Journal of Industrial Microbiology & Biotechnology

, Volume 40, Issue 10, pp 1105–1116 | Cite as

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm

  • Geevika J. Ganegama Arachchi
  • Andrew G. Cridge
  • Beatrice M. Dias-Wanigasekera
  • Cristina D. Cruz
  • Lynn McIntyre
  • Rachel Liu
  • Steve H. Flint
  • Anthony N. Mutukumira
Environmental Microbiology


Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log10 PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log10 CFU/cm2, respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log10 CFU/cm2). The phage cocktail at ≈5.8, 6.5 and 7.5 log10 PFU/cm2 eliminated Listeria contamination (≈1.5–1.7 log10 CFU/cm2) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log10 CFU/cm2) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log10 CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.


Listeria phages Biofilms Low count cells Stainless steel Low temperature 



The authors acknowledge New Zealand King Salmon Limited for funding the project, and Foundation for Research, Science and Technology, New Zealand for providing a scholarship (contract no. NZKX0902) to Geevika Ganegama Arachchi. The authors also thank Richard Smith, ex-company mentor at New Zealand King Salmon Limited; Ron Fyfe, Cawthron Institute; Graeme Fox at Sealord Group Limited and Karen Wittington at New Zealand King Salmon Limited for technical support.

Supplementary material

10295_2013_1313_MOESM1_ESM.docx (579 kb)
Supplementary material 1 (DOCX 578 kb)


  1. 1.
    Abedon ST (2009) Kinetics of phage-mediated biocontrol of bacteria. Foodborne Pathog Dis 6:807–815PubMedCrossRefGoogle Scholar
  2. 2.
    Abuladze T, Li M, Menetrez MY, Dean T, Senecal A, Sulakvelidze A (2008) Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157: H7. Appl Environ Microbiol 74:6230–6238PubMedCrossRefGoogle Scholar
  3. 3.
    Ackermann H-W (2009) Basic phage electron microscopy. In: Clokie MRJ, Kropinski AM (eds) Bacteriophages methods and protocols. Volume 1: isolation, characterization, and interaction. Humana, Hertford, pp 113–126Google Scholar
  4. 4.
    Allerberger F, Wagner M (2009) Listeriosis: a resurgent foodborne infection. Clin Microbiol Infect 16:16–23CrossRefGoogle Scholar
  5. 5.
    Bagge-Ravn D, Ng Y, Hjelm M, Christiansen JN, Johansen C, Gram L (2003) The microbial ecology of processing equipment in different fish industries-analysis of the microflora during processing and following cleaning and disinfection. Int J Food Microbiol 87:239–250PubMedCrossRefGoogle Scholar
  6. 6.
    Bernbom N, Jorgensen RL, Ng Y, Meyer R, Kingshott P, Vejborg RM, Klemm P, Besenbacher F, Gram L (2006) Bacterial adhesion to stainless steel is reduced by aqueous fish extract coatings. Biofilms 3:25–36CrossRefGoogle Scholar
  7. 7.
    Bernbom N, Ng Y, Jorgensen RL, Arpanaei A, Meyer RL, Kingshott P, Vejborg RM, Klemm P, Gram L (2009) Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films. J Appl Microbiol 106:1268–1279PubMedCrossRefGoogle Scholar
  8. 8.
    Bower C, McGuire J, Daeschel M (1995) Suppression of Listeria monocytogenes colonization following adsorption of nisin onto silica surfaces. Appl Environ Microbiol 61:992–997PubMedGoogle Scholar
  9. 9.
    Brett MSY, Short P, McLauchlin J (1998) A small outbreak of listeriosis associated with smoked mussels. Int J Food Microbiol 43:223–229PubMedCrossRefGoogle Scholar
  10. 10.
    Briandet R, Lacroix-Gueu P, Renault M, Lecart S, Meylheuc T, Bidnenko E, Steenkeste K, Bellon-Fontaine MN, Fontaine-Aupart MP (2008) Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms. Appl Environ Microbiol 74:2135–2143PubMedCrossRefGoogle Scholar
  11. 11.
    Carpentier B, Chassaing D (2004) Interactions in biofilms between Listeria monocytogenes and resident microorganisms from food industry premises. Int J Food Microbiol 97:111–122PubMedCrossRefGoogle Scholar
  12. 12.
    Chae MS, Schraft H (2000) Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int J Food Microbiol 62:103–111PubMedCrossRefGoogle Scholar
  13. 13.
    Chavant P, Gaillard Martinie B, Hébraud M (2004) Antimicrobial effects of sanitizers against planktonic and sessile Listeria monocytogenes cells according to the growth phase. FEMS Microbiol Lett 236:241–248PubMedCrossRefGoogle Scholar
  14. 14.
    Chmielewski R, Frank J (2003) Biofilm formation and control in food processing facilities. Compr Rev Food Sci Food Saf 2:22–32CrossRefGoogle Scholar
  15. 15.
    Crerar SK, Castle M, Hassel S, Schumacher D (2011) Recent experiences with Listeria monocytogenes in New Zealand and development of a food control risk-based strategy. Food Control 22:1510–1512CrossRefGoogle Scholar
  16. 16.
    Cruz CD, Fletcher GC (2011) Prevalence and biofilm-forming ability of Listeria monocytogenes in New Zealand mussel (Perna canaliculus) processing plants. Food Microbiol 28:1387–1393PubMedCrossRefGoogle Scholar
  17. 17.
    Dat NM, Hamanaka D, Tanaka F, Uchino T (2010) Surface conditioning of stainless steel coupons with skim milk solutions at different pH values and its effect on bacterial adherence. Food Control 21:1769–1773CrossRefGoogle Scholar
  18. 18.
    Di Bonaventura G, Piccolomini R, Paludi D, D’orio V, Vergara A, Conter M, Ianieri A (2008) Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity. J Appl Microbiol 104:1552–1561PubMedCrossRefGoogle Scholar
  19. 19.
    EFSA Panel on Biological Hazards (BIOHAZ) (2012) Scientific opinion on the evaluation of the safety and efficacy of ListexTM P100 for the removal of Listeria monocytogenes surface contamination of raw fish. EFSA J 10(3):2615Google Scholar
  20. 20.
    Eklund MW, Poysky FT, Paranjpye RN, Lashbrook LC, Peterson ME, Pelroy GA (1995) Incidence and sources of Listeria monocytogenes in cold-smoked fishery products and processing plants. J Food Prot 58:502–508Google Scholar
  21. 21.
    Ericsson H, Eklöw A, Danielsson-Tham M, Loncarevic S, Mentzing L, Persson I, Unnerstad H, Tham W (1997) An outbreak of listeriosis suspected to have been caused by rainbow trout. J Clin Microbiol 35:2904–2907PubMedGoogle Scholar
  22. 22.
    FDA (2006) Food additives permitted for direct addition to food for human consumption, bacteriophage preparation. 21 CFR Part 172 Fed Regis 71:47729–47732Google Scholar
  23. 23.
    Flint S, Brooks J, Bremer P (1997) The influence of cell surface properties of thermophilic Streptococci on attachment to stainless steel. J Appl Microbiol 83:508–517PubMedCrossRefGoogle Scholar
  24. 24.
    Food Standards Australia New Zealand (2013) Application A1045—bacteriophage preparation as a processing aid. Food Standards Australia New Zealand. Accessed 15 Jan 2013
  25. 25.
    Gallet R, Lenormand T, Wang IN (2012) Phenotypic stochasticity prevents lytic bacteriophage population from extinction during bacterial stationary phase. Evolution 66:3485–3494PubMedCrossRefGoogle Scholar
  26. 26.
    Gandhi M, Chikindas ML (2007) Listeria: a foodborne pathogen that knows how to survive. Int J Food Microbiol 113:1–15PubMedCrossRefGoogle Scholar
  27. 27.
    Ganegama Arachchi GJ (2013) A study of natural lytic Listeria phages with decontaminating properties for use in seafood processing plants. Unpublished doctoral dissertation, Massey University, New ZealandGoogle Scholar
  28. 28.
    Ganegama Arachchi GJ, Mutukumira AN, Dias-Wanigasekera BM, Cruz CD, McIntyre L, Young J, Hudson A, Flint SH (2012) Characterisation of Listeria-infecting bacteriophages isolated from seafood environments. In: Poster session presented at the 57th annual scientific meeting of the New Zealand Microbiological Society Inc, Dunedin, New ZealandGoogle Scholar
  29. 29.
    Gibson H, Taylor J, Hall K, Holah J (2001) Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms. J Appl Microbiol 87:41–48CrossRefGoogle Scholar
  30. 30.
    Gill JJ (2010) Practical and theoretical considerations for the use of bacteriophages in food systems. In: Sabour PM, Griffiths MW (eds) Bacteriophages in the control of food- and waterborne pathogens. ASM, Washington, DC, pp 217–235Google Scholar
  31. 31.
    Goodridge LD, Bisha B (2011) Phage-based biocontrol strategies to reduce foodborne pathogens in foods. Bacteriophage 1:130–137PubMedCrossRefGoogle Scholar
  32. 32.
    Guenther S, Huwyler D, Richard S, Loessner MJ (2009) Virulent bacteriophage for efficient biocontrol of Listeria monocytogenes in ready-to-eat foods. Appl Environ Microbiol 75:93–100PubMedCrossRefGoogle Scholar
  33. 33.
    Hagens S, Loessner MJ (2010) Bacteriophage for biocontrol of foodborne pathogens: calculations and considerations. Curr Pharm Biotechnol 11:58–68PubMedCrossRefGoogle Scholar
  34. 34.
    Herald PJ, Zottola EA (2006) Attachment of Listeria monocytogenes to stainless steel surfaces at various temperatures and pH values. J Food Sci 53:1549–1562CrossRefGoogle Scholar
  35. 35.
    Hibma AM, Jassim SAA, Griffiths MW (1997) Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage. Int J Food Microbiol 34:197–207PubMedCrossRefGoogle Scholar
  36. 36.
    Hitchins AD, Jinneman K (1998) Detection and enumeration of Listeria monocytogenes in foods. In: Bacteriological  Analytical Manual, 8th edn, Revision A. Accessed 21 July 2013
  37. 37.
    Intralytix (2013) Food safety products. ListShield™. Accessed 30 July 2012
  38. 38.
    Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay. In: Clokie MRJ, Kropinski AM (eds) Bacteriophages methods and protocols. Volume 1: isolation, characterization, and interaction. Humana, Hertford, pp 69–76Google Scholar
  39. 39.
    Lake RJ, Cressey PJ, Campbell DM, Oakley E (2009) Risk ranking for foodborne microbial hazards in New Zealand: burden of disease estimates. Risk Anal 30:743–752PubMedCrossRefGoogle Scholar
  40. 40.
    Leonard C, Virijevic S, Regnier T, Combrinck S (2010) Bioactivity of selected essential oils and some components on Listeria monocytogenes biofilms. S Afr J Bot 76:676–680CrossRefGoogle Scholar
  41. 41.
    Leriche V, Chassaing D, Carpentier B (1999) Behaviour of L. monocytogenes in an artificially made biofilm of a nisin-producing strain of Lactococcus lactis. Int J Food Microbiol 51:169–182PubMedCrossRefGoogle Scholar
  42. 42.
    Lu TK, Collins JJ (2007) Dispersing biofilms with engineered enzymatic bacteriophage. Proc Natl Acad Sci 104:11197–11202PubMedCrossRefGoogle Scholar
  43. 43.
    Lundén J, Autio T, Markkula A, Hellström S, Korkeala H (2003) Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants. Int J Food Microbiol 82:265–272PubMedCrossRefGoogle Scholar
  44. 44.
    Lunden JM, Miettinen MK, Autio TJ, Korkeala HJ (2000) Persistent Listeria monocytogenes strains show enhanced adherence to food contact surface after short contact times. J Food Prot 63:1204–1207PubMedGoogle Scholar
  45. 45.
    Meyer R, Arpanaei A, Pillai S, Bernbom N, Enghild J, Ng Y, Gram L, Besenbacher F, Kingshott P (2013) Physicochemical characterization of fish protein adlayers with bacteria repelling properties. Colloid Surf B Biointerfaces 102:504–510. doi: 10.1016/j.colsurfb.2012.08.044 CrossRefGoogle Scholar
  46. 46.
    Micreos Food Safety (2009) Micreos food safety-LISTEX™. Accessed 30 July 2012
  47. 47.
    Montanez-Izquierdo VY, Salas-Vazquez DI, Rodriguez-Jerez JJ (2012) Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces. Food Control 23:470–477CrossRefGoogle Scholar
  48. 48.
    Palmer J, Flint S, Brooks J (2007) Bacterial cell attachment, the beginning of a biofilm. J Ind Microbiol Biotechnol 34:577–588PubMedCrossRefGoogle Scholar
  49. 49.
    Pan Y, Breidt F Jr, Kathariou S (2006) Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment. Appl Environ Microbiol 72:7711–7717PubMedCrossRefGoogle Scholar
  50. 50.
    Purkrtová S, Turoňová H, Pilchová T, Demnerová K, Pazlarová J (2010) Resistance of Listeria monocytogenes biofilms to disinfectants. Czech J Food Sci 28:326–332Google Scholar
  51. 51.
    Rashid MH, Revazishvili T, Dean T, Butani A, Verratti K, Bishop-Lilly KA, Sozhamannan S, Sulakvelidze A, Rajanna C (2012) A Yersinia pestis-specific, lytic phage preparation significantly reduces viable Y. pestis on various hard surfaces experimentally contaminated with the bacterium. Bacteriophage 2:168–177PubMedCrossRefGoogle Scholar
  52. 52.
    Roy B, Ackermann HW, Pandian S, Picard G, Goulet J (1993) Biological inactivation of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium compound. Appl Environ Microbiol 59:2914–2917PubMedGoogle Scholar
  53. 53.
    Scharff RL (2012) Economic burden from health losses due to foodborne illness in the United States. J Food Prot 75:123–131PubMedCrossRefGoogle Scholar
  54. 54.
    Sillankorva S, Neubauer P, Azeredo J (2008) Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A. BMC Biotechnol 8:79. doi: 10.1186/1472-6750-8-79 PubMedCrossRefGoogle Scholar
  55. 55.
    Sillankorva S, Neubauer P, Azeredo J (2010) Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus. Biofouling 26:567–575PubMedCrossRefGoogle Scholar
  56. 56.
    Sillankorva S, Oliveira R, Vieira MJ, Sutherland I, Azeredo J (2004) Bacteriophage Φ S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms. Biofouling 20:133–138PubMedCrossRefGoogle Scholar
  57. 57.
    Skandamis PN, Yoon Y, Stopforth JD, Kendall PA, Sofos JN (2008) Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses. Food Microbiol 25:294–303PubMedCrossRefGoogle Scholar
  58. 58.
    Solanki K, Grover N, Downs P, Paskaleva EE, Mehta KK, Lee L, Schadler LS, Kane RS, Dordick JS (2013) Enzyme-based listericidal nanocomposites. Sci Rep 3:1584. doi: 10.1038/srep01584 PubMedGoogle Scholar
  59. 59.
    Soni KA, Nannapaneni R (2010) Bacteriophage significantly reduces Listeria monocytogenes on raw salmon fillet tissue. J Food Prot 73:32–38PubMedGoogle Scholar
  60. 60.
    Soni KA, Nannapaneni R (2010) Removal of Listeria monocytogenes biofilms with bacteriophage P100. J Food Prot 73:1519–1524PubMedGoogle Scholar
  61. 61.
    Soni KA, Nannapaneni R, Hagens S (2010) Reduction of Listeria monocytogenes on the surface of fresh channel catfish fillets by bacteriophage Listex P100. Foodborne Pathog Dis 7:427–434. doi: 10.1089/fpd.2009.0432 PubMedCrossRefGoogle Scholar
  62. 62.
    Sulakvelidze A, Pasternack GR (2010) Industrial and regularity issues in bacteriophage applications in food production and processing. In: Sabour PM, Griffiths MW (eds) Bacteriophages in the control of food- and waterborne pathogens. ASM, Washington, DC, pp 297–326Google Scholar
  63. 63.
    Sutherland IW, Hughes KA, Skillman LC, Tait K (2006) The interaction of phage and biofilms. FEMS Microbiol Lett 232(1):1–6CrossRefGoogle Scholar
  64. 64.
    Tanji Y, Shimada T, Yoichi M, Miyanaga K, Hori K, Unno H (2004) Toward rational control of Escherichia coli O157:H7 by a phage cocktail. Appl Microbiol Biotechnol 64:270–274PubMedCrossRefGoogle Scholar
  65. 65.
    Tham W, Ericsson H, Loncarevic S, Unnerstad H, Danielsson-Tham ML (2000) Lessons from an outbreak of listeriosis related to vacuum-packed gravad and cold-smoked fish. Int J Food Microbiol 62:173–175PubMedCrossRefGoogle Scholar
  66. 66.
    Tompkin R (2002) Control of Listeria monocytogenes in the food-processing environment. J Food Prot 65:709–725PubMedGoogle Scholar
  67. 67.
    Tresse O, Lebret V, Benezech T, Faille C (2006) Comparative evaluation of adhesion, surface properties, and surface protein composition of Listeria monocytogenes strains after cultivation at constant pH of 5 and 7. J Appl Microbiol 101:53–62PubMedCrossRefGoogle Scholar
  68. 68.
    Truelstrup Hansen L, Vogel BF (2011) Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: survival and transfer to salmon products. Int J Food Microbiol 146:88–93CrossRefGoogle Scholar
  69. 69.
    Wirtanen G, Salo S (2003) Disinfection in food processing-efficacy testing of disinfectants. Rev Environ Sci Biotechnol 2:293–306CrossRefGoogle Scholar
  70. 70.
    Wong S, Street D, Delgado SI (2000) Recalls of foods and cosmetics due to microbial contamination reported to the US Food and Drug Administration. J Food Prot 63:1113–1116PubMedGoogle Scholar
  71. 71.
    Zhao T, Doyle MP, Zhao P (2004) Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms. Appl Environ Microbiol 70:3996–4003PubMedCrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2013

Authors and Affiliations

  • Geevika J. Ganegama Arachchi
    • 1
  • Andrew G. Cridge
    • 5
  • Beatrice M. Dias-Wanigasekera
    • 2
  • Cristina D. Cruz
    • 3
  • Lynn McIntyre
    • 4
  • Rachel Liu
    • 1
  • Steve H. Flint
    • 1
  • Anthony N. Mutukumira
    • 1
  1. 1.Institute of Food Nutrition and Human HealthMassey UniversityAucklandNew Zealand
  2. 2.Food Standards Australia New Zealand (FSANZ)Canberra BCAustralia
  3. 3.The New Zealand Institute for Plant and Food Research LimitedAucklandNew Zealand
  4. 4.Harper Adams UniversityShropshireUK
  5. 5.Laboratory for Evolution and Development, Biochemistry DepartmentUniversity of OtagoDunedinNew Zealand

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