Aim: The present study was conducted to evaluate the capacity of Listeria monocytogenes (L.m), Listeria innocua (L.i), and Escherichia coli to form biofilms on polystyrene support under different parameters by performing crystal violet (CV) staining technique.

Materials and Methods: Different suspensions were prepared with single strains and with multiple combinations of strains including two serogroups of L.m (IIa and IIb), L.i, and E. coli strains at different microbial load. Selected strains and combinations were grown in biofilms for 6 days attached to polystyrene microplates under aerobic and microaerophilic conditions. The evaluation of the power of adhesion and biofilm formation was determined by CV staining followed by the measurement of optical density at 24 h, 72 h, and 6 days incubation time with and without renewal of the culture medium.

Results: All the strains tested, presented more or less adhesion power depending on the variation of the studied parameters as well as the ability to form multispecies biofilms. Their development is more important by renewing the culture medium and increasing the initial load of bacteria. The ability to adhere and form biofilms differs from one serogroup to another within the same species. In bacterial combination, strains and species of bacteria adopt different behaviors.

Conclusion: The ability to form biofilms is a key factor in the persistence of tested strains in the environment. Our study showed that L.m, L.i, and E. coli could adhere to polystyrene and form biofilms under different conditions. More researches are necessary to understand the mechanisms of biofilm formation and the influence of different parameters in their development.

Keywords: biofilm, Escherichia coli, Listeria innocua, Listeria monocytogenes, polystyrene support, variation of parameters.

1. DuPont, G.A. (1997) Understanding dental plaque; Biofilm dynamics. J. Vet. Dent., 14(3): 91-94. [PubMed]

2. Parot, S. (2007) Biofilms Electroactifs: Formation, Caracterisation et Mecanismes. These de Doctorat, Specialite Genie des Procedes et de l'Environnement. p247.

3. Bougle, C. and Leroyer, R. (2003) Anti-infective coated central venous catheters: Technical aspects and clinical studies. J. Pharm. Clin., 22(3): 159-167.

4. Clutterbuck, A.L., Woods, E.J., Knottenbelt, D.C., Clegg, P.D., Cochrane, C.A. and Percival, S.L. (2007) Biofilms and their relevance to veterinary medicine. Vet. Microbiol., 121(1-2): 1-17. [Crossref]

5. Rodriguez, A., Autio, W.R. and McLandsborough, L.A. (2008) Effect of surface roughness and stainless steel finish on Listeria monocytogenes attachment and biofilm formation. J. Food Prot., 71(1): 170-175. [Crossref]

6. Chmielewski, R.A.N. and Frank, J.F. (2003) Biofilm formation and control in food processing facilities. Compr. Rev. Food Sci. Food Saf., 2(1): 22-32. [Crossref]

7. Chylkova, T., Cadena, M., Ferreiro, A. and Pitesky, M. (2017) Susceptibility of Salmonella biofilm and planktonic bacteria to common disinfectant agents used in poultry processing. J. Food Prot., 80(7): 1072-1079. [Crossref] [PubMed]

8. I-iguez-Moreno, M., Gutierrez-Lomeli, M., Guerrero-Medina, P.J. and Avila-Novoa, M.G. (2018) Biofilm formation by Staphylococcus aureus and Salmonella spp. Under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. Braz. J. Microbiol., 49(2): 310-319. [Crossref] [PubMed] [PMC]

9. Reis-Teixeira, F.B.D., Alves, V.F. and Martinis, E.C.P. (2017) Growth, viability and architecture of biofilms of Listeria monocytogenes formed on abiotic surfaces. Braz. J. Microbiol., 48(3): 587-591. [Crossref] [PubMed] [PMC]

10. Jeon, H.R., Kwon, M.J. and Yoon, K.S. (2018) Control of Listeria innocua biofilms on food contact surfaces with slightly acidic electrolyzed water and the risk of biofilm cells transfer to duck meat. J. Food Prot., 81(4): 582-592. [Crossref] [PubMed]

11. Govindaraj, D.K., Dev, K., Sadhana, R. and Jitendra, P. (2014) Comparative evaluation of factors affecting Escherichia coli biofilms on organic leafy greenwash water contact surface. J. Food Prot. Suppl., 77(1): 44-45.

12. Naito, M., Frirdich, E., Fields, J.A., Pryjma, M., Li, J., Cameron, A., Gilbert, M., Thompson, S.A. and Gaynor, E.C. (2010) Effects of sequential Campylobacter jejuni 81-176 lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis. J. Bacteriol., 192(8): 2182-2192. [Crossref] [PubMed] [PMC]

13. Rodrigues, L.B., Santos, L.R.D., Tagliari, V.Z., Rizzo, N.N., Trenhago, G., Oliveira, A.P.D., Goetz, F. and Nascimento, V.P.D. (2010) Quantification of biofilm production on polystyrene by Listeria, Escherichia coli and Staphylococcus aureus isolated from a poultry slaughterhouse. Braz. J. Microbiol., 41(4): 1082-1085. [Crossref]

14. Lane, P.S., Walker, J.T. and Hunter, P.R. (2000) Microbiological Aspects of Biofilms and Drinking Water. CRC Press, Boca Raton, FL. p164-168.

15. Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M. and Beachey, E.H. (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol., 22(6): 996-1006. [PubMed] [PMC]

16. Stepanovic, S., Vukovic, D., Dakic, I., Savic, B. and Svabic-Vlahovic, M. (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J. Microbiol. Methods, 40(2): 175-179. [Crossref]

17. Bouayad, L., Hamdi, T.M., Naim, M., Leclercq, A. and Lecuit, M. (2015) Prevalence of Listeria spp. and molecular characterization of Listeria monocytogenes isolates from broilers at the abattoir. Foodborne Pathog. Dis., 12(7): 606-611. [Crossref] [PubMed]

18. ISO. (2004) 18593-2004. Microbiology of Food and Animal Feeding Stuffs-Horizontal Methods for Sampling Techniques from Surfaces using Contact Plates and Swabs. ISO, Geneva, Switzerland.

19. ISO. (2006) 4832: 2006. Microbiology of Food and Animal Feeding Stuffs Horizontal Method for the Enumeration of Coliform Colony Count Technique. ISO, Geneva.

20. Peeters, E., Nelis, H.J. and Coenye, T. (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J. Microbiol. Methods, 72(2): 157-165. [Crossref] [PubMed]

21. Wang, J., Ray, A.J., Hammons, S.R. and Oliver, H.F. (2015) Persistent and transient Listeria monocytogenes strains from retail deli environments vary in their ability to adhere and form biofilms and rarely have inlA premature stop codons. Foodborne Pathog. Dis., 12(2): 151-158. [Crossref] [PubMed]

22. Lemon, K.P., Higgins, D.E. and Kolter, R. (2007) Flagellar motility is critical for Listeria monocytogenes biofilm formation. J. Bacteriol., 189(12): 4418-4424. [Crossref] [PubMed] [PMC]

23. Han, R., Klu, Y.A.K. and Chen, J. (2017) Attachment and biofilm formation by selected strains of Salmonella Enterica and enterohemorrhagic Escherichia coli of fresh produce origin. J. Food Sci., 82(6): 1461-1466. [Crossref] [PubMed]

24. Stepanovic, S., Vukovic, D., Hola, V., Bonaventura, G.D., Djukic, S., Cirkovic, I. and Ruzicka, F. (2007) Quantification of biofilm in microtiter plates: Overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Acta Pathol. Microbiol. Immunol. Scand., 115(8): 891-899. [Crossref] [PubMed]

25. Le Henaff, M., Huguet, N., Richard, A., Vaillant, M., Papillon, S. and Le Gallet, A. (2016) Evaluation de l'impact de nouvelles formulations nettoyantes qui associent des derives de pin et des enzymes sur la reduction du biofilm microbien. Hygienes, 24(6): 293-297.

26. Auger, M. (2012) Formation de Biofilm in vitro par des Souches d'Escherichia coli: Impacts de la Modification des Conditions Experimentales. These de Doctorat, Universite de Nantes, France. p90.

27. Hassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A. and Iqbal, M. (2011) Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz. J. Infect. Dis., 15(4): 305-311. [Crossref]

28. Kadam, S.R., Den Besten, H.M., Van Der Veen, S., Zwietering, M.H., Moezelaar, R. and Abee, T. (2013) Diversity assessment of Listeria monocytogenes biofilm formation: Impact of growth condition, serotype and strain origin. Int. J. Food Microbiol., 165(3): 259-264. [Crossref] [PubMed]

29. Renaud, J.F., Hansen, W. and Bollet, C. (1994) Manuel de Bacteriologie Clinique. ASM Press, Washington, DC. p833-849.

30. Rocourt, J. (1988) The recognition and identification of Listeria species by classical methods. Turk. J. Infect., 2(4): 471-485.

31. Borucki, M.K., Peppin, J.D., White, D., Loge, F. and Call, D.R. (2003) Variation in biofilm formation among strains of Listeria monocytogenes. Appl. Environ. Microbiol., 69(12): 7336-7342. [Crossref] [PMC]

32. Pan, Y., Breidt, F. and Kathariou, S. (2009) Competition of Listeria monocytogenes serotype 1/2a and 4b strains in mixed culture biofilms. Appl. Environ. Microbiol., 75(18): 5846-5852. [Crossref] [PubMed] [PMC]

33. Kalmokoff, M.L., Austin, J.W., Wan, X.D., Sanders, G., Banerjee, S. and Farber, J.M. (2001) Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions. J. Appl. Microbiol., 91(4): 725-734. [Crossref]

34. Robitaille, G., Choiniere, S., Ells, T., Deschenes, L. and Mafu, A.A. (2014) Attachment of Listeria innocua to polystyrene: Effects of ionic strength and conditioning films from culture media and milk proteins. J. Food Prot., 77(3): 427-434. [Crossref] [PubMed]

35. Azam, M., Jan, A.T., Kumar, A., Siddiqui, K., Mondal, A.H. and Haq, Q.M.R. (2018) Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna. Braz. J. Microbiol., 49(3): 471-480. [Crossref] [PubMed] [PMC]

36. Jeong, D.K. and Frank, J.F. (1994) Growth of Listeria monocytogenes at 10 C in biofilms with microorganisms isolated from meat and dairy processing environments. J. Food Prot., 57(7): 576-586. [Crossref]

37. Chen, D., Zhao, T. and Doyle, M.P. (2015) Control of pathogens in biofilms on the surface of stainless steel by levulinic acid plus sodium dodecyl sulfate. Int. J. Food Microbiol., 207: 1-7. [Crossref] [PubMed]

38. Buchanan, R.L., Gorris, L.G., Hayman, M.M., Jackson, T.C. and Whiting, R.C. (2017) A review of Listeria monocytogenes: An update on outbreaks, virulence, dose-response, ecology, and risk assessments. Food Control, 75: 1-13. [Crossref]

39. Romeo, T. (2008) Bacterial Biofilms. Vol. 322. Springer Science and Business Media, New York. p293. [Crossref]

40. Giaouris, E., Heir, E., Hebraud, M., Chorianopoulos, N., Langsrud, S., Moretro, T., Habimana, O., Desvaux, M., Renier, S. and Nychas, G.J. (2014) Attachment and biofilm formation by foodborne bacteria in meat processing environments: Causes, implications, role of bacterial interactions and control by alternative novel methods. Meat Sci., 97(3): 298-309. [Crossref] [PubMed]

41. Giaouris, E., Heir, E., Desvaux, M., Hebraud, M., Moretro, T., Langsrud, S., Doulgeraki, A., Nychas, G.J., Kacaniova, M., Czaczyk, K. and Olmez, H. (2015) Intra-and inter-species interactions within biofilms of important foodborne bacterial pathogens. Front. Microbiol., 6 : 841. [Crossref] [PubMed] [PMC]

42. Larsen, M.H., Dalmasso, M., Ingmer, H., Langsrud, S., Malakauskas, M., Mader, A., Moretro, T., Mozina, S.S., Rychli, K., Wagner, M. and Wallace, R.J. (2014) Persistence of foodborne pathogens and their control in primary and secondary food production chains. Food Control, 44: 92-109. [Crossref]

43. Jay, J.M., Loessner, M.J. and Golden, D.A. (2005) Modern Food Microbiology. Aspen Publishers, Gaithersburg, MD. p751.

44. Zeraik, A.E. and Nitschke, M. (2012) Influence of growth media and temperature on bacterial adhesion to polystyrene surfaces. Braz. Arch. Biol. Technol., 55(4): 569-576. [Crossref]

45. Bonsaglia, E.C.R., Silva, N.C.C., Junior, A.F., Junior, J.A., Tsunemi, M.H. and Rall, V.L.M. (2014) Production of biofilm by Listeria monocytogenes in different materials and temperatures. Food Control, 35(1): 386-391. [Crossref]