Skip to main content
SpringerLink
Log in

Molecular Characterization and Computational Modelling of New Delhi Metallo-β-Lactamase-5 from an Escherichia coli Isolate (KOEC3) of Bovine Origin

  • Original Article
  • Published:
Indian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Emergence of antimicrobial resistance mediated through New Delhi metallo-β-lactamases (NDMs) is a serious therapeutic challenge. Till date, 16 different NDMs have been described. In this study, we report the molecular and structural characteristics of NDM-5 isolated from an Escherichia coli isolate (KOEC3) of bovine origin. Using PCR amplification, cloning and sequencing of full blaNDM gene, we identified the NDM type as NDM-5. Cloning of full gene in E. coli DH5α and subsequent assessment of antibiotic susceptibility of the transformed cells indicated possible role of native promoter in expression blaNDM-5. Translated amino acid sequence had two substitutions (Val88Leu and Met154Leu) compared to NDM-1. Theoretically deduced isoelectric pH of NDM-5 was 5.88 and instability index was 36.99, indicating a stable protein. From the amino acids sequence, a 3D model of the protein was computed. Analysis of the protein structure elucidated zinc coordination and also revealed a large binding cleft and flexible nature of the protein, which might be the reason for broad substrate range. Docking experiments revealed plausible binding poses for five carbapenem drugs in the vicinity of metal ions. In conclusion, results provided possible explanation for wide range of antibiotics catalyzed by NDM-5 and likely interaction modes with five carbapenem drugs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. World Health Organization (WHO) (2012) The evolving threat of antimicrobial resistance: options for action. WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland

  2. Makena A, Brem J, Pfeffer I, Geffen RE, Wilkins SE, Tarhonskaya H, Flashman E, Phee LM, Wareham DW, Schofield CJ (2015) Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability. J Antimicrob Chemother 70:463–469. doi:10.1093/jac/dku403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Palzkill T (2013) Metallo-β-lactamase structure and function. Ann N Y Acad Sci 1277:91–104. doi:10.1111/j.1749-6632.2012.06796.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Nordmann P, Naas T, Poirel L (2011) Global spread of carbapenemase producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1798. doi:10.3201/eid1710.110655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Berrazeg M, Diene S, Medjahed L, Parola P, Drissi M, Raoult D &Rolain J (2014) New Delhi Metallo-beta-lactamase around the world: an eReview using Google Maps. Euro Surveill 19: 20809. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20809

  6. Jones LS, Carvalho MJ, Toleman MA, White PL, Connor TR, Mushtaq A, Weeks JL, Kumarasamy KK, Raven KE, Török ME, Peacock SJ, Howe RA, Walsh TR (2014) Characterization of plasmids in extensively drug-resistant Acinetobacter strains isolated in India and Pakistan. Antimicrob Agents Chemother 59:923–929. doi:10.1128/AAC.03242-14

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lahey Clinic (2015) NDM-type ß-Lactamases. http://www.lahey.org/Studies/other.asp#table1. Accessed on 28 Oct 2015

  8. Dortet L, Poirel L, Nordmann P (2014) Worldwide dissemination of the NDM-type carbapenemases in Gram-negative bacteria. BioMed Res Int. doi:10.1155/2014/249856

    PubMed  PubMed Central  Google Scholar 

  9. Yang P, Xie Y, Feng P, Zong Z (2014) blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167. Antimicrob Agents Chemother 58:7548–7552. doi:10.1128/AAC.03911-14

    Article  PubMed  PubMed Central  Google Scholar 

  10. Hornsey M, Phee L, Wareham DW (2011) A novel variant, NDM-5, of the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom. Antimicrob Agents Chemother 55:5952–5954. doi:10.1128/AAC.05108-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rahman M, Shukla SK, Prasad KN, Ovejero CM, Pati BK, Tripathi A, Singh A, Srivastava AK, Gonzalez-Zorn B (2014) Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India. Int J Antimicrob Agents 44:30–37. doi:10.1016/j.ijantimicag.2014.03.003

    Article  CAS  PubMed  Google Scholar 

  12. Toleman MA, Bugert JJ, Nizam SA (2015) Extensively drug-resistant New Delhi metallo-β-lactamase–encoding bacteria in the environment, Dhaka, Bangladesh, 2012. Emerg Infect Dis 21:1027–1030. doi:10.3201/eid2106.141578

    Article  PubMed  PubMed Central  Google Scholar 

  13. Zhao J, Zhu Y, Li Y, Mu X, You L, Xu C, Qin P, Ma J (2014) Coexistence of SFO-1 and NDM-1 β-lactamase genes and fosfomycin resistance gene fosA3 in an Escherichia coli clinical isolate. FEMS Microbiol Lett 362:1–7. doi:10.1093/femsle/fnu018

    Article  Google Scholar 

  14. Seija V, Presentado JCM, Bado I, Ezdra RP, Batista N, Gutierrez C, Guirado M, Vidal M, Nin M, Vignoli R (2015) Sepsis caused by New Delhi metallo-β-lactamase (blaNDM-1) and qnrD-producing Morganella morganii, treated successfully with fosfomycin and meropenem: case report and literature review. Int J Infect Dis 30:20–26. doi:10.1016/j.ijid.2014.09.010

    Article  PubMed  Google Scholar 

  15. Tran HH, Ehsani S, Shibayama K, Matsui M, Suzuki S, Nguyen MB, Tran DN, Tran VP, Tran DL, Nguyen HT, Dang DA, Trinh HS, Nguyen TH, Wertheim HFL (2015) Common isolation of New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae in a large surgical hospital in Vietnam. Eur J Clin Microbiol Infect Dis 34:1247–1254. doi:10.1007/s10096-015-2345-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bhattacharya D, Dey S, Kadam S, Kalal S, Jali S, Koley H, Sinha R, Nag D, Kholkute SD, Roy S (2015) Isolation of NDM-1-producing multidrug-resistant Pseudomonas putida from a paediatric case of acute gastroenteritis, India. New Microbe New Infect 5:5–9. doi:10.1016/j.nmni.2015.02.002

    Article  CAS  Google Scholar 

  17. Pitart C, Solé M, Roca I, Román A, Moreno A, Vila J, Marco F (2015) Molecular characterization of blaNDM-5 carried on an IncFII plasmid in an Escherichia coli isolate from a non-traveler patient in Spain. Antimicrob Agents Chemother 59:659–662. doi:10.1128/AAC.04040-14

    Article  PubMed  PubMed Central  Google Scholar 

  18. Sassi A, Loucif L, Gupta SK, Dekhil M, Chettibi H, Rolain JM (2014) NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria. Antimicrob Agents Chemother 58:5606–5608. doi:10.1128/AAC.02818-13

    Article  PubMed  PubMed Central  Google Scholar 

  19. Nakano R, Nakano A, Hikosaka K, Kawakami S, Matsunaga N, Asahara M, Ishigaki S, Furukawa T, Suzuki M, Shibayama K, Ono Y (2014) First report of metallo-β-lactamase NDM-5-producing Escherichia coli in Japan. Antimicrob Agents Chemother 58:7611–7612. doi:10.1128/AAC.04265-14

    Article  PubMed  PubMed Central  Google Scholar 

  20. Cho SY, Huh HJ, Baek JY, Chung NY, Ryu JG, Ki CS, Chung DR, Lee NY, Song JH (2015) Klebsiella pneumoniae co-Producing NDM-5 and OXA-181 carbapenemases, South Korea. Emerg Infect Dis 21:1088–1089. doi:10.3201/eid2106.150048

    Article  PubMed  PubMed Central  Google Scholar 

  21. Shaheen BW, Nayak R, Boothe DM (2013) Emergence of a New Delhi metallo-β-lactamase (NDM-1)-encoding gene in clinical Escherichia coli isolates recovered from companion animals in the United States. Antimicrob Agents Chemother 57:2902–2903. doi:10.1128/AAC.02028-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cain R, Narramore S, McPhillie M, Simmons K, Fishwick CWG (2014) Applications of structure-based design to antibacterial drug discovery. Bioorgan Chem. doi:10.1016/j.bioorg.2014.05.008

    Google Scholar 

  23. Ghatak S, Singha A, Sen A, Guha C, Ahuja A, Bhattacherjee U, Das S, Pradhan NR, Puro K, Jana C, Dey TK, Prashantkumar KL, Das A, Shakuntala I, Biswas U, Jana PS (2013) Detection of New Delhi metallo-beta-Lactamase and extended-spectrum beta-lactamase genes in Escherichia coli isolated from mastitic milk samples. Transbound Emerg Dis 60:385–389. doi:10.1111/tbed.12119

    Article  CAS  PubMed  Google Scholar 

  24. EUCAST (European Committee on Antimicrobial Susceptibility Testing) (2003) Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. EUCAST Discussion Document E Dis 5: 1–7. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/MIC_testing/Edis5.1_broth_dilution.pdf. Accessed on 20 Oct 2015

  25. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26:1781–1802. doi:10.1002/jcc.20289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Grosdidier A, Zoete V, Michielin O (2011) SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res 39:W270–W277. doi:10.1093/nar/gkr366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. doi:10.1002/jcc.20084

    Article  CAS  PubMed  Google Scholar 

  28. Nordmann P, Boulanger AE, Poirel L (2012) NDM-4 metallo-β-lactamase with increased carbapenemase activity from Escherichia coli. Antimicrob Agents Chemother 56:2184–2186. doi:10.1128/AAC.05961-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press Inc, New Jersey, pp 571–607

    Chapter  Google Scholar 

  30. Marsh JA, Teichmann SA (2011) Relative solvent accessible surface area predicts protein conformational changes upon binding. Structure 19:859–867. doi:10.1016/j.str.2011.03.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang JF, Chou KC (2011) Insights from modelling the 3D structure of New Delhi metallo-β-lactamase and its binding interactions with antibiotic drugs. PLoS ONE 6:e18414. doi:10.1371/journal.pone.0018414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Laskowski RA, Luscombe NM, Swindells MB, Thornton JM (1996) Protein clefts in molecular recognition and function. Protein Sci 5:2438–2452. doi:10.1002/pro.5560051206

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Li X, Ma S (2015) Advances in the discovery of novel antimicrobials targeting the assembly of bacterial cell division protein FtsZ. Eur J Med Chem 95:1–15. doi:10.1016/j.ejmech.2015.03.026

    Article  PubMed  Google Scholar 

  34. Karumuri S, Singh PK, Shukla P (2015) In silico analog design for Terbinafine against Trichophyton rubrum: a preliminary study. Indian J Microbiol 55:333–340. doi:10.1007/s12088-015-0524-x

    Article  CAS  PubMed  Google Scholar 

  35. Mihasan M (2012) What in silico molecular docking can do for the ‘bench-working biologists’. J Biosci 37:1089–1095. doi:10.1007/s12038-012-9273-8

    Article  CAS  PubMed  Google Scholar 

  36. Green VL, Verma A, Owens RJ, Phillips SE, Carr SB (1996) Structure of New Delhi metallo-β-lactamase 1 (NDM-1). Acta Crystallogr, Sect F: Struct Biol Cryst Commun 67:1160–1164. doi:10.1107/S1744309111029654

    Article  Google Scholar 

  37. Kim Y, Tesar C, Jedrzejczak R, Babnigg G, Sacchettini J, Joachimiak A. Crystal structure of New Delhi metallo-β-lactamase-1 in complex with hydrolyzed meropenem. PDB ID: 4RBS 2014; Midwest Center for Structural Genomics

  38. Ma B, Shatsky M, Wolfson HJ, Nussinov R (2002) Multiple diverse ligands binding at a single protein site: a matter of pre-existing populations. Protein Sci 11:184–197. doi:10.1110/ps.21302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors are thankful to Director, ICAR RC for NEH Region, Meghalaya for providing necessary facilities and to Department of Biotechnology, New Delhi for partly funding the study under Twinning programme for NE (Sanction Order No. BT/363/NE/TBFJIL/12 dated March 21, 2013). Authors are thankful to Dr Manish Kakkar for his critical inputs into the manuscript.

Author information

Author notes

    Authors and Affiliations

    1. Division of Animal Health, ICAR Research Complex for NEH Region, Umiam, Meghalaya, 793103, India

      D. Purkait, A. Ahuja, U. Bhattacharjee, K. Rhetso, T. K. Dey, S. Das, R. K. Sanjukta, K. Puro, I. Shakuntala, A. Sen & S. Ghatak

    2. West Bengal University of Animal and Fishery Sciences, Belgachia, West Bengal, 700037, India

      A. Singha, C. Guha & N. R. Pradhan

    3. Division of Crop Protection, ICAR Research Complex for NEH Region, Umiam, Meghalaya, 793103, India

      A. Banerjee

    4. Department of Microbiology, School of Life Sciences, Assam University, Silchar, Assam, 788011, India

      I. Sharma

    5. Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow, India

      R. S. Bhatta

    6. Nazareth Hospital, Shillong, Meghalaya, 793003, India

      M. Mawlong

    Authors
    1. D. Purkait
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. A. Ahuja
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. U. Bhattacharjee
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. A. Singha
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. K. Rhetso
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. T. K. Dey
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. S. Das
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. R. K. Sanjukta
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. K. Puro
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. I. Shakuntala
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. A. Sen
      View author publications

      You can also search for this author in PubMed Google Scholar

    12. A. Banerjee
      View author publications

      You can also search for this author in PubMed Google Scholar

    13. I. Sharma
      View author publications

      You can also search for this author in PubMed Google Scholar

    14. R. S. Bhatta
      View author publications

      You can also search for this author in PubMed Google Scholar

    15. M. Mawlong
      View author publications

      You can also search for this author in PubMed Google Scholar

    16. C. Guha
      View author publications

      You can also search for this author in PubMed Google Scholar

    17. N. R. Pradhan
      View author publications

      You can also search for this author in PubMed Google Scholar

    18. S. Ghatak
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to S. Ghatak.

    Additional information

    D. Purkait, A. Ahuja and U. Bhattacharjee have contributed equally for this study.

    Electronic Supplementary Material

    Below is the link to the electronic supplementary material.

    12088_2016_569_MOESM1_ESM.png

    PCR detection of blaNDM-5 gene in Escherichia coli isolates (KOEC3 and transformed DH5α cells). Lane M: Molecular weight marker; Lane A: KOEC3 (positive control); Lane B: Transformed DH5α (blaNDM-5 forward and reverse primers); Lane C: Transformed DH5α (blaNDM-5 forward and T7 promoter primers); Lane D: Negative control. (PNG 342 kb)

    12088_2016_569_MOESM2_ESM.png

    Quality assessment of computed three dimensional model of NDM-5. (a) Comparison of NDM-5 computed model with non-redundant set of PDB structures. (b) Density plot of the QMEAN score of reference PDB set with NDM-5 (in red). (c) Per residue error plot NDM-5. Blue: errors < 1 Å, red: errors above 3.5 Å. (PNG 360 kb)

    Ramachandran plot analysis of NDM-5 (PNG 336 kb)

    RMSD plot of residues of NDM-5 following molecular dynamics simulation of 2.5 ns (PNG 98 kb)

    Secondary structure of NDM-5 (PNG 8622 kb)

    Zinc coordination of computed model of NDM-5. Hydrogen bondings are depicted as dashed lines. (PNG 1793 kb)

    Supplementary material 7 (DOCX 47 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Purkait, D., Ahuja, A., Bhattacharjee, U. et al. Molecular Characterization and Computational Modelling of New Delhi Metallo-β-Lactamase-5 from an Escherichia coli Isolate (KOEC3) of Bovine Origin. Indian J Microbiol 56, 182–189 (2016). https://doi.org/10.1007/s12088-016-0569-5

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s12088-016-0569-5

    Keywords

    Search

    Navigation