Effect of dry sanitizing methods on Bacillus cereus biofilm


Bacillus cereus is a relevant foodborne pathogen and biofilm producer which can contaminate and persist in the processing environment of both high and low water activity foods. Because of this, it is crucial to understand better the resistance of this pathogen biofilm to different sanitation methods. The aim of this study was to evaluate the efficacy of dry sanitizing treatments against B. cereus biofilm formed on stainless steel (SS) and polypropylene (PP). Biofilm formation was held through the static method at 25 °C. After 4 days of incubation, coupons were exposed for up to 30 min to UV-C light, dry heat, gaseous ozone, 70% ethanol, and a commercial sanitizer. Sodium hypochlorite (200 mg/l) was also tested in two different pH values (7 and 11) for comparison purposes. In general, the surface material did not influence (p > 0.05) the performance of the treatments. From 10 min of exposure, 70% ethanol and the commercial product caused the lowest reductions on both surfaces. In addition, dry heat exhibited a poor performance on PP, with reductions < 1 log CFU/cm2. UV-C light on SS and PP and ozone on PP achieved reductions around 2 log CFU/cm2 after 30 min. The same level of reduction was obtained after 5 or 10 min using sodium hypochlorite (200 mg/l). Therefore, the results showed that dry sanitizing methods are not as effective as sodium hypochlorite against B. cereus biofilms. Further studies to evaluate the efficacy of the combination of dry methods are necessary.

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Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.


  1. 1.

    Peng JS, Tsai WC, Chou CC (2002) Inactivation and removal of Bacillus cereus by sanitizer and detergent. Int J Food Microbiol 77:11–18. https://doi.org/10.1016/S0168-1605(02)00060-0

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Codex (2015) Code of Hygienic Practice for Low-Moisture Foods. CAC/RCP:75–2015

  3. 3.

    Simões M, Simões LC, Vieira MJ (2010) A review of current and emergent biofilm control strategies. LWT 43:573–583. https://doi.org/10.1016/j.lwt.2009.12.008

    CAS  Article  Google Scholar 

  4. 4.

    Glasset B, Herbin S, Guillier L, Cadel-Six S, Vignaud M, Grout J, Pairaud S, Michel V, Hennekinne J, Ramarao N, Brisabois A (2016) Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: Epidemiology and genetic characterisation. Eurosurveillance 21(48). https://doi.org/10.2807/1560-7917.ES.2016.21.48.30413

  5. 5.

    Majed R, Faille C, Kallassy M, Gohar M (2016) Bacillus cereus biofilms-same, only different. Front Microbiol 7:1–16. https://doi.org/10.3389/fmicb.2016.01054

    Article  Google Scholar 

  6. 6.

    Faille C, Bénézech T, Midelet-Bourdin G, Lequette Y, Clarisse M, Ronse G, Ronse A, Slomianny C (2014) Sporulation of Bacillus spp. within biofilms: a potential source of contamination in food processing environments. Food Microbiol 40:64–74. https://doi.org/10.1016/j.fm.2013.12.004

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Hussain MS, Oh DH (2017) Substratum attachment location and biofilm formation by Bacillus cereus strains isolated from different sources: effect on total biomass production and sporulation in different growth conditions. Food Control 77:270–280. https://doi.org/10.1016/j.foodcont.2017.02.014

    CAS  Article  Google Scholar 

  8. 8.

    Kwon M, Hussain MS, Oh DH (2017) Biofilm formation of Bacillus cereus under food-processing-related conditions. Food Sci Biotechnol 26(4):1103–1111. https://doi.org/10.1007/s10068-017-0129-8

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Huang Y, Flint SH, Palmer JS (2020) Bacillus cereus spores and toxins –The potential role of biofilms. Food Microbiol 90:103493. https://doi.org/10.1016/j.fm.2020.103493

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Wijman JG, de Leeuw PP, Moezelaar R, Zwietering MH, Abee T (2007) Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion. Appl Environ Microbiol 73(5):1481–1488. https://doi.org/10.1128/AEM.01781-06

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Hussain MS, Oh DH (2018) Impact of the isolation source on the biofilm formation characteristics of Bacillus cereus. J Microbiol Biotechnol 28(1):77–86. https://doi.org/10.4014/jmb.1707.07023

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Marchand S, De Block J, De Jonghe V, Coorevits A, Heyndrickx M, Herman L (2012) Biofilm formation in milk production and processing environments; influence on milk quality and safety. Compr Rev Food Sci Food Saf 11(2):133–147. https://doi.org/10.1111/j.1541-4337.2011.00183.x

    CAS  Article  Google Scholar 

  13. 13.

    Pagedar A, Singh J (2012) Influence of physiological cell stages on biofilm formation by Bacillus cereus of dairy origin. Int Dairy J 23(1):30–35. https://doi.org/10.1016/j.idairyj.2011.10.009

    CAS  Article  Google Scholar 

  14. 14.

    Tewari A, Abdullah S (2015) Bacillus cereus food poisoning: International and Indian perspective. J Food Sci Technol 52(5):2500–2511. https://doi.org/10.1007/s13197-014-1344-4

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    European Food Safety Authority (2009) The community summary report on foodborne outbreaks in the European Union in 2007. EFSA J 7(5):271r. https://doi.org/10.2903/j.efsa.2009.271r

    Article  Google Scholar 

  16. 16.

    Centers for Disease Control and Prevention (2012) Foodborne outbreak online database. http://www.cdc.gov/foodborneoutbreaks/. Accessed 2 Aug 2018

  17. 17.

    Gurtler JB, Doyle MP, Kornacki JL (2014) The Microbiological Safety of Low Water Activity Foods and Spices. Springer, New York

    Google Scholar 

  18. 18.

    Thomas P (2012) Long-term survival of Bacillus spores in alcohol and identification of 90% ethanol as relatively more spori/bactericidal. Curr Microbiol 64(2):130–139. https://doi.org/10.1007/s00284-011-0040-0

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Kim H, Moon MJ, Kim CY, Ryu K (2019) Efficacy of chemical sanitizers against Bacillus cereus on food contact surfaces with scratch and biofilm. Food Sci Biotechnol 28(2):581–590. https://doi.org/10.1007/s10068-018-0482-2

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Ha JIH, Ha SD (2010) Synergistic effects of ethanol and UV radiation to reduce levels of selected foodborne pathogenic bacteria. J Food Prot 73(3):556–561. https://doi.org/10.4315/0362-028X-73.3.556

    Article  PubMed  Google Scholar 

  21. 21.

    Fernandes MS, Kabuki DY, Kuaye AY (2015) Biofilms of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta and the control of these pathogens through cleaning and sanitization procedures. Int J Food Microbiol 200:97–103. https://doi.org/10.1016/j.ijfoodmicro.2015.02.004

    CAS  Article  Google Scholar 

  22. 22.

    Charaf UK, Bakich SL, Falbo DM (1999) A model biofilm for efficacy assessment of antimicrobials versus biofilm bacteria. In: Biofilms The Good, The Bad and The Ugly ed. Wimpenny J, Gilbert P, Walker J, Brading M, Bayston R BioLine, Cardiff

  23. 23.

    Oja T, Blomqvist B, Buckingham-Meyer K, Goeres D, Vuorela P, Fallarero A (2014) Revisiting an agar-based plate method: what the static biofilm method can offer for biofilm research. J Microbiol Methods 107:157–160. https://doi.org/10.1016/j.mimet.2014.10.008

    Article  PubMed  Google Scholar 

  24. 24.

    Brazilian National Standards Organization. ABNT NBR 9425 (2005) Sodium hypochlorite - Determination of active chlorine - Volumetric method. ABNT, Rio de Janeiro

  25. 25.

    British Standard - BS EN 1040 (2005) Chemical disinfectants and antiseptics. Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics. Test method and requirements (phase 1). British Standards Institution

  26. 26.

    Nicholas R, Dunton P, Tatham A, Fielding L (2013) The effect of ozone and open air factor on surface-attached and biofilm environmental Listeria monocytogenes. J Appl Microbiol 115(2):555–564. https://doi.org/10.1111/jam.12239

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Andrade NJD (2008) Higienização na Industria de Alimentos: Avaliação e controle da adesão e formação de biofilmes bacterianos. Varela, São Paulo

  28. 28.

    Sommers CH, Sites JE, Musgrove M (2010) Ultraviolet light (254 nm) inactivation of pathogens on foods and stainless steel surfaces. J Food Saf 30:470–479. https://doi.org/10.1111/j.1745-4565.2010.00220.x

    Article  Google Scholar 

  29. 29.

    Rutala, WA, Weber DJ, Healthcare Infection Control Practices Advisory Committee (HICPAC) (2008) Guideline for disinfection and sterilization in healthcare facilities, Centers for Disease Control and Prevention

  30. 30.

    Chojecka A, Tarka P, Kierzkowska A, Nitsch-Osuch A, Kanecki K (2017) Neutralization efficiency of alcohol based products used for rapid hand disinfection. Rocz Panstw Zakl Hig 68:389–394

    CAS  PubMed  Google Scholar 

  31. 31.

    Giaouris E, Chorianopoulos N, Nychas GJE (2005) Effect of Temperature, pH, and water activity on biofilm formation by Salmonella enterica enteritidis PT4 on stainless steel surfaces as indicated by the bead vortexing method and conductance measurements. J Food Prot 68(10):2149–2154. https://doi.org/10.4315/0362-028X-68.10.2149

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    European Standard EN ISO 7932 (2004) Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of presumptive Bacillus cereus - Colony-count technique at 30°C (ISO 7932:2004)

  33. 33.

    Ronner AB, Wong ACL (1993) Biofilm development and sanitizer inactivation of Listeria monocytogenes and Salmonella typhimurium on stainless steel and Buna-N rubber. J Food Prot 56:750–758. https://doi.org/10.4315/0362-028X-56.9.750

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Ryu JH, Beuchat LR (2005) Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid–based sanitizer. J Food Prot 68(12):2614–2622. https://doi.org/10.4315/0362-028X-68.12.2614

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Fernandes MS, Fujimoto G, Schneid I, Kabuki DY, Kuaye AY (2014) Enterotoxigenic profile, antimicrobial susceptibility, and biofilm formation of Bacillus cereus isolated from ricotta processing. Int Dairy J 38(1):16–23. https://doi.org/10.1016/j.idairyj.2014.03.009

    CAS  Article  Google Scholar 

  36. 36.

    Cronin UP, Wilkinson MG (2008) Physiological response of Bacillus cereus vegetative cells to simulated food processing treatments. J Food Prot 71(11):2168–2176. https://doi.org/10.4315/0362-028X-71.11.2168

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Epstein AK, Pokroy B, Seminara A, Aizenberg J (2011) Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration. P Natl Acad Sci USA 108:995–1000. https://doi.org/10.1073/pnas.1011033108/-/DCSupplemental

    CAS  Article  Google Scholar 

  38. 38.

    Almatroudi A, Tahir S, Hu H, Chowdhury D, Gosbell IB, Jensen SO, Whiteley GS, Deva AK, Glasbey T, Vickery K (2018) Staphylococcus aureus dry-surface biofilms are more resistant to heat treatment than traditional hydrated biofilms. J Hosp Infect 98:161–167. https://doi.org/10.1016/j.jhin.2017.09.007

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Bae YM, Lee SY (2012) Inhibitory effects of UV treatment and a combination of UV and dry heat against pathogens on stainless steel and polypropylene surfaces. J Food Sci 77(1):61–64. https://doi.org/10.1111/j.1750-3841.2011.02476.x

    CAS  Article  Google Scholar 

  40. 40.

    Bernbom N, Vogel BF, Gram L (2011) Listeria monocytogenes survival of UV-C radiation is enhanced by presence of sodium chloride, organic food material and by bacterial biofilm formation. Int J Food Microbiol 147(1):69–73. https://doi.org/10.1016/j.ijfoodmicro.2011.03.009

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Kuda T, Iwase T, Chaturongkasumrit Y, Takahashi H, Koyanagi T, Kimura B (2012) Resistances to UV-C irradiation of Salmonella Typhimurium and Staphylococcus aureus in wet and dried suspensions on surface with egg residues. Food Control 23(2):485–490. https://doi.org/10.1016/j.foodcont.2011.08.018

    Article  Google Scholar 

  42. 42.

    Shikano A, Kuda T, Takahashi H, Kimura B (2017) Effect of quantity of food residues on resistance to desiccation, disinfectants, and UV-C irradiation of spoilage yeasts adhered to a stainless steel surface. LWT 80:169–177. https://doi.org/10.1016/j.lwt.2017.02.020

    CAS  Article  Google Scholar 

  43. 43.

    Bucheli-Witschel M, Bassin C, Egli T (2010) UV-C inactivation in Escherichia coli is affected by growth conditions preceding irradiation, in particular by the specific growth rate. J Appl Microbiol 109(5):1733–1744. https://doi.org/10.1111/j.1365-2672.2010.04802.x

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Gayán E, Álvarez I, Condón S (2013) Inactivation of bacterial spores by UV-C light. Innov Food Sci Emerg 19:140–145. https://doi.org/10.1016/j.ifset.2013.04.007

    CAS  Article  Google Scholar 

  45. 45.

    Food and Drug Administration (2001) Guidance and regulation: food code. U.S. Department of Health and Human Services

  46. 46.

    Megahed A, Aldridge B, Lowe J (2018) The microbial killing capacity of aqueous and gaseous ozone on different surfaces contaminated with dairy cattle manure. PLoS One 13:e0196555. https://doi.org/10.1371/journal.pone.0196555

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Sharma M, Hudson JB (2008) Ozone gas is an effective and practical antibacterial agent. Am J Infect Control 36(8):559–563. https://doi.org/10.1016/j.ajic.2007.10.021

    Article  PubMed  Google Scholar 

  48. 48.

    Fukuzaki S, Urano H, Yamada S (2007) Effect of pH on the efficacy of sodium hypochlorite solution as cleaning and bactericidal agents. J Surface Finish Soc Jpn 58(8):465–465. https://doi.org/10.4139/sfj.58.465

    CAS  Article  Google Scholar 

  49. 49.

    Ryu JH, Beuchat LR (2005) Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and curli production on its resistance to chlorine. Appl Environ Microbiol 71(1):247–254. https://doi.org/10.1128/AEM.71.1.247-254.2005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    AOAC International (2013) Official Method 960.06 Germicidal and detergent sanitizing action of disinfectants. AOAC Official Methods of Analysis. Standard Methods for the Examination of Water and Wastewater (2005) 21st Ed., American Public Health Association, Washington, DC

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The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for providing the financial support for scholarship (CNPq–Process 132828/2018-9). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–Finance Code 001.


This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) (CNPq–Process 132828/2018-9). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–Finance Code 001.

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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Andréia Miho Morishita Harada. The first draft of the manuscript was written by Andréia Miho Morishita Harada and Maristela Silva Nascimento, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Maristela Silva Nascimento.

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Harada, A.M.M., Nascimento, M.S. Effect of dry sanitizing methods on Bacillus cereus biofilm. Braz J Microbiol (2021). https://doi.org/10.1007/s42770-021-00451-0

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  • Bacillus cereus
  • Biofilm
  • Dry sanitation
  • Food hygiene
  • Food contact surfaces