Journal of Microbiology

, Volume 56, Issue 6, pp 408–415 | Cite as

Metagenomic analysis reveals the prevalence and persistence of antibiotic- and heavy metal-resistance genes in wastewater treatment plant

  • Sachin Kumar Gupta
  • Hanseob Shin
  • Dukki Han
  • Hor-Gil HurEmail author
  • Tatsuya UnnoEmail author
Microbial Ecology and Environmental Microbiology


The increased antibiotic resistance among microorganisms has resulted into growing interest for investigating the wastewater treatment plants (WWTPs) as they are reported to be the major source in the dissemination of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) in the environment. In this study, we investigated the prevalence and persistence of ARGs and HMRGs as well as bacterial diversity and mobile genetic elements (MGEs) in influent and effluent at the WWTP in Gwangju, South Korea, using high-throughput sequencing based metagenomic approach. A good number of broad-spectrum of resistance genes (both ARG and HMRG) were prevalent and likely persistent, although large portion of them were successfully removed at the wastewater treatment process. The relative abundance of ARGs and MGEs was higher in effluent as compared to that of influent. Our results suggest that the resistance genes with high abundance and bacteria harbouring ARGs and MGEs are likely to persist more through the treatment process. On analyzing the microbial community, the phylum Proteobacteria, especially potentially pathogenic species belonging to the genus Acinetobacter, dominated in WWTP. Overall, our study demonstrates that many ARGs and HMRGs may persist the treatment processes in WWTPs and their association to MGEs may contribute to the dissemination of resistance genes among microorganisms in the environment.


antibiotic resistance genes heavy metal resistance genes metagenomics persistence wastewater treatment plant 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12275_2018_8195_MOESM1_ESM.pdf (589 kb)
Supplementary material, approximately 588 KB.


  1. Allen, H.K., Donato, J., Wang, H.H., Cloud-Hansen, K.A., Davies, J., and Handelsman, J. 2010. Call of the wild: antibiotic resistance genes in natural environments. Nat. Rev. Microbiol. 8, 251.CrossRefPubMedGoogle Scholar
  2. Bengtsson-Palme, J., Boulund, F., Fick, J., Kristiansson, E., and Larsson, D. 2014. Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Front. Microbiol. 5, 648.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Brown, K.D., Kulis, J., Thomson, B., Chapman, T.H., and Mawhinney, D.B. 2006. Occurrence of antibiotics in hospital, residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico. Sci. Total Environ. 366, 772–783.CrossRefPubMedGoogle Scholar
  4. Buchfink, B., Xie, C., and Huson, D.H. 2014. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 12, 59–60.CrossRefPubMedGoogle Scholar
  5. Chen, Q.L., Li, H., Zhou, X.Y., Zhao, Y., Su, J.Q., Zhang, X., and Huang, F.Y. 2017. An underappreciated hotspot of antibiotic resistance: The groundwater near the municipal solid waste landfill. Sci. Total Environ. 609, 966–973.CrossRefPubMedGoogle Scholar
  6. Czekalski, N., Gascón Díez, E., and Bürgmann, H. 2014. Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake. ISME J. 8, 1381.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Deycard, V.N., Schäfer, J., Blanc, G., Coynel, A., Petit, J.C.J., Lanceleur, L., Dutruch, L., Bossy, C., and Ventura, A. 2014. Contributions and potential impacts of seven priority substances (As, Cd, Cu, Cr, Ni, Pb, and Zn) to a major European Estuary (Gironde Estuary, France) from urban wastewater. Mar. Chem. 167, 123–134.CrossRefGoogle Scholar
  8. Forsberg, K.J., Reyes, A., Wang, B., Selleck, E.M., Sommer, M.O.A., and Dantas, G. 2012. The shared antibiotic resistome of soil bacteria and human pathogens. Science 337, 1107.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gillings, M.R., Gaze, W.H., Pruden, A., Smalla, K., Tiedje, J.M., and Zhu, Y.G. 2014. Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution. ISME J. 9, 1269.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Guo, J., Li, J., Chen, H., Bond, P.L., and Yuan, Z. 2017. Metagenomic analysis reveals wastewater treatment plants as hotspots of antibiotic resistance genes and mobile genetic elements. Water Res. 123, 468–478.CrossRefPubMedGoogle Scholar
  11. Helms, M., Vastrup, P., Gerner-Smidt, P., and Mølbak, K. 2002. Excess mortality associated with antimicrobial drug-resistant Salmonella Typhimurium. Emerg. Infect. Dis. 8, 490.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hu, Y., Yang, X., Qin, J., Lu, N., Cheng, G., Wu, N., Pan, Y., Li, J., Zhu, L., Wang, X., et al. 2013. Metagenome-wide analysis of antibiotic resistance genes in a large cohort of human gut microbiota. Nat. Commun. 4, 2151.CrossRefPubMedGoogle Scholar
  13. Huson, D.H., Beier, S., Flade, I., Górska, A., El-Hadidi, M., Mitra, S., Ruscheweyh, H.J., and Tappu, R. 2016. MEGAN community edition - Interactive exploration and analysis of large-scale microbiome sequencing data. PLoS Comput. Biol. 12, e1004957.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hyatt, D., Chen, G.L., LoCascio, P.F., Land, M.L., Larimer, F.W., and Hauser, L.J. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Ji, X., Shen, Q., Liu, F., Ma, J., Xu, G., Wang, Y., and Wu, M. 2012. Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures and agricultural soils adjacent to feedlots in Shanghai; China. J. Hazard. Mater. 235-236, 178–185.CrossRefPubMedGoogle Scholar
  16. Jia, B., Raphenya, A.R., Alcock, B., Waglechner, N., Guo, P., Tsang, K.K., Lago, B.A., Dave, B.M., Pereira, S., Sharma, A.N., 2017. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 45, D566–D573.CrossRefPubMedGoogle Scholar
  17. Kozinska, A., Pazdzior, E., Pekala, A., and Niemczuk, W. 2014. Acinetobacter johnsonii and Acinetobacter lwoffii-the emerging fish pathogens. Bull. Vet. Inst. Pulawy 58, 193–199.CrossRefGoogle Scholar
  18. Krawczyk, P.S., Lipinski, L., and Dziembowski, A. 2018. PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures. Nucleic Acids Res. 46, e35.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kristiansson, E., Fick, J., Janzon, A., Grabic, R., Rutgersson, C., Weijdegård, B., Söderström, H., and Larsson, D.J. 2011. Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS One 6, e17038.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Laffite, A., Kilunga, P.I., Kayembe, J.M., Devarajan, N., Mulaji, C.K., Giuliani, G., Slaveykova, V.I., and Poté, J. 2016. Hospital effluents are one of several sources of metal, antibiotic resistance genes, and bacterial markers disseminated in sub-saharan urban rivers. Front. Microbiol. 7, 1128.CrossRefPubMedPubMedCentralGoogle Scholar
  21. Lamba, M., Graham, D.W., and Ahammad, S.Z. 2017. Hospital wastewater releases of carbapenem-resistance pathogens and genes in urban India. Environ. Sci. Technol. 51, 13906–13912.CrossRefPubMedGoogle Scholar
  22. Leplae, R., Lima-Mendez, G., and Toussaint, A. 2010. ACLAME: A classification of mobile genetic elements, update 2010. Nucleic Acids Res. 38, D57–D61.CrossRefPubMedGoogle Scholar
  23. Li, H. and Durbin, R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Li, W., Fu, L., Niu, B., Wu, S., and Wooley, J. 2012. Ultrafast clustering algorithms for metagenomic sequence analysis. Brief. Bioinform. 13, 656–668.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Li, A.D., Li, L.G., and Zhang, T. 2015. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants. Front. Microbiol. 6, 1025.PubMedPubMedCentralGoogle Scholar
  26. Li, L.G., Xia, Y., and Zhang, T. 2017. Co-occurrence of antibiotic and metal resistance genes revealed in complete genome collection. ISME J. 11, 651–662.CrossRefPubMedGoogle Scholar
  27. Lo, C.C. and Chain, P.S.G. 2014. Rapid evaluation and quality control of next generation sequencing data with FaQCs. BMC Bioinformatics 15, 366.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lood, R., Ertürk, G., and Mattiasson, B. 2017. Revisiting antibiotic resistance spreading in wastewater treatment plants–bacteriophages as a much neglected potential transmission vehicle. Front. Microbiol. 8, 2298.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lopatkin, A.J., Meredith, H.R., Srimani, J.K., Pfeiffer, C., Durrett, R., and You, L. 2017. Persistence and reversal of plasmid-mediated antibiotic resistance. Nat. Commun. 8, 1689.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lu, J., Tian, Z., Yu, J., Yang, M., and Zhang, Y. 2018. Distribution and abundance of antibiotic resistance genes in sand settling reservoirs and drinking water treatment plants across the Yellow River, China. Water 10, 246.CrossRefGoogle Scholar
  31. Michael, I., Rizzo, L., McArdell, C.S., Manaia, C.M., Merlin, C., Schwartz, T., Dagot, C., and Fatta-Kassinos, D. 2013. Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: A review. Water Res. 47, 957–995.CrossRefPubMedGoogle Scholar
  32. Noguchi, N., Tano, J., Nasu, Y., Koyama, M., Narui, K., Kamishima, H., Saito, T., Tsuyuki, K., and Sasatsu, M. 2007. Antimicrobial susceptibilities and distribution of resistance genes for β-lactams and macrolides in Streptococcus pneumoniae isolated between 2002 and 2004 in Tokyo. Int. J. Antimicrob. Agents 29, 26–33.CrossRefPubMedGoogle Scholar
  33. Pal, C., Bengtsson-Palme, J., Kristiansson, E., and Larsson, D.G.J. 2015. Co-occurrence of resistance genes to antibiotics, biocides and metals reveals novel insights into their co-selection potential. BMC Genomics 16, 964.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pal, C., Bengtsson-Palme, J., Rensing, C., Kristiansson, E., and Larsson, D.G.J. 2014. BacMet: antibacterial biocide and metal resistance genes database. Nucleic Acids Res. 42, D737–D743.CrossRefPubMedGoogle Scholar
  35. Peng, Y., Leung, H.C.M., Yiu, S.M., and Chin, F.Y.L. 2012. IDBAUD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28, 1420–1428.CrossRefPubMedGoogle Scholar
  36. Salyers, A.A. and Amabile-Cuevas, C.F. 1997. Why are antibiotic resistance genes so resistant to elimination? Antimicrob. Agents Chemother. 41, 2321.PubMedPubMedCentralCrossRefGoogle Scholar
  37. Schmitz, F.J., Fluit, A.C., Gondolf, M., Beyrau, R., Lindenlauf, E., Verhoef, J., Heinz, H.P., and Jones, M.E. 1999. The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals. J. Antimicrob. Chemother. 43, 253–259.CrossRefPubMedGoogle Scholar
  38. Segata, N., Waldron, L., Ballarini, A., Narasimhan, V., Jousson, O., and Huttenhower, C. 2012. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat. Methods 9, 811.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Seiler, C. and Berendonk, T.U. 2012. Heavy metal driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture. Front. Microbiol. 3, 399.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Sipahi, O.R. 2008. Economics of antibiotic resistance. Expert Rev. Anti-Infect. Ther. 6, 523–539.CrossRefPubMedGoogle Scholar
  41. Tang, J., Bu, Y., Zhang, X.X., Huang, K., He, X., Ye, L., Shan, Z., and Ren, H. 2016. Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water. Ecotoxicol. Environ. Saf. 132, 260–269.CrossRefPubMedGoogle Scholar
  42. VandeWalle, J., Goetz, G., Huse, S., Morrison, H., Sogin, M., Hoffmann, R., Yan, K., and McLellan, S. 2012. Acinetobacter, Aeromonas and Trichococcus populations dominate the microbial community within urban sewer infrastructure. Environ. Microbiol. 14, 2538–2552.CrossRefPubMedPubMedCentralGoogle Scholar
  43. World Health Organization (WHO). 2014. Antimicrobial resistance: global report on surveillance. World Health Organization.Google Scholar
  44. Wu, Y., Cui, E., Zuo, Y., Cheng, W., and Chen, H. 2018. Fate of antibiotic and metal resistance genes during two-phase anaerobic digestion of residue sludge revealed by metagenomic approach. Environ. Sci. Pollut. Res. Int. doi: 10.1007/s11356-018-1598-x.Google Scholar
  45. Yang, Y., Li, B., Zou, S., Fang, H.H.P., and Zhang, T. 2014. Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach. Water Res. 62, 97–106.CrossRefPubMedGoogle Scholar
  46. Zhang, Y., Marrs, C.F., Simon, C., and Xi, C. 2009. Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Sci. Total Environ. 407, 3702–3706.CrossRefPubMedGoogle Scholar
  47. Zhang, C., Qiu, S., Wang, Y., Qi, L., Hao, R., Liu, X., Shi, Y., Hu, X., An, D., Li, Z., et al. 2013. Higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals in Beijing. PLoS One 8, e64857.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Faculty of Biotechnology, School of life sciences, SARIJeju National UniversityJejuRepublic of Korea
  2. 2.School of Earth Sciences and Environmental EngineeringGwangju Institute of Science and TechnologyGwangjuRepublic of Korea
  3. 3.Subtropical/tropical Organism Gene BankJeju National UniversityJejuRepublic of Korea

Personalised recommendations