Skip to main content

Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout

Abstract

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the β-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of β-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-β-d-galactopyranoside, CPRG) and bioluminescent (6-O-β-galactopyranosyl-luciferin, Beta-Glo®) β-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-μm pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 °C for 4 h. Bacteriophage with the respective indicator substrate was added to the device, and signal (color, luminescence) development was recorded with a digital camera, luminometer, or luminescence imaging device. It was demonstrated that as low as 40 or <10 colony-forming units (cfu) ml−1 of E. coli can be detected visually within 8 h when wild-type T4 bacteriophage or recombinant lacZ T4 bacteriophage were used in the assay, respectively. Application of the bioluminescent β-galactosidase substrate allowed reliable detection of <10 cfu ml−1 within 5.5 h. The specificity of the assay was demonstrated using a panel of microorganisms including Aeromonas hydrophila, Enterobacter cloacae, E. coli, and Salmonella Typhimurium.

Scheme for rapid E. coli assay including filtration of water sample, short incubation on the filter in a paper-based culture device, addition of bacteriophage and [beta]-galactosidase substrate, and recording/processing of the accumulated color or luminescence signal.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Yager P, Domingo GJ, Gerdes J (2008) In: Annual review of biomedical engineering, vol 10. Annual review of biomedical engineering. Annual Reviews, Palo Alto, pp 107–144

  2. Center for Disease Control and Prevention (2012) Global WASH-related diseases and contaminants. http://www.cdc.gov/healthywater/wash_diseases.html. Accessed Jan 25 2014

  3. Boubetra A, Le Nestour F, Allaert C, Feinberg M (2011) Appl Environ Microbiol 77(10):3360–3367

    CAS  Article  Google Scholar 

  4. Kleinheinz GT, Busse KM, Gorman W, McDermott CM (2012) Lake Res Manag 28(4):328–337

    CAS  Article  Google Scholar 

  5. US Environmental Protection Agency (2012) Recreational water quality control. http://water.epa.gov/scitech/swguidance/standards/criteria/health/recreation/index.cfm. Accessed Jan 25 2014

  6. Byrne B, Stack E, Gilmartin N, O’Kennedy R (2009) Sensors 9(6):4407–4445

    CAS  Article  Google Scholar 

  7. Aldus CF, van Amerongen A, Ariens RMC, Peck MW, Wichers JH, Wyatt GM (2003) J Appl Microbiol 95(2):380–389

    CAS  Article  Google Scholar 

  8. Heo J, Hua SZ (2009) Sensors 9(6):4483–4502

    CAS  Article  Google Scholar 

  9. Brunt J, Webb MD, Peck MW (2010) Appl Environ Microbiol 76(13):4143–4150

    CAS  Article  Google Scholar 

  10. Foudeh AM, Didar TF, Veres T, Tabrizian M (2012) Lab Chip 12(18):3249–3266

    CAS  Article  Google Scholar 

  11. Kronlein MR, Stedtfeld RD, Sorensen J, Bhaduri P, Stedtfeld T, Eanes S, Harichandran V, Haynes K, Stevens M, Hashsham SA (2013) Water Environ Res 85(10):889–916

    Article  Google Scholar 

  12. Mandeville R, Griffiths M, Goodridge L, McIntyre L, Ilenchuk TT (2003) Anal Lett 36(15):3241–3259

    CAS  Article  Google Scholar 

  13. Mao CB, Liu AH, Cao BR (2009) Angew Chem Int Ed 48(37):6790–6810

    CAS  Article  Google Scholar 

  14. Hagens S, Loessner MJ (2007) Appl Microbiol Biotechnol 76(3):513–519

    CAS  Article  Google Scholar 

  15. Brovko LY, Anany H, Griffiths MW (2012) Adv Food Nutr Res 67:241–288

    CAS  Article  Google Scholar 

  16. Tolba M, Minikh O, Brovko LY, Evoy S, Griffiths MW (2010) Appl Environ Microbiol 76(2):528–535

    CAS  Article  Google Scholar 

  17. Minikh O, Tolba M, Brovko LY, Griffiths MW (2010) J Microbiol Meth 82(2):177–183

    CAS  Article  Google Scholar 

  18. Wu Y, Brovko L, Griffiths MW (2001) Lett Appl Microbiol 33(4):311–315

    CAS  Article  Google Scholar 

  19. Smartt AE, Ripp S (2011) Anal Bioanal Chem 400(4):991–1007

    CAS  Article  Google Scholar 

  20. Ulitzur N, Ulitzur S (2006) Appl Environ Microbiol 72(12):7455–7459

    CAS  Article  Google Scholar 

  21. Waddell TE, Poppe C (2000) FEMS Microbiol Lett 182:285–289

    CAS  Article  Google Scholar 

  22. Sarkis GJ, Jacobs WR, Hatfull GF (1995) Mol Microbiol 15(6):1055–1067

    CAS  Article  Google Scholar 

  23. Awais R, Fukudomi H, Miyanaga K, Unno H, Tanji Y (2006) Biotech Prog 22(3):853–859

    CAS  Article  Google Scholar 

  24. Oda M, Morita M, Unno H, Tanji Y (2004) Appl Environ Microbiol 70(1):527–534

    CAS  Article  Google Scholar 

  25. Goodridge L, Griffiths M (2002) Food Res Int 35(9):863–870

    CAS  Article  Google Scholar 

  26. Khoury MK, Parker I, Aswad DW (2010) Anal Biochem 397(1):129–131

    CAS  Article  Google Scholar 

  27. Martinez AW, Phillips ST, Butte MJ, Whitesides GM (2007) Angew Chem Int Ed 46(8):1318–1320

    CAS  Article  Google Scholar 

  28. Martinez AW, Phillips ST, Whitesides GM (2008) Proc Natl Acad Sci U S A 105(50):19606–19611

    CAS  Article  Google Scholar 

  29. Ellerbee AK, Phillips ST, Siegel AC, Mirica KA, Martinez AW, Striehl P, Jain N, Prentiss M, Whitesides GM (2009) Anal Chem 81(20):8447–8452

    CAS  Article  Google Scholar 

  30. Kutter E (2009) In: Clokie MRJ, Kropinski AM (eds) Methods in molecular biology, vol 501. Methods in molecular biology. Humana Press Inc, Totowa, USA

  31. New DC, Miller-Martini DM, Wong YH (2003) Phytother Res 17(5):439–448

    CAS  Article  Google Scholar 

  32. Goodridge L (2007) USA Patent 7,244,612

    Google Scholar 

  33. Anany H, Lingohr EJ, Villegas A, Ackermann HW, She YM, Griffiths MW, Kropinski AM (2011) Virol J 8. doi:24210.1186/1743-422x-8-242

  34. Funes-Huacca M, Wu A, Szepesvari E, Rajendran P, Kwan-Wong N, Razgulin A, Shen Y, Kagira J, Campbell R, Derda R (2012) Lab Chip 12(21):4269–4278

    CAS  Article  Google Scholar 

  35. Edberg SC, Rice EW, Karlin RJ, Allen MJ (2000) J Appl Microbiol 88:106S–116S

    Article  Google Scholar 

  36. Wohlsen T, Bayliss J, Bates J, Gray B, Johnson S, Schneider P (2008) J Wat Supply Res Technol AQUA 57(8):569–576

    CAS  Article  Google Scholar 

  37. Brenner KP, Rankin CC, Sivaganesan M (1996) J Microbiol Meth 27(2–3):111–119

    CAS  Article  Google Scholar 

  38. Hallas G, Giglio S, Capurso V, Monis PT, Grooby WL (2008) J Appl Microbiol 105(4):1138–1149

    CAS  Article  Google Scholar 

  39. McLain JET, Williams CF (2008) Water Res 42(15):4041–4048

    CAS  Article  Google Scholar 

  40. Reznikoff WS (1992) Mol Microbiol 6(17):2419–2422

    CAS  Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the NSERC Sentinel, Bioactive Paper Research Network.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mansel W. Griffiths.

Additional information

Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Burnham, S., Hu, J., Anany, H. et al. Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout. Anal Bioanal Chem 406, 5685–5693 (2014). https://doi.org/10.1007/s00216-014-7985-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-7985-3

Keywords

  • Bacteriophage T4
  • Escherichia coli
  • β-Galactosidase
  • Paper-based device
  • Colorimetric assay
  • Bioluminescence assay