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Current Microbiology

, Volume 73, Issue 4, pp 474–482 | Cite as

Crystal Violet and XTT Assays on Staphylococcus aureus Biofilm Quantification

  • Zhenbo XuEmail author
  • Yanrui Liang
  • Shiqi Lin
  • Dingqiang Chen
  • Bing Li
  • Lin Li
  • Yang Deng
Article

Abstract

Staphylococcus aureus (S. Aureus) is a common food-borne pathogenic microorganism. Biofilm formation remains the major obstruction for bacterial elimination. The study aims at providing a basis for determining S. aureus biofilm formation. 257 clinical samples of S. aureus isolates were identified by routine analysis and multiplex PCR detection and found to contain 227 MRSA, 16 MSSA, 11 MRCNS, and 3 MSCNS strains. Two assays for quantification of S. aureus biofilm formation, the crystal violet (CV) assay and the XTT (tetrazolium salt reduction) assay, were optimized, evaluated, and further compared. In CV assay, most isolates formed weak biofilm 74.3 %), while the rest formed moderate biofilm (23.3 %) or strong biofilm (2.3 %). However, most isolates in XTT assay showed weak metabolic activity (77.0 %), while the rest showed moderate metabolic activity (17.9 %) or high metabolic activity (5.1 %). In this study, we found a distinct strain-to-strain dissimilarity in terms of both biomass formation and metabolic activity, and it was concluded from this study that two assays were mutual complementation rather than being comparison.

Keywords

Extracellular Polymeric Substance Crystal Violet Latex Agglutination Test Crystal Violet Assay Food Poisoning Case 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We gratefully acknowledge the National 973-Plan of China (2012CB720800), the National Natural Science Foundation of China (31201362 & 31101278), the International Science & Technology Cooperation Program (2013B051000014), the National Science and Technology Support Program (2012BAD37B01), the National Outstanding Doctoral Dissertation Funding (201459), the Guangdong Outstanding Doctoral Dissertation Funding (K3140030) andthe Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University (Dr. Zhenbo Xu, 2015).

References

  1. 1.
    Beenken KE, Dunman PM, McAleese F, Macapagal D, Murphy E, Projan SJ, Blevins JS, Smeltzer MS (2004) Global gene expression in Staphylococcus aureus biofilms. J Bacteriol 186(14):4665–4684CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Cattelan N, Dubey P, Arnal L, Yantorno OM, Deora R (2016) Bordetella biofilms: a lifestyle leading to persistent infections. Pathogens Dis 74(1):ftv108CrossRefGoogle Scholar
  3. 3.
    Christensen GD, Simpson W, Younger J, Baddour L, Barrett F, Melton D, Beachey E (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):996–1006PubMedPubMedCentralGoogle Scholar
  4. 4.
    Claessens J, Roriz M, Merckx R, Baatsen P, Van Mellaert L, Van Eldere J (2015) Inefficacy of vancomycin and teicoplanin in eradicating and killing Staphylococcus epidermidis biofilms in vitro. Int J Antimicrob Agents 45(4):368–375CrossRefPubMedGoogle Scholar
  5. 5.
    Corrigan RM, Rigby D, Handley P, Foster TJ (2007) The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation. Microbiology 153(8):2435–2446CrossRefPubMedGoogle Scholar
  6. 6.
    Costerton JW (1999) Introduction to biofilm. Int J Antimicrob Agents 11(3):217–221CrossRefPubMedGoogle Scholar
  7. 7.
    Cramton SE, Gerke C, Schnell NF, Nichols WW, Götz F (1999) The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect Immun 67(10):5427–5433PubMedPubMedCentralGoogle Scholar
  8. 8.
    Cucarella C, Solano C, Valle J, Amorena B, Lasa Í, Penadés JR (2001) Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 183(9):2888–2896CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    da Silva Meira QG, de Medeiros Barbosa I, Athayde AJAA, de Siqueira-Júnior JP, de Souza EL (2012) Influence of temperature and surface kind on biofilm formation by Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers. Food Control 25(2):469–475CrossRefGoogle Scholar
  10. 10.
    Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15(2):167–193CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Flemming H-C, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8(9):623–633PubMedGoogle Scholar
  12. 12.
    Gill SR, Fouts DE, Archer GL, Mongodin EF, DeBoy RT, Ravel J, Paulsen IT, Kolonay JF, Brinkac L, Beanan M (2005) Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol 187(7):2426–2438CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2(2):95–108CrossRefPubMedGoogle Scholar
  14. 14.
    Kong K-F, Vuong C, Otto M (2006) Staphylococcus quorum sensing in biofilm formation and infection. Int J Med Microbiol 296(2):133–139CrossRefPubMedGoogle Scholar
  15. 15.
    Kumar CG, Anand S (1998) Significance of microbial biofilms in food industry: a review. Int J Food Microbiol 42(1):9–27CrossRefPubMedGoogle Scholar
  16. 16.
    Lim Y, Shin HJ, Kwon AS, Reu JH, Park G, Kim J (2013) Predictive genetic risk markers for strong biofilm-forming Staphylococcus aureus: fnbB gene and SCCmec type III. Diagn Microbiol Infect Dis 76(4):539–541CrossRefPubMedGoogle Scholar
  17. 17.
    Mizan MFR, Jahid IK, Ha S-D (2015) Microbial biofilms in seafood: a food-hygiene challenge. Food Microbiol 49:41–55CrossRefPubMedGoogle Scholar
  18. 18.
    O’Neill E, Pozzi C, Houston P, Smyth D, Humphreys H, Robinson DA, O’Gara JP (2007) Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from device-related infections. J Clin Microbiol 45(5):1379–1388CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72(2):157–165CrossRefPubMedGoogle Scholar
  20. 20.
    Pitts B, Hamilton MA, Zelver N, Stewart PS (2003) A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods 54(2):269–276CrossRefPubMedGoogle Scholar
  21. 21.
    Potera C (1999) Forging a link between biofilms and disease. Science 283(5409):1837–1839CrossRefPubMedGoogle Scholar
  22. 22.
    Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48(17):4827–4833PubMedGoogle Scholar
  23. 23.
    Silva WJd, Seneviratne J, Parahitiyawa N, Rosa EAR, Samaranayake LP, Cury AADB (2008) Improvement of XTT assay performance for studies involving Candida albicans biofilms. Braz Den J 19(4):364–369Google Scholar
  24. 24.
    Simões M, Simões LC, Vieira MJ (2010) A review of current and emergent biofilm control strategies. LWT-Food Sci Technol 43(4):573–583CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Zhenbo Xu
    • 1
    • 2
    • 4
    Email author
  • Yanrui Liang
    • 1
  • Shiqi Lin
    • 1
  • Dingqiang Chen
    • 3
  • Bing Li
    • 1
  • Lin Li
    • 1
  • Yang Deng
    • 1
  1. 1.College of Food Science and TechnologySouth China University of TechnologyGuangzhouChina
  2. 2.Department of Biomedical ScienceUniversity of MarylandBaltimoreUSA
  3. 3.Department of Laboratory MedicineFirst Affiliated Hospital of Guangzhou Medical UniversityGuangzhouChina
  4. 4.College of Light Industry and Food ScienceSouth China University of TechnologyGuangzhouChina

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