Quantitative evaluation of biofilm extracellular DNA by fluorescence-based techniques
- 297 Downloads
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.
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.
- 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. https://doi.org/10.1007/s12223-018-0603-6 CrossRefGoogle Scholar
- Doyle ME, Ph D (2001) Virulence characteristics of Listeria monocytogenes. Fri Briefings 1–13 available online https://fri.wisc.edu/files/Briefs_File/virulencelmono.pdf. Accessed 3 Sept 2018
- 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. https://doi.org/10.1073/pnas.1218898110 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1128/Aem.03173-14 CrossRefGoogle Scholar
- Harmsen M, Lappann M, Knøchel S (2010) Role of extracellular DNA during biofilm formation by Listeria monocytogenes. 76:2271–2279. https://doi.org/10.1128/AEM.02361-09
- 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. https://doi.org/10.1016/j.ijfoodmicro.2013.05.025 CrossRefPubMedGoogle Scholar
- Khajotia SS, Smart KH, Pilula M, Thompson DM (2013) Concurrent quantification of cellular and extracellular components of biofilms. Jove-J Vis ExpGoogle Scholar
- 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. https://doi.org/10.1128/AEM.01065-06
- Rajendran R et al. (2014) Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation BMC Microbiol 14:ARTN 303 https://doi.org/10.1186/s12866-014-0303-6
- 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. https://doi.org/10.5301/ijao.5000057 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1073/pnas.0610226104 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1038/s41598-018-20485-z CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1007/s11999-017-5266-0 CrossRefPubMedPubMedCentralGoogle Scholar