Folia Microbiologica

, Volume 64, Issue 4, pp 567–577 | Cite as

Quantitative evaluation of biofilm extracellular DNA by fluorescence-based techniques

  • Martina BoháčováEmail author
  • Jarmila Pazlarová
  • Viviana Fuchsová
  • Tereza Švehláková
  • Kateřina Demnerová
Original Article


The formation of a hardly removable biofilm in food processing and clinical settings calls for a deeper understanding of composition of the matrix that protects the biofilm cells, as the crucial matrix component is extracellular DNA (eDNA), participating in adhesion, aggregation and penetration reduction, yet serving as a horizontal gene transfer reservoir. Therefore, we evaluated eDNA release from the biofilm of two pathogens, Listeria monocytogenes and Staphylococcus aureus, with respect to their origin under different culturing condition. Primarily, the biofilms were observed by confocal laser scanning microscopy (CLSM) under conditions mimicking the food processing environment and human body. The eDNA was quantitatively characterised based on its area by IMARIS. Next, the eDNA content and biofilm formation were quantified by spectrophotometry. Data from both sets of experiments were statistically evaluated. The eDNA release varied between the microorganism, culturing conditions and the origin of strains. Independent of the method used, the clinical strains of S. aureus released more eDNA than the food related strains at 37 °C. eDNA content can be crucial discriminating matrix component between food related and clinical strains. Deeper understanding of the eDNA role in such a phenomenon could facilitate the design of effective strategy for biofilm disruption.



We would like to acknowledge data processing consultations with Ing. Robert Šulák.

Funding information

This work received financial support from specific university research MSMT No. 20-SVV/2016 and the Czech Science Foundation GAČR 17-15936S.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12223_2019_681_MOESM1_ESM.docx (15 kb)
ESM 1 (DOCX 15 kb)


  1. Alibayov B, Zdenkova K, Purkrtova S, Demnerova K, Karpiskova R (2014) Detection of some phenotypic and genotypic characteristics of Staphylococcus aureus isolated from food items in the Czech Republic. Ann Microbiol 64:1587–1596. CrossRefGoogle Scholar
  2. Antunes LCM, Ferreira RBR (2011) Biofilms and bacterial virulence. Rev Med Microbiol 22:12–16. CrossRefGoogle Scholar
  3. Barbosa J, Borges S, Camilo R, Magalhães R, Ferreira V, Santos I, Silva J, Almeida G, Teixeira P (2013) Biofilm formation among clinical and food isolates of listeria monocytogenes. Int J Microbiol 2013:524975. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bockelmann U, Janke A, Kuhn R, Neu TR, Wecke J, Lawrence JR, Szewzyk U (2006) Bacterial extracellular DNA forming a defined network-like structure. FEMS Microbiol Lett 262:31–38. CrossRefPubMedGoogle Scholar
  5. Boháčová M, Zdeňková K, Tomáštíková Z, Fuchsová V, Demnerová K, Karpíšková R, Pazlarová J (2018) Monitoring of resistance genes in Listeria monocytogenes isolates and their presence in the extracellular DNA of biofilms: a case study from the Czech Republic. Folia Microbiol 63:653–664. CrossRefGoogle Scholar
  6. Brackman G et al (2016) The quorum sensing inhibitor hamamelitannin increases antibiotic susceptibility of Staphylococcus aureus biofilms by affecting peptidoglycan biosynthesis and eDNA release. Sci Rep-Uk 6:ARTN 20321. CrossRefGoogle Scholar
  7. Bridier A, Dubois-Brissonnet F, Boubetra A, Thomas V, Briandet R (2010) The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM method. J Microbiol Methods 82:64–70. CrossRefPubMedGoogle Scholar
  8. Carpentier B, Cerf O (2011) Review—persistence of Listeria monocytogenes in food industry equipment and premises. Int J Food Microbiol 145:1–8. CrossRefPubMedGoogle Scholar
  9. Chiang WC, Nilsson M, Jensen PO, Hoiby N, Nielsen TE, Givskov M, Tolker-Nielsen T (2013) Extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 57:2352–2361. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Das T, Krom BP, van der Mei HC, Busscher HJ, Sharma PK (2011) DNA-mediated bacterial aggregation is dictated by acid-base interactions. Soft Matter 7:2927–2935. CrossRefGoogle Scholar
  11. Das T, Sehar S, Koop L, Wong YK, Ahmed S, Siddiqui KS, Manefield M (2014) Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation. PLoS One 9:e91935. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Doyle ME, Ph D (2001) Virulence characteristics of Listeria monocytogenes. Fri Briefings 1–13 available online Accessed 3 Sept 2018
  13. EFSA E (2017) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. EFSA J 15:e05077. CrossRefGoogle Scholar
  14. Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633. CrossRefPubMedGoogle Scholar
  15. Gloag ES, Turnbull L, Huang A, Vallotton P, Wang H, Nolan LM, Mililli L, Hunt C, Lu J, Osvath SR, Monahan LG, Cavaliere R, Charles IG, Wand MP, Gee ML, Prabhakar R, Whitchurch CB (2013) Self-organization of bacterial biofilms is facilitated by extracellular DNA. Proc Natl Acad Sci U S A 110:11541–11546. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Guilbaud M, Piveteau P, Desvaux M, Brisse S, Briandet R (2015) Exploring the diversity of Listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype. Appl Environ Microbiol 81:1804–1810. CrossRefGoogle Scholar
  17. Harmsen M, Lappann M, Knøchel S (2010) Role of extracellular DNA during biofilm formation by Listeria monocytogenes. 76:2271–2279.
  18. Jakubovics NS, Shields RC, Rajarajan N, Burgess JG (2013) Life after death: the critical role of extracellular DNA in microbial biofilms. Lett Appl Microbiol 57:467–475. CrossRefPubMedGoogle Scholar
  19. Janakiraman V (2008) Listeriosis in pregnancy: diagnosis, treatment, and prevention. Rev Obstet Gynecol 1:179–185PubMedPubMedCentralGoogle Scholar
  20. Johansson J, Mandin P, Renzoni A, Chiaruttini C, Springer M, Cossart P (2002) An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110:551–561. CrossRefPubMedGoogle Scholar
  21. Kadam SR, den Besten HMW, van der Veen S, Zwietering MH, Moezelaar R, Abee T (2013) Diversity assessment of Listeria monocytogenes biofilm formation: impact of growth condition, serotype and strain origin. Int J Food Microbiol 165:259–264. CrossRefPubMedGoogle Scholar
  22. Khajotia SS, Smart KH, Pilula M, Thompson DM (2013) Concurrent quantification of cellular and extracellular components of biofilms. Jove-J Vis ExpGoogle Scholar
  23. Konkel ME, Tilly K (2000) Temperature-regulated expression of bacterial virulence genes. Microbes Infect 2:157–166CrossRefPubMedGoogle Scholar
  24. Lee JH, Kim YG, Lee K, Kim SC, Lee J (2015) Temperature-dependent control of Staphylococcus aureus biofilms and virulence by thermoresponsive oligo (N-vinylcaprolactam). Biotechnol Bioeng 112:716–724. CrossRefPubMedGoogle Scholar
  25. Lemon KP, Freitag NE, Kolter R (2010) The virulence regulator PrfA promotes biofilm formation by Listeria monocytogenes. J Bacteriol 192:3969–3976. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Meyer C, Fredriksson-Ahomaa M, Kleta S, Ellerbroek L, Thiel S, Martlbauer E (2012) Occurrence of L. monocytogenes in ready-to-eat poultry products available on the German market. Food Res Int 48:944–947. CrossRefGoogle Scholar
  27. Montanaro L, Poggi A, Visai L, Ravaioli S, Campoccia D, Speziale P, Arciola CR (2011) Extracellular DNA in biofilms. Int J Artif Organs 34:824–831. CrossRefPubMedGoogle Scholar
  28. Ng M et al (2014) Induction of MRSA biofilm by low-dose beta-lactam antibiotics: specificity, prevalence and dose-response effects. Dose Response 12:152–161CrossRefPubMedGoogle Scholar
  29. Nguyen UT, Burrows LL (2014) DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms. Int J Food Microbiol 187:26–32. CrossRefPubMedGoogle Scholar
  30. Nilsson RE, Ross T, Bowman JP (2011) Variability in biofilm production by Listeria monocytogenes correlated to strain origin and growth conditions. Int J Food Microbiol 150:14–24. CrossRefPubMedGoogle Scholar
  31. Okshevsky M, Meyer RL (2013) The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Crit Rev Microbiol 41:341–352. CrossRefPubMedGoogle Scholar
  32. Okshevsky M, Meyer RL (2014) Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms. J Microbiol Methods 105:102–104. CrossRefPubMedGoogle Scholar
  33. Oniciuc EA, Cerca N, Nicolau AI (2016) Compositional analysis of biofilms formed by Staphylococcus aureus isolated from food sources. Front Microbiol 7:390. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pan Y, Jr FB, Kathariou S, Breidt F (2006) Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment. 72:7711–7717.
  35. Rajendran R et al. (2014) Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation BMC Microbiol 14:ARTN 303
  36. Ravaioli S, Campoccia D, Visai L, Pirini V, Cangini I, Corazzari T, Maso A, Poggio C, Pegreffi F, Montanaro L, Arciola CR (2011) Biofilm extracellular-DNA in 55 Staphylococcus epidermidis clinical isolates from implant infections. Int J Artif Organs 34:840–846. CrossRefPubMedGoogle Scholar
  37. Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE, Smeltzer MS, Bayles KW (2007) The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc Natl Acad Sci U S A 104:8113–8118. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Rode TM, Langsrud S, Holck A, Moretro T (2007) Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions. Int J Food Microbiol 116:372–383. CrossRefPubMedGoogle Scholar
  39. Steinberger RE, Holden PA (2005) Extracellular DNA in single- and multiple-species unsaturated biofilms. Appl Environ Microbiol 71:5404–5410. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, Mizunoe Y (2018) Broad impact of extracellular DNA on biofilm formation by clinically isolated methicillin-resistant and -sensitive strains of Staphylococcus aureus. Sci Rep 8:2254. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Tang L, Schramm A, Neu TR, Revsbech NP, Meyer RL (2013) Extracellular DNA in adhesion and biofilm formation of four environmental isolates: a quantitative study. FEMS Microbiol Ecol 86:394–403. CrossRefPubMedGoogle Scholar
  42. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG (2015) Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28:603–661. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tresse O, Shannon K, Pinon A, Malle P, Vialette M, Midelet-Bourdin G (2007) Variable adhesion of listeria monocytogenes isolates from food-processing facilities and clinical cases to inert surfaces. J Food Prot 70:1569–1578CrossRefPubMedGoogle Scholar
  44. van Hal SJ, Jensen SO, Vaska VL, Espedido BA, Paterson DL, Gosbell IB (2012) Predictors of mortality in Staphylococcus aureus bacteremia. Clin Microbiol Rev 25:362–386. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wu J, Xi C (2009) Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. Appl Environ Microbiol 75:5390–5395. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Yin JM, Liu ZT, Zhao SC, Guo YJ (2013) Diagnosis, management, and prevention of prosthetic joint infections. Front Biosci 18:1349–1357CrossRefGoogle Scholar
  47. Zatorska B, Groger M, Moser D, Diab-Elschahawi M, Lusignani LS, Presterl E (2017) Does extracellular DNA production vary in staphylococcal biofilms isolated from infected implants versus controls? Clin Orthop Relat Res 475:2105–2113. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Authors and Affiliations

  1. 1.Faculty of Food and Biochemical TechnologyUniversity of Chemistry and Technology, PraguePragueCzech Republic

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