zn significantly impacted the structure of total bacterial community（P values were all less than 0.05）.4.Effectsofcontentsofantibiotics,heavymetalsand physicochemical properties on the distribution of ARGs and MGEsPearson correlation and Spearman rank correlation showed that contents of 4antibiotics（SDZ,NOR,ROX,TC）were correlated with relative abundances of 26genes（including 25 ARGs and 1 MGE）.Contents of 8 antibiotics were correlated with relative abundances of 33 genes（including 30 ARGs and 3 MGEs）.Contents of5 physicochemical properties（TN,TP,OM,pH,MC）were correlated with relative abundances of 36 genes（including 34 ARGs and 2 MGEs）.5.Effects of bacterial community on the distribution of ARGs and MGEsTotal numbers of culturable bacteria were related with relative abuandances of 7ARGs（mphA-01,adeA,mexE,mex F,oprJ,vat E-01,yceL/mdtH-01）.Abuandances of39 bacterial phyla were correlated with relative abuandances of 71 genes（including67 ARGs and 4 MGEs）（P values were all less than 0.05）.6.Comparison of effects of antibiotics,heavy metals and bacterial community structure on the distribution of ARGs and MGEsRelative abundances of 87 ARGs were related to contents of heavy metals,physicochemical properties,total numbers of culturable bacteria,or abundances of total bacterial phyla.Relative abundances of 36 ARGs were affected by one single factor of 5 factors,and relative abundances of 51 ARGs were affected by two or more than two factors.Among them,numbers of ARGs affected by abundances of total bacterial phyla were the largest,up to 25（25/87,28.74%）.Relative abundances of other 42 ARGs were impacted by abundances of total bacterial phyla and other factors.Total of 67 ARGs relative abundances were affected by total bacterial phyla abundances,accounting for 77.02%of 87 ARGs.Numbers of ARGs affected by contents of antibiotic,heavy metals,physicochemical properties were 4,1,3,which far less than numbers impacted by combined factors（21,29,31）.Conclusions:1.FCA resistance genes are found with the highest positive rate in this heavy metal polluted soils,followed by aminoglycoside resistance genes and sulfonamides resistance genes.And sulfonamides resistance genes are found with the highest total relative abundances.The distribution of ARGs in agricultural soils is affected by abundances of bacterial phyla,contents of antibiotics,heavy metals,physicochemical properties and total numbers of culturable bacteria,in which numbers of ARGs affected by abundances of bacterial phyla are the largest.2.The distribution of certain ARGs are impacted by contents of soil antibiotics,heavy metals,physicochemical properties,and total numbers of culturable bacteria alone,while these factors may influence the distribution of the ARGs by affecting the total bacterial community structure.