Journal of Industrial Microbiology & Biotechnology

, Volume 37, Issue 11, pp 1111–1119 | Cite as

The role of surface charge and hydrophobicity in the attachment of Anoxybacillus flavithermus isolated from milk powder

  • J. S. Palmer
  • S. H. Flint
  • J. Schmid
  • J. D. Brooks
Original Paper


The aim of the present study was to investigate the attachment mechanisms that enable the thermophile Anoxybacillus flavithermus (B12) to attach to stainless-steel surfaces. Passing a B12 culture through a column of stainless-steel chips, collecting the first cells to pass through, re-culturing, and repeating the process six times, resulted in the isolation of a mutant, labeled X7, with tenfold reduced ability to attach to stainless steel as well as a reduced ability to attach to plastic. A comparison of bacterial cell-surface properties indicated that X7 was less hydrophobic than its parental strain B12. Cell-surface charge measurements also suggest that X7 had a lower net-negative surface charge. Disruption of extracellular polysaccharides and DNA appeared to have no effect on the attachment process. Removal of surface proteins caused a reduction in attachment of both B12 and X7, suggesting surface protein involvement in attachment.


Anoxybacillus flavithermus Biofilm Dairy Surface charge Hydrophobicity 


  1. 1.
    Ahimou F, Denis FA, Touhami A, Dufrene YF (2002) Probing microbial cell surface charges by atomic force microscopy. Langmuir 18:9937–9941CrossRefGoogle Scholar
  2. 2.
    Ahimou F, Poquot M, Thonart P, Rouxhet PG (2001) Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilus. J Microbiol Methods 45:119–126CrossRefPubMedGoogle Scholar
  3. 3.
    Allesen-Holm M, Bundvig Barken K, Yang L, Klausen M, Webb JS, Kjelleberg S, Molin S, Givskov M, Tolker-Nielsen T (2006) A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59:1114–1128CrossRefPubMedGoogle Scholar
  4. 4.
    Arrizubieta MJ, Toledo-Arana A, Amorena B, Penadés JR, Lasa I (2004) Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus. J Bacteriol 186:7490–7498CrossRefPubMedGoogle Scholar
  5. 5.
    Briandet R, Meylheuc T, Maher C, Bellon-Fontaine MN (1999) Listeria monocytogenes Scott a: cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under different environmental growth conditions. Appl Environ Microbiol 65:5328–5333PubMedGoogle Scholar
  6. 6.
    Brugnoni LI, Lozano JE, Cubitto MA (2007) Potential of yeast isolated from apple juice to adhere to stainless-steel surfaces in the apple juice processing industry. Food Res Int 40:332–340CrossRefGoogle Scholar
  7. 7.
    Busscher HJ, van de Belt-Gritter B, Dijkstra RJB, Norde N, van der Mei HC (2008) Streptococcus mutans and Streptococcus intermedius adhesion to fibronectin films are oppositely influenced by ionic strength. Langmuir 24:10968–10973CrossRefPubMedGoogle Scholar
  8. 8.
    Busscher HJ, van de Belt-Gritter B, van der Mei HC (1995) Implications of microbial adhesion to hydrocarbons for evaluating cell surface hydrophobicity 1. Zeta potentials of hydrocarbon droplets. Colloids Surf B: Biointerfaces 5:111–116CrossRefGoogle Scholar
  9. 9.
    Calabi E, Calabi F, Phillips AD, Fairweather NF (2002) Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues. Infect Immun 70:5770–5778CrossRefPubMedGoogle Scholar
  10. 10.
    Cowan MM, Mikx FHM, Busscher HJ (1994) Electrophoretic mobility and haemagglutination of Treponema denticola ATCC33520. Colloids Surf B: Biointerfaces 2:407–410CrossRefGoogle Scholar
  11. 11.
    Cucarella C, Solano C, Valle J, Amorena B, Lasa I, Penades JR (2001) Bap a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 183:2888–2896CrossRefPubMedGoogle Scholar
  12. 12.
    Dall L, Herndon B (1989) Quantitative assay of glycocalyx produced by viridans group streptococci that cause endocarditis. J Clin Microbiol 27:2039–2041PubMedGoogle Scholar
  13. 13.
    Davies DG, Geesey GG (1995) Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. Appl Environ Microbiol 61:860–867PubMedGoogle Scholar
  14. 14.
    Doyle RJ (2000) Contribution of the hydrophobic effect to microbial infection. Microbes Infect 2:391–400CrossRefPubMedGoogle Scholar
  15. 15.
    Dubreuil JD, Logan SM, Cubbage S, Ni Eidhin D, McCubbin WD, Kay CM, Beveridge TJ, Ferris FG, Trust TJ (1988) Structural and biochemical analyses of a surface array protein of Campylobacter fetus. J Bacteriol 170:4165–4173PubMedGoogle Scholar
  16. 16.
    Dunne MW (2002) Bacterial adhesion—seen any good biofilms lately? Clin Microbiol Rev 15:155–166CrossRefPubMedGoogle Scholar
  17. 17.
    Flint SH, Brooks JD, Bremer PJ (1997) The influence of cell surface properties of thermophilic Streptococci on attachment to stainless steel. J Appl Microbiol 83:508–517CrossRefPubMedGoogle Scholar
  18. 18.
    Flint SH, Brooks JD, Bremer PJ (2000) Properties of the stainless-steel substrate influencing the adhesion of thermo-resistant streptococci. J Food Eng 43:235–242CrossRefGoogle Scholar
  19. 19.
    Flint SH, Palmer J, Bloemen K, Brooks J, Crawford R (2001) The growth of Bacillus stearothermophilus on stainless steel. J App Microbiol 90:151–157CrossRefGoogle Scholar
  20. 20.
    Flint SH, Ward LJH, Walker KMR (2001) Functional grouping of thermophilic Bacillus strains using amplification profiles of the 16S–23S internal spacer region. Syst Appl Microbiol 24:539–548CrossRefPubMedGoogle Scholar
  21. 21.
    Gilbert P, Evans DJ, Evans E, Duguid IG, Brown MRW (1991) Surface characteristics and adhesion of E.coli and Staphylococcus epidermidis. J Appl Bacteriol 71:72–77PubMedGoogle Scholar
  22. 22.
    Heilmann C, Gerke C, Perdreau-Remington F, Götz F (1996) Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun 64:277–282PubMedGoogle Scholar
  23. 23.
    Heilmann C, Götz F (1998) Further characterization of Staphlococcus epidermidis transposon mutants deficient in primary attachment or intercellular adhesion. Zentralblatt für Bakteriologie 287:69–83PubMedGoogle Scholar
  24. 24.
    Hermansson M (1999) The DLVO theory in microbial adhesion. Colloids Surf B: Biointerfaces 14:105–119CrossRefGoogle Scholar
  25. 25.
    Hood SK, Zottola EA (1995) Biofilms in food processing. Food Control 6:9–18CrossRefGoogle Scholar
  26. 26.
    Kervella M, Pagès JM, Pei Z, Grollier G, Blaser MJ, Fauchère J-L (1993) Isolation and characterization of two Campylobacter glycine-extracted proteins that bind to HeLa cell membranes. Infect & Immun 61:3440–3448Google Scholar
  27. 27.
    Kolenbrander PE (1982) Isolation and characterization of coaggregation-defective mutants of Actinomyces viscosus, Actinomyces naeslundii, and Streptococcus sanguis. Infect & Immun 37:1200–1208Google Scholar
  28. 28.
    Kuchma SL, O’Toole GA (2000) Surface-induced and biofilm induced changes in gene expression. Curr Opin Biotechnol 11:429–433CrossRefPubMedGoogle Scholar
  29. 29.
    Lejeune P (2003) Contamination of abiotic surfaces: what a colonizing bacterium sees and how to blur it. Trends Microbiol 11:179–184CrossRefPubMedGoogle Scholar
  30. 30.
    Liu Y, Yang S, Li Y, Xu H, Qin L, Tay J (2004) The influence of cell and substratum surface hydrophobicities on microbial attachment. J Bacteriol 110:251–256Google Scholar
  31. 31.
    Loo CY, Corliss DA, Ganeshkumar N (2000) Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J Bacteriol 182:1374–1382CrossRefPubMedGoogle Scholar
  32. 32.
    Marshall KC, Stout R, Mitchell R (1971) Mechanisms of the initial events in the absorption of marine bacteria to surfaces. J Gen Microbiol 68:337–348Google Scholar
  33. 33.
    Mosoni P, Gaillard-Martinie B (2001) Characterization of a spontaneous adhesion-defective mutant of Ruminococcus albus strain 20. Arch Microbiol 176:52–61CrossRefPubMedGoogle Scholar
  34. 34.
    Mozes N, Rouxhet PG (1987) Methods for measuring hydrophobicity of micro-organisms. J Microbiol Method 6:99–112CrossRefGoogle Scholar
  35. 35.
    Murphy PM, Lynch D, Kelly PM (1999) Growth of thermophilic spore forming bacilli in milk during the manufacture of low heat powders. Int J Dairy Techn 52:45–50CrossRefGoogle Scholar
  36. 36.
    Ong YL, Razatos A, Georgiou G, Sharma MM (1999) Adhesion forces between E.coli bacteria and biomaterial surfaces. Langmuir 15:2719–2725CrossRefGoogle Scholar
  37. 37.
    Parkar SG, Flint SH, Palmer JS, Brooks JD (2001) Factors influencing attachment of thermophilic bacilli to stainless steel. J Appl Microbiol 90:901–908CrossRefPubMedGoogle Scholar
  38. 38.
    Paul JH, Jeffrey WH (1985) Evidence for separate adhesion mechanisms for hydrophilic and hydrophobic surfaces in Vibrio proteolytica. Appl Environ Microbiol 50:431–437PubMedGoogle Scholar
  39. 39.
    Pedersen K (1980) Electrostatic interaction chromatography, a method for assaying the relative surface charges of bacteria. FEMS Microbiol Lett 12:365–367CrossRefGoogle Scholar
  40. 40.
    Peng JS, Tsai WC, Chou CC (2001) Surface characteristics of Bacillus cereus and its adhesion to stainless steel. Int J Food Microbiol 65:105–111CrossRefPubMedGoogle Scholar
  41. 41.
    Perni S, Aldsworth TG, Jordan SJ, Fernandes I, Barbosa M, Sol M, Tenreiro RP, Chambel L, Zilhão I, Barata B, Adrião A, Faleiro ML, Andrew PW, Shama G (2007) The resistance to detachment of dairy strains of Listeria monocytogenes from stainless steel by shear stress is related to the fluid dynamic characteristics of the location of isolation. Int J Food Microbiol 116:384–390CrossRefPubMedGoogle Scholar
  42. 42.
    Planchon S, Gaillard-Martinie B, Leroy S, Bellon-Fontaine MN, Fada S, Talon R (2007) Surface properties and behaviour on abiotic surfaces of Staphylococcus carnosus, a genetically homogeneous species. Food Microbiol 24:44–51CrossRefPubMedGoogle Scholar
  43. 43.
    Ronimus RS, Parker LE, Morgan HW (1997) The utilization of RAPD-PCR for identifying thermophilic and mesophilic Bacillus species. FEMS Microbiol Lett 147:75–79CrossRefPubMedGoogle Scholar
  44. 44.
    Rosenberg M, Gutnick D, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29–33CrossRefGoogle Scholar
  45. 45.
    Rückert A, Ronimus RS, Morgan HW (2004) A RADP-based survey of thermophilic bacilli in milk powders from different countries. Int J Food Microbiol 96:263–272CrossRefPubMedGoogle Scholar
  46. 46.
    Smyth CJ, Jonsson P, Olsson E, Soderland O, Rosengren J, Hjerten Adstrom T (1978) Differences in hydrophobic surface characteristics of porcine enteropathogenic E. coli with or without K88 antigen as revealed by hydrophobic interaction chromatography. Infect & Immun 22:462–472Google Scholar
  47. 47.
    Steinberger RE, Holden PA (2005) Extracellular DNA in single and multiple-species unsaturated biofilms. Appl Environ Microbiol 71:5404–5410CrossRefPubMedGoogle Scholar
  48. 48.
    Teixeira P, Lopes Z, Azeredo J, Oliveira R, Vieira MJ (2005) Physico-chemical surface characterization of a bacterial population isolated from a milking machine. Food Microbiol 22:247–251CrossRefGoogle Scholar
  49. 49.
    Tsuneda S, Aikawa H, Hayashi H, Yuasa A, Hirata A (2003) Extracellular polymeric substances responsible for bacterial adhesion onto solid surface. FEMS Microbiol Lett 223:287–292CrossRefPubMedGoogle Scholar
  50. 50.
    Ukuku DO, Fett WF (2002) Relationship of cell surface change and hydrophobicity to strength of attachment of bacterial to cantaloupe rind. J Food Prot 65:1093–1099PubMedGoogle Scholar
  51. 51.
    Van der Mei HC, Busscher HJ, Bos R, de Vries J, Boonaert CJP, Dufrene YF (2000) Direct probing by atomic force microscopy of the cell surface softness of an fibrillated and nonfibrillated oral streptococcal strain. Biophys J 78:2668–2674CrossRefGoogle Scholar
  52. 52.
    Van der Mei HC, De Vries J, Buscher H (1993) Hydrophobic and electrostatic cell surface properties of thermophilic dairy Streptococci. Appl Environ Microbiol 59:4305–4312PubMedGoogle Scholar
  53. 53.
    Whitchurch CB, Toker-Nielsen T, Raps PC, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295:1487CrossRefPubMedGoogle Scholar
  54. 54.
    Ziebuhr W, Krimmer V, Rachid S, Lößner I, Götz F, Hacker J (1999) A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 32:345–356CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  • J. S. Palmer
    • 1
  • S. H. Flint
    • 1
  • J. Schmid
    • 2
  • J. D. Brooks
    • 3
  1. 1.Institute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand
  2. 2.Institute of Molecular BiosciencesMassey UniversityPalmerston NorthNew Zealand
  3. 3.School of Applied SciencesAuckland University of TechnologyAucklandNew Zealand

Personalised recommendations