Journal of Soils and Sediments

, Volume 19, Issue 2, pp 641–651 | Cite as

High-throughput characterization of antibiotic resistome in soil amended with commercial organic fertilizers

  • Xue Zhou
  • Min QiaoEmail author
  • Jian-Qiang Su
  • Yong-Guan Zhu
Soils, Sec 2 • Global Change, Environ Risk Assess, Sustainable Land Use • Research Article



The use and abuse of antibiotics in the livestock industry are believed to have contributed to the enhancement of antibiotic resistance in agricultural settings through the application of animal manure. The use of manure-based commercial organic fertilizers (COFs) shows promise for the abatement of potential dissemination of antibiotic resistance. This study aims to assess the effects of repeated COF applications on the soil antibiotic resistome and bacterial communities and to reveal potential correlations.

Materials and methods

High-throughput quantitative PCR was used to characterize the shift of the antibiotic resistance gene (ARG) contents after repeated COF applications. Illumina sequencing was employed to investigate changes in the bacterial community composition.

Results and discussion

Applications of COFs increased the diversity and abundance of a wide spectrum of ARGs and mobile genetic elements (MGEs) in the soil. The first and second COF applications enhanced the proliferation of genes encoding resistance to beta lactam and vancomycin. Procrustes analysis and mantel test revealed that ARG profiles were significantly correlated with bacterial community composition, indicating that the microbial community composition might be a determinant of the soil resistome. Network analysis further demonstrated significant associations between ARGs and MGEs, highlighting the likelihood of the horizontal gene transfer of ARGs in the COF-amended soils.


COFs are vast ARG reservoirs that should be categorized as ARG contamination sources in agriculturally related environments. The application of COFs increased the diversity, abundance, and potential horizontal transfer of ARGs in soil.


Antibiotic resistome Bacterial community composition Commercial organic fertilizers  Soil 



This research was funded by the National Natural Science Foundation of China (41571130063, 21210008).

Supplementary material

11368_2018_2064_MOESM1_ESM.docx (5.2 mb)
ESM 1 (DOCX 5353 kb)


  1. Baker-Austin C, Wright MS, Stepanauskas R, McArthur J (2006) Co-selection of antibiotic and metal resistance. Trends Microbiol 14:176–182CrossRefGoogle Scholar
  2. Baldi E, Toselli M (2014) Mineralization dynamics of different commercial organic fertilizers from agro-industry organic waste recycling: an incubation experiment. Plant Soil Environ 60:93–99CrossRefGoogle Scholar
  3. Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E, Walsh F, Bürgmann H, Sørum H, Norström M, Pons M-N (2015) Tackling antibiotic resistance: the environmental framework. Nat Rev Microbiol 13:310–317CrossRefGoogle Scholar
  4. Berg J, Brandt KK, Al-Soud WA, Holm PE, Hansen LH, Sørensen SJ, Nybroe O (2012) Selection for Cu-tolerant bacterial communities with altered composition, but unaltered richness, via long-term Cu exposure. Appl Environ Microbiol 78:7438–7446CrossRefGoogle Scholar
  5. Binh CT, Heuer H, Kaupenjohann M, Smalla K (2009) Diverse aadA gene cassettes on class 1 integrons introduced into soil via spread manure. Res Microbiol 160:427–433CrossRefGoogle Scholar
  6. Binh CTT, Heuer H, Kaupenjohann M, Smalla K (2008) Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. FEMS Microbiol Ecol 66:25–37CrossRefGoogle Scholar
  7. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336CrossRefGoogle Scholar
  8. Crofts TS, Gasparrini AJ, Dantas G (2017) Next-generation approaches to understand and combat the antibiotic resistome. Nat Rev Microbiol 15:422–434CrossRefGoogle Scholar
  9. D'costa VM, McGrann KM, Hughes DW, Wright GD (2006) Sampling the antibiotic resistome. Science 311:374–377CrossRefGoogle Scholar
  10. Ding G-C, Radl V, Schloter-Hai B, Jechalke S, Heuer H, Smalla K, Schloter M (2014) Dynamics of soil bacterial communities in response to repeated application of manure containing sulfadiazine. PLoS One 9:e92958CrossRefGoogle Scholar
  11. Dolliver H, Gupta S, Noll S (2008) Antibiotic degradation during manure composting. J Environ Qual 37:1245–1253CrossRefGoogle Scholar
  12. Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998CrossRefGoogle Scholar
  13. Enright MC (2003) The evolution of a resistant pathogen–the case of MRSA. Curr Opin Pharmacol 3:474–479CrossRefGoogle Scholar
  14. Fang H, Wang H, Cai L, Yu Y (2014) Prevalence of antibiotic resistance genes and bacterial pathogens in long-term manured greenhouse soils as revealed by metagenomic survey. Environ Sci Technol 49:1095–1104CrossRefGoogle Scholar
  15. Forsberg KJ, Patel S, Gibson MK, Lauber CL, Knight R, Fierer N, Dantas G (2014) Bacterial phylogeny structures soil resistomes across habitats. Nature 509:612–616CrossRefGoogle Scholar
  16. Gandhi M, Chikindas ML (2007) Listeria: a foodborne pathogen that knows how to survive. Int J Food Microbiol 113:1–15CrossRefGoogle Scholar
  17. Ghosh S, LaPara TM (2007) The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J 1:191–203CrossRefGoogle Scholar
  18. Gou M, Hu H-W, Zhang Y-J, Wang J-T, Hayden H, Tang Y-Q, He J-Z (2018) Aerobic composting reduces antibiotic resistance genes in cattle manure and the resistome dissemination in agricultural soils. Sci Total Environ 612:1300–1310CrossRefGoogle Scholar
  19. Gullberg E, Cao S, Berg OG, Ilbäck C, Sandegren L, Hughes D, Andersson DI (2011) Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathog 7:e1002158CrossRefGoogle Scholar
  20. Heuer H, Focks A, Lamshöft M, Smalla K, Matthies M, Spiteller M (2008) Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil. Soil Biol Biochem 40:1892–1900CrossRefGoogle Scholar
  21. Hochberg YBY (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc B (Methodological) 57:289–300CrossRefGoogle Scholar
  22. Hu H, Wang JT, Jing L, Shi X, Ma Y, Chen D, He JZ (2016) Long-term nickel contamination increases the occurrence of antibiotic resistance genes in agricultural soils. Environ Sci Technol 51:790CrossRefGoogle Scholar
  23. Heuer H, Solehati Q, Zimmerling U, Kleineidam K, Schloter M, Müller T, Focks A, Thiele-Bruhn S, Smalla K (2011) Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine. Appl Environ Microbiol 77:2527–2530CrossRefGoogle Scholar
  24. Hughner RS, McDonagh P, Prothero A, Shultz CJ, Stanton J (2007) Who are organic food consumers? A compilation and review of why people purchase organic food. J Consum Behav 6:94–110CrossRefGoogle Scholar
  25. Jechalke S, Focks A, Rosendahl I, Groeneweg J, Siemens J, Heuer H, Smalla K (2014) Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs. FEMS Microbiol Ecol 87:78–88CrossRefGoogle Scholar
  26. Jechalke S, Kopmann C, Rosendahl I, Groeneweg J, Weichelt V, Krögerrecklenfort E, Brandes N, Nordwig M, Ding G-C, Siemens J (2013) Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs. Appl Environ Microbiol 79:1704–1711CrossRefGoogle Scholar
  27. Jiang X, Ellabaan MMH, Charusanti P, Munck C, Blin K, Tong Y, Weber T, Sommer MO, Lee SY (2017) Dissemination of antibiotic resistance genes from antibiotic producers to pathogens. In: Nat Commun 8:ncomms15784, vol 8Google Scholar
  28. Joachim S (2006) Review of history and recent development of organic farming worldwide. Agric Sci China 5:169–178CrossRefGoogle Scholar
  29. Kala DR, Rosenani AB, Fauziah CI, Ahmad SH, Radziah O, Rosazlin A (2011) Commercial organic fertilizers and their labeling in Malaysia. Malays J Soil Sci 15:147–157Google Scholar
  30. Knapp CW, Dolfing J, Ehlert PA, Graham DW (2009) Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environ Sci Technol 44:580–587CrossRefGoogle Scholar
  31. Kyselková M, Kotrbová L, Bhumibhamon G, Chroňáková A, Jirout J, Vrchotová N, Schmitt H, Elhottová D (2015) Tetracycline resistance genes persist in soil amended with cattle feces independently from chlortetracycline selection pressure. Soil Biol Biochem 81:259–265CrossRefGoogle Scholar
  32. Lamendella R, Santo Domingo JW, Ghosh S, Martinson J, Oerther DB (2011) Comparative fecal metagenomics unveils unique functional capacity of the swine gut. BMC Microbiol 11:103CrossRefGoogle Scholar
  33. Li B, Yang Y, Ma L, Ju F, Guo F, Tiedje JM, Zhang T (2015) Metagenomic and network analysis reveal wide distribution and co-occurrence of environmental antibiotic resistance genes. ISME J 9:2490–2502CrossRefGoogle Scholar
  34. Li L-G, Xia Y, Zhang T (2017) Co-occurrence of antibiotic and metal resistance genes revealed in complete genome collection. ISME J 11:651–662CrossRefGoogle Scholar
  35. Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul WJ, Stedtfeld TM, Chai B, Cole JR (2012) In-feed antibiotic effects on the swine intestinal microbiome. PNAS 109:1691–1696CrossRefGoogle Scholar
  36. Lu HJ, Ye ZQ, Zhang XL, Lin XY, Ni WZ (2011) Growth and yield responses of crops and macronutrient balance influenced by commercial organic manure used as a partial substitute for chemical fertilizers in an intensive vegetable cropping system. Phys Chem Earth 36:387–394CrossRefGoogle Scholar
  37. Lubelski J, Konings WN, Driessen AJ (2007) Distribution and physiology of ABC-type transporters contributing to multidrug resistance in bacteria. Microbiol Mol Biol Rev 71:463–476CrossRefGoogle Scholar
  38. Magoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963CrossRefGoogle Scholar
  39. Muurinen J, Stedtfeld R, Karkman A, Pärnänen K, Tiedje J, Virta M (2017) Influence of manure application on the environmental resistome under Finnish agricultural practice with restricted antibiotic use. Environ Sci Technol 51:5989–5999CrossRefGoogle Scholar
  40. Nõlvak H, Truu M, Kanger K, Tampere M, Espenberg M, Loit E, Raave H, Truu J (2016) Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil. Sci Total Environ 562:678–689CrossRefGoogle Scholar
  41. Oksanen J, Blanchet F, Kindt R, Legendre P, Minchin P, O’Hara R, Simpson G, Solymos P, Stevens M, Wagner H (2014) vegan: community ecology package. R package version 2.2-0.
  42. Ouyang W-Y, Huang F-Y, Zhao Y, Li H, Su J-Q (2015) Increased levels of antibiotic resistance in urban stream of Jiulongjiang River, China. Appl Microbiol Biotechnol 99:5697–5707CrossRefGoogle Scholar
  43. Peng S, Wang Y, Zhou B, Lin X (2015) Long-term application of fresh and composted manure increase tetracycline resistance in the arable soil of eastern China. Sci Total Environ 506:279–286CrossRefGoogle Scholar
  44. Prazak AM, Murano EA, Mercado I, Acuff GR (2002) Antimicrobial resistance of Listeria monocytogenes isolated from various cabbage farms and packing sheds in Texas. J Food Prot 65:1796–1799CrossRefGoogle Scholar
  45. Qiao M, Ying G-G, Singer AC, Zhu Y-G (2018) Review of antibiotic resistance in China and its environment. Environ Int 110:160–172CrossRefGoogle Scholar
  46. Reichenbach H (2006) The genus Lysobacter. In: The prokaryotes. Springer, pp 939–957Google Scholar
  47. Seiler C, Berendonk TU (2012) Heavy metal driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture. Front Microbiol 3:399CrossRefGoogle Scholar
  48. Su J-Q, Wei B, Ou-Yang W-Y, Huang F-Y, Zhao Y, Xu H-J, Zhu Y-G (2015) Antibiotic resistome and its association with bacterial communities during sewage sludge composting. Environ Sci Technol 49:7356–7363CrossRefGoogle Scholar
  49. Su R-F, Li X-L, Zhang WY, Fang QY, Tang YW, Tang JG (2008) Effects of commercial organic fertilizer on rice growth, yield and grain quality. Acta Agric Shanghai 24:127–130Google Scholar
  50. Team RC (2014) R: a language and environment for statistical computing; R Foundation for Statistical Computing:Vienna, Austria.
  51. Tien Y-C, Li B, Zhang T, Scott A, Murray R, Sabourin L, Marti R, Topp E (2017) Impact of dairy manure pre-application treatment on manure composition, soil dynamics of antibiotic resistance genes, and abundance of antibiotic-resistance genes on vegetables at harvest. Sci Total Environ 581:32–39CrossRefGoogle Scholar
  52. Udikovic-Kolic N, Wichmann F, Broderick NA, Handelsman J (2014) Bloom of resident antibiotic-resistant bacteria in soil following manure fertilization. PNAS 111:15202–15207CrossRefGoogle Scholar
  53. Wang F-H, Qiao M, Su J-Q, Chen Z, Zhou X, Zhu Y-G (2014) High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation. Environ Sci Technol 48:9079–9085CrossRefGoogle Scholar
  54. Wang H, Sangwan N, Li H-Y, Su J-Q, Oyang W-Y, Zhang Z-J, Gilbert JA, Zhu Y-G, Ping F, Zhang H-L (2017) The antibiotic resistome of swine manure is significantly altered by association with the Musca domestica larvae gut microbiome. ISME J 11:100–111CrossRefGoogle Scholar
  55. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267CrossRefGoogle Scholar
  56. Wright MS, Peltier GL, Stepanauskas R, McArthur JV (2006) Bacterial tolerances to metals and antibiotics in metal-contaminated and reference streams. FEMS Microbiol Ecol 58:293–302CrossRefGoogle Scholar
  57. Xie W-Y, McGrath SP, Su J-Q, Hirsch PR, Clark IM, Shen Q, Zhu Y-G, Zhao F-J (2016) Long-term impact of field applications of sewage sludge on soil antibiotic resistome. Environ Sci Technol 50:12602–12611CrossRefGoogle Scholar
  58. Xie WY, Yang XP, Li Q, Wu LH, Shen QR, Zhao FJ (2016b) Changes in antibiotic concentrations and antibiotic resistome during commercial composting of animal manures. Environ Pollut 219:182–190CrossRefGoogle Scholar
  59. Yuan H-Y, Liu P-P, Wang N, Li X-M, Zhu Y-G, Khan ST, Alkhedhairy AA, Sun G-X (2017) The influence of soil properties and geographical distance on the bacterial community compositions of paddy soils enriched on SMFC anodes. J Soils Sediments 18:517–525CrossRefGoogle Scholar
  60. Zhang X, Dong Y, Wang H, Shen D (2007) Structure of livestock and variation of fecal nitrogen pollution load in China. Environ Sci 28:1311–1318CrossRefGoogle Scholar
  61. Zhou X, Qiao M, Wang F-H, Zhu Y-G (2017) Use of commercial organic fertilizer increases the abundance of antibiotic resistance genes and antibiotics in soil. Environ Sci Pollut Res 24:701–710CrossRefGoogle Scholar
  62. Zhu Y-G, Gillings M, Simonet P, Stekel D, Banwart S, Penuelas J (2017a) Microbial mass movements. Science 357:1099–1100CrossRefGoogle Scholar
  63. Zhu Y-G, Johnson TA, Su J-Q, Qiao M, Guo G-X, Stedtfeld RD, Hashsham SA, Tiedje JM (2013) Diverse and abundant antibiotic resistance genes in Chinese swine farms. PNAS 110:3435–3440CrossRefGoogle Scholar
  64. Zhu Y-G, Reid BJ, Meharg AA, Banwart SA, Fu B-J (2017b) Optimizing Peri-URban Ecosystems (PURE) to re-couple urban-rural symbiosis. Sci Total Environ 586:1085–1090CrossRefGoogle Scholar
  65. Zhu Y-G, Zhao Y, Li B, Huang C-L, Zhang S-Y, Yu S, Chen Y-S, Zhang T, Gillings MR, Su J-Q (2017c) Continental-scale pollution of estuaries with antibiotic resistance genes. Nat Microbiol 2:16270CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Xue Zhou
    • 1
    • 2
  • Min Qiao
    • 1
    • 2
    Email author
  • Jian-Qiang Su
    • 3
  • Yong-Guan Zhu
    • 1
    • 3
  1. 1.State Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental SciencesChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Key Lab of Urban Environment and Health, Institute of Urban EnvironmentChinese Academy of SciencesXiamenChina

Personalised recommendations