Skip to main content
SpringerLink
Log in

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

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

Abstract

New Delhi metallo-β-lactamase-1 gene (bla NDM-1 ) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of β-lactam ring, and provides resistance against major classes of β-lactam antibiotics. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla NDM-1 . Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6–25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla NDM-1 gene.

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
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Yong D, Toleman M, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR (2009) Characterization of a new metallo-beta-lactamase gene, bla NDM-1 , and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53:5046–5054. doi:10.1128/AAC.00774-09

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Walsh TR, Weeks J, Livermore DM, Toleman MA (2011) Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 11:355–362. doi:10.1016/S1473-3099(11)70059-7

    Article  PubMed  Google Scholar 

  3. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharajan S, Mustaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfar W, Livermore DM, Woodford N (2010) Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10:597–602. doi:10.1016/S1473-3099(10)70143-2

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Poirel L, Lagrutta E, Taylor P, Pham J, Nordmann P (2010) Emergence of metallo-β-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia. Antimicrob Agents Chemother 54:4914–4916. doi:10.1128/AAC.00878-10

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Struelens MJ, Monnet DL, Magiorakos AP, Santos O’Connor F, Giesecke J, the European NDM-1 Survey Participants (2010) New Delhi metallo-beta-lactamase 1–producing Enterobacteriaceae: emergence and response in Europe. Euro Surveill 15(46). http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19716

  6. Ho PL, Lo WU, Yeung MK, Lin CH, Chow KH, Ang I, Tong AHY, Bao TYJ, Lok S, Lo JYC (2011) Complete sequencing of pNDM-HK encoding NDM-1 carbapenemase from a multidrug-resistant Escherichia coli strain isolated in Hong Kong. PLoS One 6:17989. doi:10.1371/journal.pone.0017989

    Article  Google Scholar 

  7. Mulvey MR, Grant JM, Plewes K, Roscoe D, Boyd DA (2011) New Delhi metallo-β-lactamase in Klebsiella pneumoniae and Escherichia coli, Canada. Emerging Infect Dis 17:103–106. doi:10.3201/eid1701.101358

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Nordmann P, Gniadkowski M, Giske CG, Poirel L, Woodford N, Miriagou V (2012) Identification and screening of carbapenemase-producing Enterobacteriaceae. Clin Microbiol Infect 18:432–438. doi:10.1111/j.1469-0691.2012.03815.x

    Article  CAS  PubMed  Google Scholar 

  9. Naas T, Ergani A, Carrër A, Nordmann P (2011) Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples. Antimicrob Agents Chemother 55:4038–4043. doi:10.1128/AAC.01734-10

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Ong DCT, Koh TH, Syahidah N, Krishnan P, Tan TY (2011) Rapid detection of the bla NDM-1 gene by real-time PCR. J Antimicrob Chemother 66:1647–1649. doi:10.1093/jac/dkr184

    Article  CAS  PubMed  Google Scholar 

  11. Bonnin RA, Naas T, Poirel L, Nordmann P (2012) Phenotypic, biochemical, and molecular techniques for detection of metallo-β-lactamase NDM in Acinetobacter baumannii. J Clin Microbiol 50:1419–1421. doi:10.1128/JCM.06276-11

    Article  PubMed Central  PubMed  Google Scholar 

  12. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:63. doi:10.1093/nar/28.12.e63

    Article  Google Scholar 

  13. Goto M, Honda E, Ogura A, Nomoto A, Hanaki KI (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46:167–172. doi:10.2144/000113072

    Article  CAS  PubMed  Google Scholar 

  14. Minami M, Ohta M, Ohkura T, Ando T, Torii K, Hasegawa T, Goto H (2006) Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori. J Clin Microbiol 44:4032–4037. doi:10.1128/JCM.00898-06

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Bista BR, Ishwad C, Wadowsky RM, Manna P, Randhawa PS, Gupta G, Adikari M, Tyagi R, Gasper G, Vats A (2007) Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus. J Clin Microbiol 45:1581–1587. doi:10.1128/JCM.01024-06

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Hill J, Beriwal S, Chandra I, Paul VK, Kapil A, Singh T, Wadowsky RM, Singh V, Goyal A, Jahnukainen T, Johnson JR, Tarr P, Vats A (2008) Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. J Clin Microbiol 46:2800–2804. doi:10.1128/JCM.00152-08

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Huang CH, Lai GH, Lee MS, Lin WH, Lien YY, Hsueh SC, Kao JY, Chang WT, Lu TC, Lin WN, Chen HJ, Lee MS (2009) Development and evaluation of Loop Mediated Isothermal Amplification assay for rapid detection of chicken anemia virus. J Appl Microbiol 108:917–924. doi:10.1111/j.1365-2672.2009.04481.x

    Article  PubMed  Google Scholar 

  18. Kawai Y, Miyashita N, Kishi F, Tabuchi M, Oda K, Yamaguchi T, Ouchi K (2009) Development and evaluation of a loop-mediated isothermal amplification method for the rapid detection of Chlamydophila pneumoniae. Eur J Clin Microbiol Infect Dis 28:801–805. doi:10.1007/s10096-009-0710-z

    Article  CAS  PubMed  Google Scholar 

  19. Kurosaki Y, Sakuma T, Fukuma A, Fujinami Y, Kawamoto K, Kamo N, Maikino S, Yasuda J (2009) A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification. J Appl Microbiol 107:1947–1956. doi:10.1111/j.1365-2672.2009.04379.x

    Article  CAS  PubMed  Google Scholar 

  20. Liu W, Zou D, Li Y, Wang X, He X, Wei X, Shao C, Li X, Shang W, Yu K, Liu D, Li Y, Guo J, Yin Z, Yuan J (2012) Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification. J Clin Microbiol 50:1580–1585. doi:10.1128/JCM.06647-11

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Kitade Y, Ootsuka S, Iitsuka O, Saga N (2003) Effect of DMSO on PCR of Porphyra yezoensis (Rhodophyta) gene. J Appl Phycol 15:555–557

    Article  CAS  Google Scholar 

  22. Kang J, Lee MS, Gorenstein DG (2005) The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods 64:147–151

    Article  CAS  PubMed  Google Scholar 

  23. Arbeli Z, Fuentes CL (2007) Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol Lett 272:269–275. doi:10.1111/j.1574-6968.2007.00764.x

    Article  CAS  PubMed  Google Scholar 

  24. Huang T, Chen T, Chen Y, Lauderdale T, Liao T, Lee T, Chen C, Liu Y, Lin A, Chang Y, Wu K, Kirby R, Lai J, Tan M, Siu L, Chang C, Fung C, Tsai S (2013) Copy number change of the NDM-1 sequence in a multidrug-resistant Klebsiella pneumoniae clinical isolate. PLoS One 8:62774. doi:10.1371/journal.pone.0062774

    Article  Google Scholar 

  25. Lee D, Kim EJ, Kilgore PE, Kim SA, Takahashi H, Ohnishi M, Anh DD, Dong BQ, Kim JS, Tomon J, Miyamoto S, Notomi T, Kim DW, Seki M (2015) Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid. PLoS One 10:e0122922. doi:10.1371/journal.pone.0122922

    Article  PubMed Central  PubMed  Google Scholar 

  26. Wang X, Seo D, Lee MH, Choi C (2014) Comparison of conventional PCR, multiplex PCR, and loop- mediated isothermal amplification assays for rapid detection of Arcobacter species. J Clin Microbiol 52:557–563. doi:10.1128/JCM.02883-13

    Article  PubMed Central  PubMed  Google Scholar 

  27. Ohtsuka K, Yanagawa K, Takatori K, Hara-Kudo Y (2005) Detection of Salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplifi cation, and characterization of Salmonella isolates. Appl Environ Microbiol 71:6730–6735. doi:10.1128/AEM.71.11.6730-6735.2005

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JS (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52:303–306. doi:10.1373/clinchem.2005.057901

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Ms. M. Sulaika, Mr. Md. Ahmar Ahmad and Mrs. Brinda Chandar for their help in this study. We also thank SRM University for their financial support for this study.

Conflict of interest

No conflict of interest declared.

Author information

Authors and Affiliations

  1. Genomics Laboratory, Department of Genetic Engineering, SRM University, Kattankulathur, Chennai, 603203, Tamil Nadu, India

    P. Rathinasabapathi, Deepak S. Hiremath, Rex Arunraj & M. Parani

Authors
  1. P. Rathinasabapathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deepak S. Hiremath
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rex Arunraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Parani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Parani.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rathinasabapathi, P., Hiremath, D.S., Arunraj, R. et al. Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1 . Indian J Microbiol 55, 400–405 (2015). https://doi.org/10.1007/s12088-015-0540-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12088-015-0540-x

Keywords

Search

Navigation