Представляется интересным, что наиболее интенсивно устойчивость к тетрациклинам формировалась в образце П+К+ОТС, что, вероятно, связано с наличием прямого фактора «давления среды» [28]. К концу эксперимента образцах П+К+ОТС и П+К+АРГ наблюдалось плавное снижение уровня резистентности.
В образце П+К наличие генов устойчивости к тетрациклинам ґєґ(М) и Є(Х) ожидаемо не было выявлено. В дальнейшем оба вида генов были обнаружены в почве, но в меньших количествах, чем в образцах с двумя другими видами компоста. Появление генов устойчивости в почве, обработанной чистым компостом, вероятно, обусловлено тем, что в исходных навозах, использованных для компостирования, такие гены присутствовали [29]. Они элиминировались, видимо, в связи с неблагоприятными для их бактерий-носителей условиями во время компостирования, такими как высокая температура, изменения рН, появление продуктов разложения органического вещества [27]. Однако, по всей вероятности, элиминирование не произошло полностью, а лишь до уровня, находящегося ниже предела обнаружения используемого метода - ПЦР в реальном времени. При попадании в благоприятные для себя (почвенные) условия бактерии, носители генов устойчивости, начали размножаться и, возможно, передавать способность к антибиотикорезистентности другим видам, вследствие чего гены устойчивости были обнаружены в почве [23].
Заключение
В работе показано, что при удобрении почвы как условно чистым компостом, так и компостами, содержащими ОТС и гены антибиотикорезистентности, наблюдается увеличение количества бактериальных и грибных штаммов по сравнению с необработанной контрольной почвой. Удобрение почвы компостом с ОТС, вероятно, вызвало сначала значительное элиминирование чувствительных к нему бактерий, а затем рост численности тех видов, которые приобрели устойчивость. Присутствие в компосте и антибиотика, и генов антибиотикорези- стентности привело к увеличению в почве бактерий, носителей устойчивости к тетрациклинам. Причем наиболее значительно количество генов устойчивости к тетрациклинам увеличилось в почве с компостом, содержащим ОТС. На более поздних стадиях инкубирования гены устойчивости к тетрациклинам были отмечены для почвы, обработанной и условно чистым компостом.
Литература
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31. Naquin A., Shrestha A., Sherpa M., Nathaniel R., Boopathy R. Presence of antibiotic resistance genes in a sewage treatment plant in Thibodaux, Louisiana, USA. Bioresour. Technol., 2015, vol. 188, pp. 79-83. doi: 10.1016/j.biortech.2015.01.052.
32. Sarmah A.K., Meyer M.T., Boxall A.B. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere, vol. 65, no. 5, pp. 725-759. doi: 10.1016/j.chemosphere.2006.03.026.
33. Thiele-Bruhn S., Beck I.C. Effects of sulfonamide and tetracycline antibiotics on soil microbial activity and microbial biomass. Chemosphere, 2005, vol. 59, no. 4, pp. 457-465. doi: 10.1016/j.chemosphere.2005.01.023.
34. Fang H., Han Y., Yin Y., Pan X., Yu Y. Variations in dissipation rate, microbial function and antibiotic resistance due to repeated introductions of manure containing sulfadiazine and chlortetracycline to soil. Chemosphere, 2014, vol. 96, pp. 51-56. doi: 10.1016/j.chemosphere.2013.07.016.
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36. Hammesfahr U., Heuer H., Manzke B., Smalla K., Thiele-Bruhn S. Impact of the antibiotic sulfadiazine and pig manure on the microbial community structure in agricultural soils. Soil Biol. Bio- chem., 2008, vol. 40, no. 7, pp. 1583-1591. doi: 10.1016/j.soilbio.2008.01.010.
37. Reichel R., Rosendahl I., Peeters E.T.H.M., Focks A., Groeneweg J., Bierl R., Schlichting A., Amelung W., Thiele-Bruhn S. Effects of slurry from sulfadiazine-(SDZ) and difloxacin-(DIF) medicated pigs on the structural diversity of microorganisms in bulk and rhizosphere soil. Soil Biol. Biochem., 2013, vol. 62, pp. 82-91. doi: 10.1016/j.soilbio.2013.03.007.
38. Zhang T., Zhang M., Zhang X., Fang H.H. Tetracycline resistance genes and tetracycline resistant lactose-fermenting enterobacteriaceae in activated sludge of sewage treatment plants. Environ. Sci. Technol., 2009, vol. 43, no. 10, pp. 3455-3460. doi: 10.1021/es803309m.
39. Wu X., Wei Y., Zheng J., Zhao X., Zhong W. The behavior of tetracyclines and their degradation products during swine manure composting. Bioresour. Technol., 2011, vol. 102, no. 10, pp. 59245931. doi: 10.1016/j.biortech.2011.03.007.
40. Kemper N. Veterinary antibiotics in the aquatic and terrestrial environment. Ecol. Indic., 2008, vol. 8, no. 1, pp. 1-13. doi: 10.1016/j.ecolind.2007.06.002.
41. Dolliver H., Noll S.L. Antibiotic degradation during manure composting. J. Environ. Qual., 2008, vol. 37, no. 3, pp. 1245-1253. doi: 10.2134/jeq2007.0399.
42. Ramaswamy J., Prasher S.O., Patel R.M., Hussain S.A., Barrington S.F. The effect of composting on the degradation of a veterinary pharmaceutical. Bioresour. Technol., 2010, vol. 101, no. 7, pp. 2294-2299. doi: 10.1016/j.biortech.2009.10.089.
43. Arikan O.A., Sikora L.J., Mulbry W., Khan S.U., Foster G.D. Composting rapidly reduces levels of extractable oxytetracycline in manure from therapeutically treated beef calves. Bioresour. Technol., vol. 98, no. 1, pp. 169-176. doi: 10.1016/j.biortech.2005.10.041.
44. Kim K., Owens G., Ok Y.S., Park W.K., Lee D.B., Kwon S.I. Decline in extractable antibiotics in manure-based composts during composting. Waste Manage., 2012, vol. 32, no. 1, pp. 110-116. doi: 10.1016/j.wasman.2011.07.026.
45. Li B., Zhang T., Xu Z., Fang H.H. Rapid analysis of 21 antibiotics of multiple classes in municipal wastewater using ultra performance liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta, 2009, vol. 645, nos. 1-2, pp. 64-72. doi: 10.1016/j.aca.2009.04.042.
46. ISO 17601:2016 Soil quality -- Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil, 2016, pp. 1-31.
47. Zhang W., Huang M., Qi F., Sun P., Van Ginkel S. Effect of trace tetracycline concentrations on the structure of a microbial community and the development of tetracycline resistance genes in sequencing batch reactors. Bioresour. Technol., 2013, vol. 150, no. 1, pp. 9-14. doi: 10.1016/j.biortech.2013.09.081.
48. Arslan E.I., Obek E., Kirbag S. Determination of the effect of compost on soil microorganisms. Int. J. Sci. Technol., 2008, vol. 3, no. 2, pp. 151-159.
49. Kim K.D., Nemec S., Musson G. Effects of composts and soil amendments on soil microflora and Phytophthora root and crown rot of bell pepper. Crop Prot., 1997, vol. 16, no. 2, pp. 165-172. doi: 10.1016/S0261-2194(96)00074-9.
50. Zhen Z., Liu H., Wang N., Guo L., Meng J., Ding N., Wu G., Jiang G. Effects of manure compost application on soil microbial community diversity and soil microenvironments in a temperate cropland in China. PLoS One, 2014, vol. 9, no. 10, art. e108555, pp. 1-12. doi: 10.1371/joumal.pone.0108555.
51. Danon M., Franke-Whittle I.H., Insam H., Chen Y., Hadar Y. Molecular analysis of bacterial community succession during prolonged compost curing. FEMS Microbiol. Ecol., 2008, vol. 65, no. 1, pp. 133-144. doi: 10.1111/j.1574-6941.2008.00506.x.
52. Xu S., Amarakoon I.D., Zaheer R., Smith A., Sura S., Wang G., Reuter T., Zvomuya F., Cessna A.J., Larney F.J., McAllister T.A. Dissipation of antimicrobial resistance genes in compost originating from cattle manure after direct oral administration or post-excretion fortification of antimicrobials. J. Environ. Sci. Health, 2018, vol. 53, no. 4, pp. 373-384. doi: 10.1080/10934529.2017.1404337.
53. Cheng W., Li J., Wu Y., Xu L., Su C., Qian Y., Zhu Y.G, Chen H. Behavior of antibiotics and antibiotic resistance genes in eco-agricultural system: A case study. J. Hazard. Mater., 2016, vol. 304, pp. 18-25. doi: 10.1016/j.jhazmat.2015.10.037.
54. Venglovsky J., Sasakova N., Placha I. Pathogens and antibiotic residues in animal manures and hygienic and ecological risks related to subsequent land application. Bioresour. Technol., 2009, vol. 100, no. 22, pp. 5386-5391. doi: 10.1016/j.biortech.2009.03.068.
55. Wang F.H., Qiao M., Chen Z., Su J.Q., Zhu Y.G. Antibiotic resistance genes in manure-amended soil and vegetables at harvest. J. Hazard. Mater., 2015, vol. 299, no. 3, pp. 215-221. doi: 10.1016/j.jhazmat.2015.05.028.
56. Xie W.-Y., Shen Q., Zhao F.J. Antibiotics and antibiotic resistance from animal manures to soil: A review. Eur. J. Soil Sci., 2017, vol. 69, no. 1, pp. 181-195. doi: 10.1111/ejss.12494.
57. Chen W., Liu W.L., Pan N., Jiao W., Wang M. Oxytetracycline on functions and structure of soil microbial community. J. Soil Sci. Plant Nutr., 2013, vol. 13, no. 4, pp. 967-975. doi: 10.4067/S0718-95162013005000076.
58. Qian X. Sun W., Gu J., Wang X.J., Sun J.J., Yin Y.N., Duan M.L. Variable effects of oxytetracy cline on antibiotic resistance gene abundance and the bacterial community during aerobic composting of cow manure. J. Hazard. Mater., 2016, vol. 315, pp. 61-69. doi: 10.1016/j.jhazmat.2016.05.002.
Abstract
Spreading of Antibiotic Resistance as a Result of Soil Fertilization by Manure Composts Containing Oxytetracycline and Antibiotic-Resistant Genes
N.V. Danilova a, P.A. Kuryntseva a , M.Sh. Tagirov b , P. Yu. Galitskaya a , S. Yu. Selivanovskaya a Kazan Federal University, Kazan, 420008 Russia hTatar Scientific Research Institute of Agriculture, FRC Kazan Scientific Center, Russian Academy of Sciences, Kazan, 420059 Russia
Due to the active usage of antibacterial drugs in animal husbandry, antibiotic residues and antibioticresistance genes enter the soil when it is fertilized with manure and compost. In this work, we estimated the spread of antibiotic-resistance genes in the soil when it was fertilized with composts containing oxytetracycline (OTC) and tetracycline-resistance genes. The following mixtures were studied for 35 days: the soil with “clean” compost, the soil with compost and 300 mg kg-1 OTC, the soil with compost and antibiotic-resistance genes. In the soil treated with composts, the number of bacteria and fungi increased, with the greatest changes in the number of bacteria observed for the soil with compost containing OTC. The addition of compost with the antibiotic and the resistance genes tet(M) and tet(X) to the soil led to the formation of antibiotic resistance in soil microorganisms with the highest number of gene copies for 5-7 days: an average of 3.31-106 and 7.77-106 copies g-1, respectively. Both genes, tet(M) and tet(X), but in smaller numbers, were detected in the soil with “clean” compost on days 14-21, respectively.
Keywords: antibiotics, oxytetracycline, antibiotic resistance, antibiotic-resistance genes, compost, real-time PCR
Figure Captions
Fig. 1. The number of bacteria (a) and fungi (b) in the mixtures of soil and compost containing OTC and tetracycline-resistance genes.
Fig. 2. The ratio of the number of bacteria and fungi in the mixtures of soil and compost containing OTC and tetracycline-resistance genes.
Fig. 3. The content of the tetracycline-resistance genes tet(M) (a) and tet(X) (b) in the mixtures of soil and compost containing OTC and tetracycline-resistance genes.