Analytical and Bioanalytical Chemistry

, Volume 408, Issue 15, pp 4169–4178 | Cite as

Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection

  • Ziyuan Wang
  • Danhui Wang
  • Amanda J. Kinchla
  • David A. Sela
  • Sam R. Nugen
Research Paper

Abstract

Escherichia coli O157:H7 is a ubiquitous pathogen which can be linked to foodborne outbreaks worldwide. In addition to the significant illnesses, hospitalizations, and deaths resulting from the outbreaks, there can be severe economic consequences to farmers, food manufacturers, and municipalities. A rapid detection assay which can validate sanitation and water quality would prove beneficial to these situations. Here, we report a novel bacteriophage-mediated detection of E. coli O157:H7 which utilizes the specific recognition between phages and their host cell as well as the natural lysis component of the infection cycle for DNA release. Carboxylic acid-functionalized magnetic beads were conjugated with bacteriophage and used to separate and concentrate E. coli O157:H7. The effects of bead incubation time, salinity, pH, and temperature on the bio-magnetic separation were investigated and compared to an antibody-based counterpart. The conditions of 0.01 M PBS, pH 7.0, and 20 min of reaction at 37 °C were found to be optimal. The capture efficiency of the coupled assay was approximately 20 % higher than that of antibody-based separation under extreme conditions. The resulting bead-phage-bacteria complexes were quantitatively detected by real-time PCR (qPCR). Our results demonstrated that the use of phage-based magnetic separation coupled with qPCR improved the sensitivity of detection by 2 orders of magnitude compared that without phage-based pre-concentration. Specificity and selectivity of the assay system was evaluated, and no cross-reactivity occurred when Salmonella typhimurium, Staphylococcus aureus, and Pseudomonas aeruginosa were tested. The total assay time was less than 2 h.

Keywords

Escherichia coli O157:H7 Bacteriophage Magnetic separation Real-time PCR Water testing E. coli 

Supplementary material

216_2016_9511_MOESM1_ESM.pdf (187 kb)
ESM. 1(PDF 187 kb)

References

  1. 1.
    Besser RE, Lett SM, Weber J, et al. An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-pressed apple cider. JAMA. 1993;269(17):2217–20.CrossRefGoogle Scholar
  2. 2.
    Tarr PI. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin Infect Dis Official Publ Infect Dis Soc Am. 1995;20(1):1–8. quiz 9-10.CrossRefGoogle Scholar
  3. 3.
    Blackall DP, Marques MB. Hemolytic uremic syndrome revisited: Shiga toxin, factor H, and fibrin generation. Am J Clin Pathol. 2004;121(Suppl):S81–8.Google Scholar
  4. 4.
    Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, et al. Food-related illness and death in the United States. Emerg Infect Dis. 1999;5(5):607–25.CrossRefGoogle Scholar
  5. 5.
    Bell BP, Goldoft M, Griffin PM, Davis MA, Gordon DC, Tarr PI, et al. A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. Jama. 1994;272(17):1349–53.CrossRefGoogle Scholar
  6. 6.
    Hilborn ED, Mermin JH, Mshar PA, et al. A multistate outbreak of Escherichia coli O157:H7 infections associated with consumption of mesclun lettuce. Arch Intern Med. 1999;159(15):1758–64.CrossRefGoogle Scholar
  7. 7.
    Frenzen PD, Drake A, Angulo FJ. Economic cost of illness due to Escherichia coli O157 infections in the United States. J Food Prot. 2005;68(12):2623–30.Google Scholar
  8. 8.
    Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al. Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis. 2011;17(1):7–15.CrossRefGoogle Scholar
  9. 9.
    Solomon EB, Yaron S, Matthews KR. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl Environ Microbiol. 2002;68(1):397–400.CrossRefGoogle Scholar
  10. 10.
    Islam M, Doyle MP, Phatak SC, Millner P, Jiang X. Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water. J Food Prot. 2004;67(7):1365–70.Google Scholar
  11. 11.
    Szalanski AL, Owens CB, McKay T, Steelman CD. Detection of Campylobacter and Escherichia coli O157:H7 from filth flies by polymerase chain reaction. Med Vet Entomol. 2004;18(3):241–6.CrossRefGoogle Scholar
  12. 12.
    March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol. 1986;23(5):869–72.Google Scholar
  13. 13.
    Vuddhakul V, Patararungrong N, Pungrasamee P, Jitsurong S, Morigaki T, Asai N, et al. Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand. 2000;2000-01-01 00:00:00. 343-7 p.Google Scholar
  14. 14.
    Johnson RP, Durham RJ, Johnson ST, MacDonald LA, Jeffrey SR, Butman BT. Detection of Escherichia coli O157:H7 in meat by an enzyme-linked immunosorbent assay. EHEC-Tek Appl Environ Microbiol. 1995;61(1):386–8.Google Scholar
  15. 15.
    Zhu P, Shelton DR, Karns JS, Sundaram A, Li S, Amstutz P, et al. Detection of water-borne E. coli O157 using the integrating waveguide biosensor. Biosensors Bioelectronics. 2005;21(4):678–83.CrossRefGoogle Scholar
  16. 16.
    Wilkes JG, Tucker RK, Montgomery JA, Cooper WM, Sutherland JB, Buzatu DA. Reduction of food matrix interference by a combination of sample preparation and multi-dimensional gating techniques to facilitate rapid, high sensitivity analysis for Escherichia coli serotype O157 by flow cytometry. Food Microbiol. 2012;30(1):281–8.CrossRefGoogle Scholar
  17. 17.
    Donhauser SC, Niessner R, Seidel M. Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica, and Campylobacter jejuni by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis. Anal Chem. 2011;83(8):3153–60.CrossRefGoogle Scholar
  18. 18.
    Wang Y, Ye Z, Si C, Ying Y. Monitoring of Escherichia coli O157:H7 in food samples using lectin based surface plasmon resonance biosensor. Food Chemistry.136(3–4):1303-8Google Scholar
  19. 19.
    Gordillo R, Rodríguez A, Werning ML, Bermúdez E, Rodríguez M. Quantification of viable Escherichia coli O157:H7 in meat products by duplex real-time PCR assays. Meat Sci. 2014;96(2, Part A):964–70.CrossRefGoogle Scholar
  20. 20.
    Yang H, Qu L, Wimbrow AN, Jiang X, Sun Y. Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR. Int J Food Microbiol. 2007;118(2):132–8.CrossRefGoogle Scholar
  21. 21.
    Brussow H, Hendrix RW. Phage genomics: small is beautiful. Cell. 2002;108(1):13–6.CrossRefGoogle Scholar
  22. 22.
    Lin L, Hong W, Ji X, Han J, Huang L, Wei Y. Isolation and characterization of an extremely long tail Thermus bacteriophage from Tengchong hot springs in China. J Basic Microbiol. 2010;50(5):452–6.CrossRefGoogle Scholar
  23. 23.
    Prigent M, Leroy M, Confalonieri F, Dutertre M, DuBow MS. A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the Sahara Desert. Extremophiles life Under Extreme Conditions. 2005;9(4):289–96.CrossRefGoogle Scholar
  24. 24.
    Sawstrom C, Lisle J, Anesio AM, Priscu JC, Laybourn-Parry J. Bacteriophage in polar inland waters. Extremophiles Life Under extreme Conditions. 2008;12(2):167–75.CrossRefGoogle Scholar
  25. 25.
    Jończyk E, Kłak M, Międzybrodzki R, Górski A. The influence of external factors on bacteriophages—review. Folia Microbiologica. 2011;56(3):191–200.CrossRefGoogle Scholar
  26. 26.
    Wang Z, Wang D, Chen J, Sela DA, Nugen SR. Development of a novel bacteriophage based biomagnetic separation method as an aid for sensitive detection of viable Escherichia coli. Analyst. 2016;141(3):1009–16.CrossRefGoogle Scholar
  27. 27.
    Smartt AE, Ripp S. Bacteriophage reporter technology for sensing and detecting microbial targets. Anal Bioanal Chem. 2011;400(4):991–1007.CrossRefGoogle Scholar
  28. 28.
    Martelet A, L'Hostis G, Nevers MC, Volland H, Junot C, Becher F, et al. Phage amplification and immunomagnetic separation combined with targeted mass spectrometry for sensitive detection of viable bacteria in complex food matrices. Anal Chem. 2015;87(11):5553–60.CrossRefGoogle Scholar
  29. 29.
    Chang TC, Ding HC, Chen S. A conductance method for the identification of Escherichia coli O157:H7 using bacteriophage AR1. J Food Prot. 2002;65(1):12–7.Google Scholar
  30. 30.
    Balasubramanian S, Sorokulova IB, Vodyanoy VJ, Simonian AL. Lytic phage as a specific and selective probe for detection of Staphylococcus aureus—a surface plasmon resonance spectroscopic study. Biosens Bioelectron. 2007;22(6):948–55.CrossRefGoogle Scholar
  31. 31.
    Singh A, Poshtiban S, Evoy S. Recent advances in bacteriophage based biosensors for food-borne pathogen detection. Sensors. 2013;13(2):1763.CrossRefGoogle Scholar
  32. 32.
    Carter CD, Parks A, Abuladze T, Li M, Woolston J, Magnone J, et al. Bacteriophage cocktail significantly reduces Escherichia coli O157:H7 contamination of lettuce and beef, but does not protect against recontamination. Bacteriophage. 2012;2(3):178–85.CrossRefGoogle Scholar
  33. 33.
    Bekal S, Brousseau R, Masson L, Prefontaine G, Fairbrother J, Harel J. Rapid identification of Escherichia coli pathotypes by virulence gene detection with DNA microarrays. J Clin Microbiol. 2003;41(5):2113–25.CrossRefGoogle Scholar
  34. 34.
    Yu MX, Slater MR, Ackermann HW. Isolation and characterization of Thermus bacteriophages. Arch Virol. 2006;151(4):663–79.CrossRefGoogle Scholar
  35. 35.
    Handa H, Gurczynski S, Jackson MP, Auner G, Mao G. Recognition of Salmonella typhimurium by immobilized phage P22 monolayers. Surf Sci. 2008;602(7):1392–400.CrossRefGoogle Scholar
  36. 36.
    Gupta K, Lee Y, Yin J. Extremo-phage: in vitro selection of tolerance to a hostile environment. J Mol Evol. 1995;41(2):113–4.CrossRefGoogle Scholar
  37. 37.
    Fluit AC, Torensma R, Visser MJ, Aarsman CJ, Poppelier MJ, Keller BH, et al. Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay. Appl Environ Microbiol. 1993;59(5):1289–93.Google Scholar
  38. 38.
    Rakhuba DV, Kolomiets EI, Dey ES, Novik GI. Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell. Polish journal of microbiology/Polskie Towarzystwo Mikrobiologow = The Polish Society of Microbiologists. 2010;59(3):145–55.Google Scholar
  39. 39.
    Nanduri V, Sorokulova IB, Samoylov AM, Simonian AL, Petrenko VA, Vodyanoy V. Phage as a molecular recognition element in biosensors immobilized by physical adsorption. Biosens Bioelectron. 2007;22(6):986–92.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ziyuan Wang
    • 1
  • Danhui Wang
    • 1
  • Amanda J. Kinchla
    • 1
  • David A. Sela
    • 1
  • Sam R. Nugen
    • 1
  1. 1.Department of Food ScienceUniversity of MassachusettsAmherstUSA

Personalised recommendations