Isolation of broad-host-range bacteriophages against food- and patient-derived Shiga toxin-producing Escherichia coli

Document Type : Research Articles


1 Ferdowsi University of Mashhad

2 Department of Faculty of Veterinary Medicine

3 Mashhad university of medical sciences


This study aimed to isolate bacteriophages specific to Shiga toxin-producing Escherichia coli (E. coli) strains, particularly EHEC O157:H7, in order to develop a collection of phages against different E. coli pathotypes isolated from northeast of Iran. Eighteen samples were screened without any preliminary enrichment and also with small scale enrichment using E. coli 12900, which did not result in the phage recovery. Seven samples were prepared with an extensive enrichment. Of them, 5 samples produced plaques. Eventually, seven phages out of thirteen isolated phages were selected for phage host range investigation. Results of the spotting host range assay demonstrated that 22 pathogenic E. coli strains and isolates (54%) were susceptible to at least one of the phages. Phage Ecol-MHD1 was polyvalent against E. coli and Salmonella isolates. The other phages were specific to E. coli pathotypes. In conclusion, the phages isolated in this study can be suggested as preventive or therapeutic candidates against foodborne E. coli infections in humans.


Main Subjects

. Havelaar AH, Kirk MD, Torgerson PR, Gibb HJ, Hald T, Lake RJ, et al. World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med. 2015;12(12):1–23.
2. Marder, MPH EP, Griffin PM, Cieslak PR, Dunn J, Hurd S, Jervis R, et al. preliminary incidence and trends of infections with pathogens transmitted commonly through food — Foodborne diseases active surveillance network, 10 U.S. Sites, 2006–2017. MMWR Morb Mortal Wkly Rep. 2018;67(11):324–8.
3. EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018;16(12).
4. Questions and Answers | E. coli | CDC [Internet]. [cited 2020 Jan 4]. Available from:
5. Grant J, Wendelboe AM, Wendel A, Jepson B, Torres P, Smelser C, et al. Spinach-associated Escherichia coli O157:H7 outbreak, Utah and New Mexico, 2006. Emerg Infect Dis. 2008;14(10):1633–6.
6. Orth D, Wurzner R. What makes an enterohemorrhagic Escherichia coli? Clin Infect Dis. 2006;43(9):1168–9.
7. Escherichia coli (E. coli) | FDA [Internet]. [cited 2020 Jan 4]. Available from:
8. Anany H, Brovko LY, El-Arabi T, Griffiths MW. Bacteriophages as antimicrobials in food products: History, biology and application. Handbook of natural antimicrobials for food safety and quality. 2014; 69–87.
9. Kazi M, Annapure US. Bacteriophage biocontrol of foodborne pathogens. J Food Sci Technol. 2016;53(3):1355–62.
10. Kon K, Rai M. Antibiotic resistance: mechanisms and new antimicrobial approaches. 2016. 1–413 p.
11. Sharma S. pengawet Food Preservatives and their harmful effects. Int J Sci Res Publ. 2015;5(4):5–6.
12. Fister S, Robben C, Witte AK, Schoder D, Wagner M, Rossmanith P. Influence of environmental factors on phage-bacteria interaction and on the efficacy and infectivity of phage P100. Front Microbiol. 2016;7(7):1152.
13. Mills S, Ross RP, Hill C. Bacteriocins and bacteriophage; a narrow-minded approach to food and gut microbiology. FEMS Microbiol Rev. 2017;41(1):S129–53.
14. Ross A, Ward S, Hyman P. More is better: Selecting for broad host range bacteriophages. Front Microbiol. 2016;7(9):1352.
15. Khan Mirzaei M, Nilsson AS. Isolation of Phages for Phage Therapy: A comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy. Schuch R, editor. PLoS One. 2015;10(3):1–13.
16. Hungaro HM, Mendonça RCS, Gouvêa DM, Vanetti MCD, Pinto CL de O. Use of bacteriophages to reduce Salmonella in chicken skin in comparison with chemical agents. Food Res Int. 2013;52(1):75–81.
17. Abedon ST. Information phage therapy research should report. Pharmaceuticals. 2017;10(4):43.
18. Abedon ST. Editorial the ‘Nuts and Bolts’ of phage therapy. Curr Pharm Biotechnol. 2010;11(1):1.
19. Garcia P, Martinez B, Obeso JM, Rodriguez A. Bacteriophages and their application in food safety. Lett Appl Microbiol. 2008;47(6):479–85.
20. Moye ZD, Woolston J, Sulakvelidze A. Bacteriophage applications for food production and processing. Viruses. 2018;10(4):1–22.
21. Bourdin G, Schmitt B, Guy LM, Germond JE, Zuber S, Michot L, et al. Amplification and purification of T4-Like Escherichia coli phages for phage therapy: From laboratory to pilot scale. Appl Environ Microbiol. 2014;80(4):1469–76.
22. Furfaro LL, Payne MS, Chang BJ. Bacteriophage therapy: clinical trials and regulatory hurdles. Front Cell Infect Microbiol. 2018;8(10):376.
23. Nagel TE, Chan BK, De Vos D, El-Shibiny A, Kang’ethe EK, Makumi A, et al. The developing world urgently needs phages to combat pathogenic bacteria. Front Microbiol. 2016;7(6):882.
24. Djie-Maletz A, Reither K, Danour S, Anyidoho L, Saad E, Danikuu F, et al. High rate of resistance to locally used antibiotics among enteric bacteria from children in Northern Ghana. J Antimicrob Chemother. 2008;61(6):1315–8.
25. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: A molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10(9):597–602.
26. Oot RA, Raya RR, Callaway TR, Edrington TS, Kutter EM, Brabban AD. Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces. Lett Appl Microbiol. 2007;45(4):445–53.
27. Kutter E. Phage host range and efficiency of plating. In: Clokie MRJ, Kropinski AM, editors. Bacteriophages: Methods in molecular biology. Berlin/Heidelberg: Humana Press; 2009. p. 141–9.
28. Debartolomeis J, Cabelli VJ. Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages. Appl Environ Microbiol. 1991;57(5):1301–5.
29. Yildirim Z, Sakin T, Çoban F. Isolation of anti-Escherichia coli O157:H7 bacteriophages and determination of their host ranges. Turkish J Agric - Food Sci Technol. 2018;6(9):1200.
30. Chibani-Chennouf S, Sidoti J, Bruttin A, Kutter E, Sarker S, Bru¨ssow H. In vitro and in vivo bacteriolytic activities of Escherichia coli phages: implications for phage therapy. Antimicrob Agents Chemother. 2004;48(7):2558–69.
31. Carlson K. Working with bacteriophages: common techniques and methodological approaches. Vol. 1. CRC press Boca Raton, FL; 2005.
32. Niu YD, McAllister TA, Xu Y, Johnson RP, Stephens TP, Stanford K. Prevalence and impact of bacteriophages on the presence of Escherichia coli O157:H7 in feedlot cattle and their environment. Appl Environ Microbiol. 2009;75(5):1271–8.
33. Viazis S, Akhtar M, Feirtag J, Brabban AD, Diez-Gonzalez F. Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli. J Appl Microbiol. 2011;110(5):1323–31.
34. Gabisonia T, Loladze M, Chakhunashvili N, Katamadze T, Tamarashvili N, Nadiradze M, et al. New bacteriophage cocktail against antibiotic resistant Escherichia coli. Bull Georg Natl Acad Sci. 2018;12(3):95–102.
35. Hudson JA, Billington C, Cornelius AJ, Wilson T, On SLW, Premaratne A, et al. Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef. Food Microbiol. 2013;36(1):14–21.
36. Montso PK, Mlambo V, Ateba CN. Characterization of lytic bacteriophages infecting multidrug-resistant shiga toxigenic atypical Escherichia coli O177 strains isolated from cattle feces. Front Public Heal. 2019;7(11):355.
37. O’Flynn G, Ross RP, Fitzgerald GF, Coffey A. Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. Appl Environ Microbiol. 2004;70(6):3417–24.
38. Majdani R. Isolation of lytic bacteriophages against pathogenic Escherichia coli strains in poultry in the northwest of Iran. Arch Razi Inst. 2016;71(4):235–44.
39. Beheshti Maal K, Soleimani Delfan A, Salmanizadeh S. Isolation and identification of two novel Escherichia coli bacteriophages and their application in wastewater treatment and coliform’s phage therapy. Jundishapur J Microbiol. 2015;8(3):e14945.
40. Shahrbabak SS, Khodabandehlou Z, Shahverdi AR, Skurnik M, Ackermann HW, Varjosalo M, et al. Isolation, characterization and complete genome sequence of Phaxi: A phage of Escherichia coli O157:H7. Microbiol (United Kingdom). 2013;159(8):1629–38.
41. Park M, Lee J-H, Shin H, Kim M, Choi J, Kang D-H, et al. Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7. Appl Environ Microbiol. 2012;78(1):58–69.
42. Yildirim Z, Sakіn T, Çoban F. Isolation of lytic bacteriophages infecting Salmonella Typhimurium and Salmonella Enteritidis. Acta Biol Hung. 2018;69(3):350–69.
43. Gill JJ, Hyman P. Phage choice, isolation, and preparation for phage therapy. Curr Pharm Biotechnol. 2010;11(1):2–14.
44. Van Twest R, Kropinski AM. Bacteriophage enrichment from water and soil. In: Bacteriophages: Methods in molecular biology. Humana Press; 2009. p. 15–21.
45. Akhtar M, Viazis S, Diez-Gonzalez F. Isolation, identification and characterization of lytic, wide host range bacteriophages from waste effluents against Salmonella enterica serovars. Food Control. 2014;38(1):67–74.
46. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by double agar overlay plaque assay. In: Bacteriophages: Methods in molecular biology. Humana Press; 2009. p. 69–76.