鸡粪有机肥对土壤中抗生素抗性基因和整合酶基因的影响
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摘要
为探究施用有机肥对土壤中抗性基因和整合酶基因分布的影响及其与土壤环境因子的相关关系,利用高通量PCR技术监测了鸡粪有机肥施用120 d后土壤中四环素类抗性基因、大环内酯类抗性基因、整合酶基因和土壤理化性质的变化情况。研究结果表明,施肥土壤的电导率、pH、有机质含量均明显增加,而土壤氧化还原电位由217.27 mV降低到154.47 mV。施肥土壤中钾、氮、磷、铅、铜和锌含量均上升。施加鸡粪有机肥120 d后,土壤中tetM、tetQ、tetW、tetG和tetX 5种四环素类抗性基因相对丰度分别增加了2.90、0.97、6.80、0.98和0.94倍,而erm35、ermB、ermT、ermX和ermF 5种大环内酯类抗性基因相对丰度分别增加了0.98、136.68、0.95、2.89和2.89倍;但erm36相对丰度降低了0.75倍,其中施肥后土壤中ermB丰度最高而erm36丰度最低。施用鸡粪有机肥后,土壤中intI1和intI3的丰度分别降低了4.71×10-5和2.57×10-7,而intI2的丰度增加了3.74×10~(-6)。Network分析发现intI3与除ermB外的基因均呈显著负相关关系(R=-1.00,P<0.05)。
In order to investigate the effects of the application of chicken manure organic fertilizer on the distribution of antibiotic resistance genes and integrase genes in soil and their correlations with environmental factors, high-throughput PCR was used to monitor the variations of tetracycline resistance genes, macrolactone resistance genes and integrase genes in soil, as well as the soil physicochemical properties, after 120 d of chicken manure organic fertilizer application. The results showed that the electrical conductivity(EC), pH, and organic matter contents of manure applied soil increased, while oxidation-reduction potential(ORP) decreased from 217.27 mV to 154.47 mV. The contents of nitrogen, phosphorus, potassium, Cu, Zn, and Pb increased in soil. The relative abundances of five types of tetracycline resistance genes: tetM, tetQ, tetW, tetG and tetX,increased by 2.90, 0.97, 6.80, 0.98 and 0.94 times after 120 d of fertilization, respectively. The abundances of five types of macrolactone resistance genes: erm35, ermB, ermT, ermX, and ermF, increased by 0.98, 136.68,0.95, 2.89 and 2.89 times, but the abundance of erm36 decreased by 0.75 times, of which the highest abundance of ermB and the lowest one of erm36 occurred in the fertilized soils. After application of chicken manure organic fertilizer, the relative abundances of intI1 and intI3 in soil decreased by 4.71 × 10-5 and 2.57 × 10-7,respectively, while the relative abundance of intI2 increased by 3.74 × 10~(-6). Network analysis showed that intI3 was negatively correlated with the genes except for ermB(R=-1.00, P < 0.05).
In order to investigate the effects of the application of chicken manure organic fertilizer on the distribution of antibiotic resistance genes and integrase genes in soil and their correlations with environmental factors, high-throughput PCR was used to monitor the variations of tetracycline resistance genes, macrolactone resistance genes and integrase genes in soil, as well as the soil physicochemical properties, after 120 d of chicken manure organic fertilizer application. The results showed that the electrical conductivity(EC), pH, and organic matter contents of manure applied soil increased, while oxidation-reduction potential(ORP) decreased from 217.27 mV to 154.47 mV. The contents of nitrogen, phosphorus, potassium, Cu, Zn, and Pb increased in soil. The relative abundances of five types of tetracycline resistance genes: tetM, tetQ, tetW, tetG and tetX,increased by 2.90, 0.97, 6.80, 0.98 and 0.94 times after 120 d of fertilization, respectively. The abundances of five types of macrolactone resistance genes: erm35, ermB, ermT, ermX, and ermF, increased by 0.98, 136.68,0.95, 2.89 and 2.89 times, but the abundance of erm36 decreased by 0.75 times, of which the highest abundance of ermB and the lowest one of erm36 occurred in the fertilized soils. After application of chicken manure organic fertilizer, the relative abundances of intI1 and intI3 in soil decreased by 4.71 × 10-5 and 2.57 × 10-7,respectively, while the relative abundance of intI2 increased by 3.74 × 10~(-6). Network analysis showed that intI3 was negatively correlated with the genes except for ermB(R=-1.00, P < 0.05).
引文
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[14] WANG K, MAO H, LI X K. Functional characteristics and influence factors of microbial community in sewage sludge com?posting with inorganic bulking agent[J]. Bioresource Technology, 2018, 249:527-535.
[15] LIAO H P, LU X M, RENSING C, et al. Hyperthermophilic composting accelerates the removal of antibiotic resistance genes and mobile genetic elements in sewage sludge[J]. Environmental Science&Technology, 2018, 52(1):266-276.
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[17] DUAN M, GU J, WANG X, et al. Effects of genetically modified cotton stalks on antibiotic resistance genes, intI1, and intI2during pig manure composting[J]. Ecotoxicology Environmental Safety, 2018, 147:637-642.
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[19] ACCINELLI C, KOSKINEN W C, BECKER J M, et al. Environmental fate of two sulfonamide antimicrobial agents in soil[J].Journal of Agricultural and Food Chemistry, 2007, 55(7):2677-2682.
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[21]吴传栋.基于碳源调控的污泥堆肥氮素转化及氨同化作用机制研究[D].哈尔滨:哈尔滨工业大学, 2018.
[22] ASGHARPOUR F, AMIN MARASHI S M, MOULANA Z. Molecular detection of class 1, 2 and 3 integrons and some antimi?crobial resistance genes in Salmonella infantis isolates[J]. Iranian Journal of Microbiology, 2018, 10(2):104-110.
[23] CHEN J, YU Z T, MICHEL F C, et al. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems[J].Applied and Environmental Microbiology, 2007, 73(14):4407-4416.
[24]钱勋.好氧堆肥对畜禽粪便中抗生素抗性基因的削减条件探索及影响机理研究[D].杨凌:西北农林科技大学, 2016.
[25] TELLA M, DOELSCH E, LETOURMY P, et al. Investigation of potentially toxic heavy metals in different organic wastes used to fertilize market garden crops[J]. Waste Management, 2013, 33(1):184-192.
[26]孙红霞,张花菊,徐亚铂,等.猪饲料、粪便、沼渣和沼液中重金属元素含量的测定分析[J].黑龙江畜牧兽医, 2017, 5(9):285-287.
[27] NICHOLSON F A, SMITH S R, ALLOWAY B J, et al. An inventory of heavy metals inputs to agricultural soils in England and Wales[J]. Water&Environment Journal, 2006, 311(1/2/3):205-219.
[28] HAN X M, HU H W, CHEN Q L, et al. Antibiotic resistance genes and associated bacterial communities in agricultural soils amended with different sources of animal manures[J]. Soil Biology and Biochemistry, 2018, 126:91-102.
[29] LUO L, MA Y B, ZHANG S Z, et al. An inventory of trace element inputs to agricultural soils in China[J]. Journal of Environ?mental Management, 2009, 90(8):2524-2530.
[30]李谦.畜禽粪便及有机肥中的重金属、抗生素和抗性基因及其在堆肥过程中的变化[D].南京:南京农业大学, 2006.
[31]彭双,王一明,林先贵.连续施用发酵猪粪对土壤中四环素抗性基因数量的影响[J].中国环境科学, 2015, 35(4):1173-1180.
[32] PENG S, FENG Y Z, WANG Y M, et al. Prevalence of antibiotic resistance genes in soils after continually applied with dif?ferent manure for 30 years[J]. Journal of Hazardous Materials, 2017, 340:16-25.
[33] SANDBERG K D, LAPARA T M. The fate of antibiotic resistance genes and class 1 integrons following the application of swine and dairy manure to soils[J]. FEMS Microbiology Ecology, 2016, 92(2):1-7.
[34] ROBERTS M C. Update on macrolide-lincosamide-streptogramin, ketolide, and oxazolidinone resistance genes[J]. FEMS Mi?crobiology Letters, 2008, 282(2):147-159.
[2] LIN H, ZHANG J, CHEN H, et al. Effect of temperature on sulfonamide antibiotics degradation, and on antibiotic resistance determinants and hosts in animal manures[J]. Science of the Total Environment, 2017, 607-608:725-732.
[3]苏建强,黄福义,朱永官.环境抗生素抗性基因研究进展[J].生物多样性, 2013, 21(4):481-487.
[4] ZHANG L, GU J, WANG X, et al. Fate of antibiotic resistance genes and mobile genetic elements during anaerobic co-diges?tion of Chinese medicinal herbal residues and swine manure[J]. Bioresource Technology, 2017, 250:799-805.
[5] ZHU Y G, ZHAO Y, LI B, et al. Continental-scale pollution of estuaries with antibiotic resistance genes[J]. Nature Microbiol?ogy, 2017, 2:16270.
[6] LI J J, XIN Z H, ZHANG Y Z, et al. Long-term manure application increased the levels of antibiotics and antibiotic resis?tance genes in a greenhouse soil[J]. Applied Soil Ecology, 2017, 121:193-200.
[7] ELIZABETH R, CHANDA D D, CHAKRAVARTY A, et al. Association of glycerol kinase gene with class 3 integrons:A nov?el cassette array within Escherichia coli[J]. Indian Journal of Medical Microbiology, 2018, 36(1):104-107.
[8] WANG F H, QIAO M, CHEN Z, et al. Antibiotic resistance genes in manure-amended soil and vegetables at harvest[J]. Jour?nal of Hazardous Materials, 2015, 299:215-221.
[9] ZHANG Y J, HU H W, GOU M, et al. Temporal succession of soil antibiotic resistance genes following application of swine,cattle and poultry manures spiked with or without antibiotics[J]. Environmental Pollution, 2017, 231(2):1621-1632.
[10] MAZEL D. Integrons:Agents of bacterial evolution[J]. Nature Reviews Microbiology, 2006, 4(8):608-620.
[11] MOHADESEH Z Y, GILDA E, HENGAMEH Z, et al. Prevalence of class 1, 2 and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Yazd, Iran[J]. Iranian Journal of Microbiology, 2018, 10:300-306.
[12]何芳,罗阳,浣成,等.高温堆肥技术在我国畜禽粪便污染治理中的应用[J].安徽农业科学, 2018, 46(17):41-43.
[13] WANG K, CHU C, LI X K, et al. Succession of bacterial community function in cow manure composing[J]. Bioresource Tech?nology, 2018, 267:63-70.
[14] WANG K, MAO H, LI X K. Functional characteristics and influence factors of microbial community in sewage sludge com?posting with inorganic bulking agent[J]. Bioresource Technology, 2018, 249:527-535.
[15] LIAO H P, LU X M, RENSING C, et al. Hyperthermophilic composting accelerates the removal of antibiotic resistance genes and mobile genetic elements in sewage sludge[J]. Environmental Science&Technology, 2018, 52(1):266-276.
[16] ZHANG J, SUI Q W, TONG J, et al. Soil types influence the fate of antibiotic-resistant bacteria and antibiotic resistance genes following the land application of sludge composts[J]. Environment International, 2018, 118:34-43.
[17] DUAN M, GU J, WANG X, et al. Effects of genetically modified cotton stalks on antibiotic resistance genes, intI1, and intI2during pig manure composting[J]. Ecotoxicology Environmental Safety, 2018, 147:637-642.
[18] ZHAO X, WANG J, ZHU L, et al. Field-based evidence for enrichment of antibiotic resistance genes and mobile genetic ele?ments in manure-amended vegetable soils[J]. Science of the Total Environment, 2018, 654:906-913.
[19] ACCINELLI C, KOSKINEN W C, BECKER J M, et al. Environmental fate of two sulfonamide antimicrobial agents in soil[J].Journal of Agricultural and Food Chemistry, 2007, 55(7):2677-2682.
[20] LIANG Y, MENG P, WANG D, et al. Improvement of soil ecosystem multifunctionality by dissipating manure-induced antibi?otics and resistance genes[J]. Environmental Science&Technology, 2017, 51(9):4988-4998.
[21]吴传栋.基于碳源调控的污泥堆肥氮素转化及氨同化作用机制研究[D].哈尔滨:哈尔滨工业大学, 2018.
[22] ASGHARPOUR F, AMIN MARASHI S M, MOULANA Z. Molecular detection of class 1, 2 and 3 integrons and some antimi?crobial resistance genes in Salmonella infantis isolates[J]. Iranian Journal of Microbiology, 2018, 10(2):104-110.
[23] CHEN J, YU Z T, MICHEL F C, et al. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems[J].Applied and Environmental Microbiology, 2007, 73(14):4407-4416.
[24]钱勋.好氧堆肥对畜禽粪便中抗生素抗性基因的削减条件探索及影响机理研究[D].杨凌:西北农林科技大学, 2016.
[25] TELLA M, DOELSCH E, LETOURMY P, et al. Investigation of potentially toxic heavy metals in different organic wastes used to fertilize market garden crops[J]. Waste Management, 2013, 33(1):184-192.
[26]孙红霞,张花菊,徐亚铂,等.猪饲料、粪便、沼渣和沼液中重金属元素含量的测定分析[J].黑龙江畜牧兽医, 2017, 5(9):285-287.
[27] NICHOLSON F A, SMITH S R, ALLOWAY B J, et al. An inventory of heavy metals inputs to agricultural soils in England and Wales[J]. Water&Environment Journal, 2006, 311(1/2/3):205-219.
[28] HAN X M, HU H W, CHEN Q L, et al. Antibiotic resistance genes and associated bacterial communities in agricultural soils amended with different sources of animal manures[J]. Soil Biology and Biochemistry, 2018, 126:91-102.
[29] LUO L, MA Y B, ZHANG S Z, et al. An inventory of trace element inputs to agricultural soils in China[J]. Journal of Environ?mental Management, 2009, 90(8):2524-2530.
[30]李谦.畜禽粪便及有机肥中的重金属、抗生素和抗性基因及其在堆肥过程中的变化[D].南京:南京农业大学, 2006.
[31]彭双,王一明,林先贵.连续施用发酵猪粪对土壤中四环素抗性基因数量的影响[J].中国环境科学, 2015, 35(4):1173-1180.
[32] PENG S, FENG Y Z, WANG Y M, et al. Prevalence of antibiotic resistance genes in soils after continually applied with dif?ferent manure for 30 years[J]. Journal of Hazardous Materials, 2017, 340:16-25.
[33] SANDBERG K D, LAPARA T M. The fate of antibiotic resistance genes and class 1 integrons following the application of swine and dairy manure to soils[J]. FEMS Microbiology Ecology, 2016, 92(2):1-7.
[34] ROBERTS M C. Update on macrolide-lincosamide-streptogramin, ketolide, and oxazolidinone resistance genes[J]. FEMS Mi?crobiology Letters, 2008, 282(2):147-159.