Biofilm Formation by Salmonella Enteritidis at Different Incubation Temperatures


  • Laura Beatriz Rodrigues Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Bruna Webber Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Rafael Levandowski Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Sarah Souza Gehlen Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Luciana Ruschel dos Santos Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Fernando Pilotto Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Eduardo Cesar Tondo Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.
  • Vladimir Pinheiro do Nascimento Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil.



Background: The genus Salmonella, associated with poultry products, is considered the leading cause of foodborne outbreaks in humans in many countries. In Brazil, Salmonella Enteritidis (SE) is the serovar remains as one most frequently isolated from humans, and it is also a major serovar found in animals, food, animal feed, and environmental samples, despite all the efforts to control this pathogen. Also, the bacterium is able to form biofilms on different surfaces, protecting cells from both cleaning and sanitizing procedures in the food industries. This study aimed to verify the ability of Salmonella Enteritidis isolates to form biofilm on polystyrene at different incubation temperatures.

Materials, Methods & Results: A total of 171 SE samples were isolated from foodborne outbreaks (foods and stool cultures) and poultry products between 2003 and 2010. The biofilm-forming ability of samples was measured at four different temperatures (3°C, 9ºC, 25ºC, and 36ºC), for 24 h, simulating temperatures usually found in poultry slaughterhouses. Later, 200 μL of each bacterial suspension was inoculated, in triplicate, onto 96-well, flat-bottomed sterile polystyrene microtiter plates, washed, after that, the biofilm was fixed with methanol. The plates were dried at ambient temperature, stained with 2% Hucker’s crystal violet. Afterwards, absorbance was read using an ELISA plate reader and the optical density (OD) of each isolate was obtained by the arithmetic mean of the absorbance of three wells and this value was compared with the mean absorbance of negative controls (ODnc). The following classification was used for the determination of biofilm formation: no biofilm production, weak biofilm production, moderate biofilm production and strong biofilm production. Results demonstrated all isolates from stool cultures and foods involved in foodborne outbreaks, at least one of the four temperatures tested, were able to form biofilm, even at 3°C, undescribed as possible for the growth of SE. SE strains from poultry products also formed biofilm at least at one of the temperatures.

Discussion: the prevention of biofilms formation is very important, once they can be difficult to remove from utensils and food equipment surfaces, becoming a chronic source of microbial contamination of foods, possible dissemination of diseases, and increase of resistance to cleaning and sanitization procedures. A high ability for biofilm formation on plastic surfaces was observed. We may consider that Salmonella has the capacity to bind to surfaces, with relevant impacts on public health. Although biofilm formation could be affected by temperature, most of the SE isolates analyzed in our study were strong biofilm producers at all temperatures, including at 3°C, a temperature used for food preservation and until then not acknowledged as worrisome regarding the development of Salmonella spp. There is a common sense that maintenance of food at low temperatures, particularly below 5°C, is safer to consumers as low temperatures reduce microbial multiplication. However, our results show that the growth of SE in its sessile form is possible under refrigeration. These findings lead to the assumption that the ability of SE to form biofilms, especially at low temperatures, is related to its endurance in inhospitable environments, eventually infecting humans, and that may be one of the factors associated with the high prevalence of this serovar in outbreaks of foodborne diseases. To our knowledge, this is the first publication about biofilm formation by Salmonella Enteritidis at 3ºC.


Download data is not yet available.


Barnhart M.M. & Chapman M.R. 2006. Curli biogenesis and function. Annual Review of Microbiology. 60: 131-147.

Brazil. 1998. Portaria n° 210, de 10 de novembro de 1998. Regulamento Técnico da Inspeção Tecnológica e Higiênico-Sanitária de Carne de Aves. Brasília: Ministério da Agricultura, Pecuária e Abastecimento, 46p.

Cardoso A.L.S.P., Tessari E.N.C., Castro A.G.M. & Kanashiro A.M.I. 2000. Pesquisa de Salmonella spp., coliformes totais, coliformes fecais e mesófilos em carcaças e produtos derivados de frango. Arquivos do Instituto Biológico. 67: 25-30.

Carpentier B. & Cerf O. 1993. Biofilms and their consequences with particular reference to hygiene in the food industry. Journal of Applied Microbiology. 75: 99-511.

Carpentier B. 1997. Sanitary quality of meat chopping board surfaces: a bibliographical study. Food Microbiology. 14: 31-37.

Costalunga S. & Tondo E.C. 2002. Salmonellosis in Rio Grande do Sul, 1997 to 1999. Brazilian Journal of Microbiology. 33: 342-346.

Costerton J.W., Cheng K.J., Geesey G.G., Ladd T.I., Nickel J.C., Dasgupta M. & Marrie T.J. 1987. Bacterial biofilms in nature and disease. Annual Review of Microbiology. 41: 435-464.

Costerton J.W., Cheng K.J. & Geesey G.G. 1995. Bacterial biofilms in nature and disease. Annual Review of Microbiology. 49: 711-745.

Davey M.E. & O’Toole G.A. 2000. Microbial biofilms: from ecology to molecular genetics. Microbiology and Molecular Biology Reviews. 64: 847-867.

Donlan R.M. & Costerton J.M. 2002. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews. (15): 167-193.

Donlan R.M. 2002. Biofilms: microbial life on surfaces. Emerging Infectious Diseases. 8: 881-890.

Ebrahimi A., Hemati M., Dehkordi S.H., Bahadoran S., Khoshnood S., Khubani S., Faraj M. D. & Alni R.H. 2014. Chlorhexidine digluconate effects on planktonic growth and biofilm formation in some field isolates of animal bacterial pathogens. Jundishapur Journal of Natural Pharmaceutical Products. 9: 14298.

Galinari E., Nóbrega J.E., Andrade N.J. & Ferreira C.L.L.F. 2014. Microbiological aspects of the biofilm on wooden utensils used to make a Brazilian artisanal cheese. Brazilian Journal of Microbiology. 45: 713-720.

Gast R.K. 2008. Salmonella infections – Paratyphoid Infections. In: Saif Y.M., Fadly A.M., Glisson J.R., McDougald L.R., Nolan L.K., Swayne D.A. (Eds). Diseases of poultry. 12th edn. Ames: Blackwell Publishing Professional, pp.465-516.

Geimba M.P., Tondo E.C., Oliveira F.A., Canal C.W. & Brandelli A. 2004. Serological characterization and prevalence of spvR genes in Salmonella sp. isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul, south of Brazil. Journal of Food Protection. 67: 1229-1233.

Jessens B. & Lammert L. 2003. Biofilm and disinfection in meat processing plants. International Biodeterioration and Biodegradation. 51: 265-269.

Kumar G.C. & Anand S.K. 1998. Significance of microbial biofilm in food industry: a review. International Journal of Food Microbiology. 42: 9-27.

Lianou A. & Koutsoumanis K.P. 2012. Strain variability of the biofilm-forming ability of Salmonella enterica under various environmental conditions. International Journal of Food Microbiology. 160: 171-178.

Lima E.D.S.C.D., Pinto P.S.D.A., Santos J.L.D., Vanetti M.C.D., Bevilacqua P.D., Almeida L.P.D., Pinto M.S. & Dias F.S. 2004. Isolation of Salmonella and Staphylococcus aureus at swine slaughtering as subsidy for HACCP, the hazard analysis and critical control point system. Pesquisa Veterinária Brasileira. 24: 185-190.

Malheiros P.S., Paula C.M.D. & Tondo E.C. 2007. Cinética de crescimento de Salmonella Enteritidis envolvida em surtos alimentares no RS: uma comparação com linhagens de outros sorovares. Ciência e Tecnologia de Alimentos. 27: 751-755.

Marinho A.R., Martins P.D., Ditmer E.M., d'Azevedo P.A., Frazzon J., Van Der Sand S.T. & Frazzon A.P.G. 2013. Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food. Brazilian Journal of Microbiology. 44: 423-426.

Mead G.C. 1989. Hygienic problems and control of process contamination. In: Mead G.C. (Ed). Processing of poultry. New York: Elsevier, pp.360-368.

Melo P.C., Ferreira L.M., Nader A.F., Zafalon L.F., Vicente H.I.G. & Souza V. 2013. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis. Brazilian Journal of Microbiology. 44: 119-124.

Morey A. & Singh M. 2012. Low-temperature survival of Salmonella spp. in a model food system with natural microflora. Foodborne Pathogens and Disease. 9: 218-223.

Parizzi S.Q.F., Andrade N.J., Silva C.A.S. & Soares N.D.F.F. 2004. Bacterial adherence to different inert surfaces evaluated by epifluorescence microscopy and plate count method. Brazilian Archives of Biology and Technology. 47: 77-83.

Pitts B., Hamilton M.A., Zelver N. & Stewart P.S. 2003. A microtiter-plate screening method for biofilm disinfection and removal. Journal of Microbiological Methods. 54: 269-276.

Rode T.M., Langsrud S., Holck A. & Moretro T. 2007. Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions. International Journal of Food Microbiology. 116: 372-383.

Rodrigues L.B., Santos L.R.D., Rizzo N.N., Tagliari V.Z., Oliveira A.P.D., Trenhago G., Rodegheri S.C., Taglitti R.M., Dickel E.L. & Nascimento V. P.D. 2009. Avaliação da hidrofobicidade e da formação de biofilme em poliestireno por Salmonella Heidelberg isoladas de abatedouro avícola. Acta Scientiae Veterinariae. 37: 225-230.

Rodrigues L.B., Santos L.R.D., Rizzo N.N., Tagliari V.Z., Trenhago G., Oliveira A.P., Ferreira D., Pilotto F. & Nascimento V.P.D. 2013. Salmonella and Listeria from stainless steel, polyurethane and polyethylene surfaces in the cutting room of a poultry slaughterhouse. Acta Scientiae Veterinariae. 41: 1164.

Seixas R., Gabriel M., Machado J., Tavares L., Bernardo F. & Oliveira M. 2014. Effect of simulated gastrointestinal conditions on biofilm formation by Salmonella 1,4,[5],12:i:-. Scientific World Journal. ID (153956): 5.

Seixas R., Machado J., Bernardo F., Vilela C. & Oliveira M. 2014. Biofilm formation by Salmonella enterica serovar 1,4,[5],12:i:- Portuguese isolates: a phenotypic, genotypic, and sociogeographic analysis. Current Microbiology. 68: 670-677.

Steenackers H., Hermans K., Vanderleyden J. & Keersmaecker S.C.J. 2012. Salmonella biofilms: An overview on occurrence, structure, regulation and eradication. Food Research International. 45: 502-531.

Stepanovic S., Irkovic I.C., Ranin L. & Svabic-Vlahovic M. 2004. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Letters in Applied Microbiology. 38: 428-432.

Stepanovic S., Vukovic D., Dakic I., Savic B. & Vlahovic M.S.A. 2000. Modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods. 40: 175-179.

Stepanović S., Vuković D., Hola V., Bonaventura G.D., Djukić S., Ćirković I. & Ruzicka F. 2007. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 71: 687-690.

Wagner V.R., Silveira J.B. & Tondo E.C. 2013. Salmonelloses in the State of Rio Grande do Sul, southern Brazil, 2002 to 2004. Brazilian Journal of Microbiology. 44: 723-729.

Wang H., Ding S., Wang G., Xu X. & Zhou G. 2013. In situ characterization and analysis of Salmonella biofilm formation under meat processing environments using a combined microscopic and spectroscopic approach. International Journal of Food Microbiology. 167: 293-302.

WHO. 2017. World Health Organization. Health Topics: Salmonella. Disponível em: <>. [Accessed online in November 2017].

WHO. 2013. World Health Organization. Global Network Global Foodborne Infections Network. Global Salm Surv. Disponível em: <>[Accessed online in April 2016].

Yang Y., Kumar A., Zheng Q. & Yuk H.G. 2015. Preacclimation alters Salmonella Enteritidis surface properties and its initial attachment to food contact surfaces. Colloids and Surfaces B: Biointerfaces. 128: 577-585.



How to Cite

Rodrigues, L. B., Webber, B., Levandowski, R., Gehlen, S. S., Ruschel dos Santos, L., Pilotto, F., Tondo, E. C., & do Nascimento, V. P. (2019). Biofilm Formation by Salmonella Enteritidis at Different Incubation Temperatures. Acta Scientiae Veterinariae, 47(1).




Most read articles by the same author(s)

> >>