Thermophilic anaerobic digestion is an effective treatment for reducing cefazolin-resistant bacteria and ESBL-producers in dairy manure

  • Masahiro Iwasaki
  • Masazumi Miyake
  • Hideaki Maseda
  • Guangdou Qi
  • Zhifei Pan
  • Ikko Ihara
  • Kazutaka UmetsuEmail author


The application of thermophilic anaerobic digestion (TAD) treatments to dairy manure in a biogas plant was evaluated to investigate whether the prominent countermeasure was sufficient for the dissemination of antimicrobial-resistant (AMR) bacteria in the dairy industry. To determine the changes in the number of AMR bacteria in dairy manure after TAD, cefazolin-resistant (CEZ-R) and ampicillin-resistant (AMP-R) bacteria in dairy manure and digestate were quantified by plate spread methods performed with combinations of antimicrobials added to agar plates and selective and differential agar plates. In addition, extended-spectrum β-lactamase (ESBL)-producing bacteria were also quantified to evaluate the effect of TAD on plasmid-borne resistant bacteria. CEZ-R and AMP-R bacteria were widely reduced after TAD compared with the susceptible bacteria against these antimicrobials. The classification into E. coli, other coliforms, and non-coliform bacteria revealed that CEZ-R and AMP-R coliform bacteria were more significantly reduced than non-coliform bacteria by TAD. Moreover, ESBL-producing bacteria were reduced significantly among CEZ-R bacteria. From these results, TAD appears to be a useful treatment to counter the dissemination of AMR bacteria in dairy manure by the preferential elimination of AMR bacteria.


Anaerobic digestion Antimicrobial-resistant bacteria Cefazolin Digestate Extended-spectrum β-lactamase 



This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 10670499).


  1. 1.
    Roca I, Akova M, Baquero F, Carlet J, Cavaleri M, Coenen S, Cohen J, Findlay D, Gyssens I, Heure OE, Kahlmeter G, Kruse H, Laxminarayan R, Liébana E, López-Cerero L, MacGowan A, Martins M, Rodríguez-Baño J, Rolain JM, Segovia C, Sigauque B, Tacconelli E, Wellington E, Vila J (2015) The global threat of antimicrobial resistance: science for intervention. New Microbes New Infect 6:22–29. CrossRefGoogle Scholar
  2. 2.
    Asai T, Kojima A, Harada K, Ishihara K, Takahashi T, Tamura Y (2005) Correlation between the usage volume of veterinary therapeutic antimicrobials and resistance in Escherichia coli isolated from the feces of food-producing animals in Japan. Jpn J Infect Dis 58:369–372Google Scholar
  3. 3.
    Furuya EY, Franklin DL (2006) Antimicrobial-resistant bacteria in the community setting. Nat Rev Microbiol 4:36–45. CrossRefGoogle Scholar
  4. 4.
    The National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry, and Fisheries (2013) Report of the Japanese veterinary antimicrobial resistance monitoring system—2008 to 2011. Accessed 23 Mar 2017
  5. 5.
    Iwasaki M, Yamashiro T, Beneragama N, Nishida T, Kida K, Ihara I, Takahashi J, Umetsu K (2011) The effect of temperature on survival of pathogenic bacteria in biogas plants. Anim Sci J 82:707–712. CrossRefGoogle Scholar
  6. 6.
    Brinas L, Moreno MA, Zarazaga M, Porrero C, Saenz Y, García M, Dominguez L, Torres C (2003) Detection of CMY-2, CTX-M-14, and SHV-12 β-lactamases in Escherichia coli fecal-sample isolates from healthy chickens. Antimicrob Agents Chemother 47:2056–2058CrossRefGoogle Scholar
  7. 7.
    Jacoby GA (2006) β-Lactamase nomenclature. Antimicrob Agents Chemother 50:1123–1129. CrossRefGoogle Scholar
  8. 8.
    Batchelor M, Threlfall EJ, Liebana E (2005) Cephalosporin resistance among animal-associated Enterobacteria: a current perspective. Expert Rev Anti Infect Ther 3:403–417. CrossRefGoogle Scholar
  9. 9.
    Carattoli A (2008) Animal reservoirs for extended spectrum β-lactamase producers. Clin Microbiol Infect 14(Suppl 1):117–123. CrossRefGoogle Scholar
  10. 10.
    Asuming-Brempong S, Aferi NK (2014) Isolation of phosphate solubilizing bacteria from tropical soil. Glob Adv Res J Agric Sci 3:8–15Google Scholar
  11. 11.
    Lowe SE, Jain MK, Zeikus JG (1993) Biology, ecology, and biotechnological applications of anaerobic bacteria adapted to environmental stresses in temperature, pH, salinity, or substrates. Microbiol Rev 57:451–509Google Scholar
  12. 12.
    Sahlstrom L (2003) A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresour Technol 87:161–166. CrossRefGoogle Scholar
  13. 13.
    Desbois AP, Smith VJ (2010) Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 85:1629–1642. CrossRefGoogle Scholar
  14. 14.
    Aldsworth TG, Sharman RL, Dodd CER (1999) Bacterial suicide through stress. Cell Mol Life Sci 56:378–383. CrossRefGoogle Scholar
  15. 15.
    Palleroni NJ (2005) Genus I. Pseudomonas. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey’s Manual of Systematic Bacteriology, vol 2, 2nd edn. Springer, East Lansing, pp 323–379Google Scholar
  16. 16.
    Qi G, Pan Z, Sugawa Y, Andriamanohiarisoamanana FJ, Yamashiro T, Iwasaki M, Kawamoto K, Ihara I, Umetsu K (2018) Comparative fertilizer properties of digestates from mesophilic and thermophilic anaerobic digestion of dairy manure: focusing on plant growth promoting bacteria (PGPB) and environmental risk. J Mater Cycles Waste. Google Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Masahiro Iwasaki
    • 1
  • Masazumi Miyake
    • 2
  • Hideaki Maseda
    • 3
  • Guangdou Qi
    • 1
  • Zhifei Pan
    • 2
  • Ikko Ihara
    • 4
  • Kazutaka Umetsu
    • 1
    Email author
  1. 1.Department of Agro-Environmental ScienceObihiro University of Agriculture and Veterinary MedicineObihiroJapan
  2. 2.Department of Animal and Food HygieneObihiro University of Agriculture and Veterinary MedicineObihiroJapan
  3. 3.Department of Bioresource Chemistry and TechnologyTokushima UniversityTokushimaJapan
  4. 4.Department of Agricultural Engineering and Socio-EconomicsKobe UniversityKobeJapan

Personalised recommendations