Hostname: page-component-78c5997874-dh8gc Total loading time: 0 Render date: 2024-11-17T14:52:38.586Z Has data issue: false hasContentIssue false
  • English
  • Français

Visualization of Microbiological Processes Underlying Stress Relaxation in Pseudomonas aeruginosa Biofilms

Published online by Cambridge University Press:  13 March 2014

Brandon W. Peterson ,
Henk J. Busscher ,
Prashant K. Sharma  and
Henny C. van der Mei
Brandon W. Peterson
Affiliation:
University of Groningen and University Medical Center Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Henk J. Busscher
Affiliation:
University of Groningen and University Medical Center Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Prashant K. Sharma
Affiliation:
University of Groningen and University Medical Center Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Henny C. van der Mei*
Affiliation:
University of Groningen and University Medical Center Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
*
*Corresponding author.h.c.van.der.mei@umcg.nl
Get access

Abstract

Bacterial biofilms relieve themselves from external stresses through internal rearrangement, as mathematically modeled in many studies, but never microscopically visualized for their underlying microbiological processes. The aim of this study was to visualize rearrangement processes occurring in mechanically deformed biofilms using confocal-laser-scanning-microscopy after SYTO9 (green-fluorescent) and calcofluor-white (blue-fluorescent) staining to visualize bacteria and extracellular-polymeric matrix substances, respectively. We apply 20% uniaxial deformation to Pseudomonas aeruginosa biofilms and fix deformed biofilms prior to staining, after allowing different time-periods for relaxation. Two isogenic P. aeruginosa strains with different abilities to produce extracellular polymeric substances (EPS) were used. By confocal-laser-scanning-microscopy all biofilms showed intensity distributions for fluorescence from which rearrangement of EPS and bacteria in deformed biofilms were derived. For the P. aeruginosa strain producing EPS, bacteria could not find new, stable positions within 100 s after deformation, while EPS moved toward deeper layers within 20 s. Bacterial rearrangement was not seen in P. aeruginosa biofilms deficient in production of EPS. Thus, EPS is required to stimulate bacterial rearrangement in mechanically deformed biofilms within the time-scale of our experiments, and the mere presence of water is insufficient to induce bacterial movement, likely due to its looser association with the bacteria.

Keywords

extracellular polymeric substances (EPS)biofilm structureviscoelastic relaxationMaxwell elements
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 3 , June 2014 , pp. 912 - 915
Copyright
© Microscopy Society of America 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Boyd, A. & Chakrabarty, A.M. (1995). Pseudomonas aeruginosa biofilms: Role of the alginate exopolysaccharide. J Ind Microbiol 15, 162–168.Google Scholar
Busscher, H.J., Jager, D., Finger, G., Schaefer, N. & Van der Mei, H.C. (2010). Energy transfer, volumetric expansion, and removal of oral biofilms by non-contact brushing. Eur J Oral Sci 118, 177–182.Google Scholar
Cense, A.W., Peeters, E.A., Gottenbos, B., Baaijens, F.P., Nuijs, A.M. & Van Dongen, M.E.H. (2006). Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device. J Microbiol Methods 67, 463–472.Google Scholar
Colvin, K.M., Gordon, V.D., Murakami, K., Borlee, B.R., Wozniak, D.J., Wong, G.C.L. & Parsek, M.R. (2011). The Pel polysaccharide can serve as a structural and protective role in biofilm matrix of Psuedomonas aeruginosa . PLoS Pathog 7, e1001264.Google Scholar
Dauletbaev, N., Fischer, P., Aulbach, B., Gross, J., Kusche, W., Thyroff-Friesinger, U., Wagner, T.O. & Bargon, J. (2009). A phase II study on safety and efficacy of high-dose N-acetylcysteine in patients with cystic fibrosis. Eur J Med Res 14, 352–358.CrossRefGoogle ScholarPubMed
Flemming, H.C., Neu, T.R. & Wozniak, D.J. (2007). The EPS matrix: the “house of biofilm cells”. J Bacteriol 189, 7945–7947.Google Scholar
Flemming, H.C. & Wingender, J. (2010). The biofilm matrix. Nat Rev Microbiol 8, 623–633.Google Scholar
Gomez-Suarez, C., Pasma, J., Van der Borden, A.J., Wingender, J., Flemming, H.C., Busscher, H.J. & Van der Mei, H.C. (2002). Influence of extracellular polymeric substances on deposition and redeposition of Pseudomonas aeruginosa to surfaces. Microbiology 148, 1161–1169.Google Scholar
He, Y., Peterson, B.W., Jongsma, M.A., Ren, Y., Sharma, P.K., Busscher, H.J. & Van der Mei, H.C. (2013). Stress relaxation analysis facilitates a quantitative approach towards antimicrobial penetration into biofilms. PLoS One 8, e63750.Google Scholar
Klapper, I., Rupp, C.J., Cargo, R., Purvedorj, B. & Stoodley, P. (2002). Viscoelastic fluid description of bacterial biofilm material properties. Biotechnol Bioeng 80, 289–296.Google Scholar
Korstgens, V., Flemming, H.C., Wingender, J. & Borchard, W. (2001). Uniaxial compression measurement device for investigation of the mechanical stability of biofilms. J Microbiol Methods 46, 9–17.Google Scholar
Lau, P.C., Dutcher, J.R., Beveridge, T.J. & Lam, J.S. (2009). Absolute quantitation of bacterial biofilm adhesion and viscoelasticity by microbead force spectroscopy. Biophys J. 96, 2935–2948.CrossRefGoogle ScholarPubMed
Lieleg, O., Caldara, M., Baumgartel, R. & Ribbeck, K. (2011). Mechanical robustness of Pseudomonas aeruginosa biofilms. Soft Matter 7, 3307–3314.Google Scholar
Mclean, J.S., Ona, O.N. & Majors, P.D. (2008). Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy. ISME J 2, 121–131.Google Scholar
Mrsny, R.J., Lazazzera, B.A., Daugherty, A.L., Schiller, N.L. & Patapoff, T.W. (1994). Addition of a bacterial alginate lyase to purulent CF sputum in vitro can result in the disruption of alginate and modification of sputum viscoelasticity. Pulm Pharmacol 7, 357–366.Google Scholar
Peterson, B.W., Busscher, H.J., Sharma, P.K. & Van der Mei, H.C. (2012). Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro . Biofouling 28, 913–920.Google Scholar
Picioreanu, C., Van Loosdrecht, M.C. & Heijnen, J.J. (2000). Effect of diffusive and convective substrate transport on biofilm structure formation: a two-dimensional modeling study. Biotechnol Bioeng 69, 504–515.3.0.CO;2-S>CrossRefGoogle ScholarPubMed
Shaw, T., Winston, M., Rupp, C.J., Klapper, I. & Stoodley, P. (2004). Commonality of elastic relaxation times in biofilms. Phys Rev Lett 93, 098102.Google Scholar
Stoodley, P., Lewandowski, Z., Boyle, J.D. & Lappin-Scott, H.M. (1999). Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: An in situ investigation of biofilm rheology. Biotechnol Bioeng 65, 83–92.Google Scholar
Strathmann, M., Wingender, J. & Flemming, H.C. (2002). Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa . J Microbiol Methods 50, 237–248.Google Scholar
Towler, B.W., Cunningham, A., Stoodley, P. & McKittrick, L. (2007). A model of fluid-biofilm interaction using a Burger material law. Biotechnol Bioeng 96, 259–271.Google Scholar