AFM analysis of the extracellular polymeric substances (EPS) released during bacterial attachment on polymeric surfaces

Extracellular polymeric substances (EPS) secreted by bacteria have a key role in adhesion and aggregation of bacterial cells on solid surfaces. In the present study, atomic force microscopy (AFM) has been used to study the adhesion propensity of bacterial strain St. guttiformis, and the ultrastructure and distribution of the EPS materials, on hydrophobic poly(tert-butylmethacrylate)(PtBMA) and hydrophilic polystyrene maleic acid (PSMA) surfaces. The results showed that bacterial attachment to the PSMA surface over incubation periods of 24-72 h was insignificant, whereas there was a strong propensity for the bacterial cells to attach to the PtBMA surface, forming multi-layered biofilms. For the PSMA surface, planktonic EPS adsorbed onto the polymeric surface and formed a continuous surface layer. For the PtBMA surface, non-contact mode imaging revealed that capsular EPS on the cell surface exhibited granular structures with the lateral dimensions of 30-50 nm and the vertical roughness of 7-10 nm. Lateral force imaging showed inter-connected elongated features which had lower frictional property compared to the surrounding EPS matrix, suggesting possible segregation of hydrophobic fractions of the EPS materials. The planktonic EPS adsorbed onto the PtBMA surface also showed similar nanometer-scale granular structures and could form stacks up to 150 nm in height. However, lateral force imaging did not show frictional differences, as in the case of capsular EPS. This is attributed to possible differences in the composition of the two EPS materials, and/or greater deformation of the planktonic EPS in the contact imaging mode which may obscure the fine surface features.

[1]  S. Vansteenkiste,et al.  Scanning probe microscopy of biomedical interfaces , 1998 .

[2]  F. Denes,et al.  12‐crown‐4–ether and tri(ethylene glycol) dimethyl–ether plasma‐coated stainless steel surfaces and their ability to reduce bacterial biofilm deposition , 2001 .

[3]  L. Melo,et al.  Biofilms--science and technology , 1992 .

[4]  A. Zehnder,et al.  DLVO and steric contributions to bacterial deposition in media of different ionic strengths , 1999 .

[5]  M. Fletcher,et al.  An Electron-microscopic Demonstration of an Acidic Polysaccharide Involved in the Adhesion of a Marine Bacterium to Solid Surfaces , 1973 .

[6]  L. V. Evans,et al.  Biofilms : recent advances in their study and control , 2000 .

[7]  Gerber,et al.  Atomic force microscope. , 1986, Physical review letters.

[8]  J. Wimpenny,et al.  Heterogeneity in biofilms. , 2000, FEMS microbiology reviews.

[9]  G. Leggett,et al.  Scanning force microscopy of bulk-filled uniaxially oriented poly(ethylene terephthalate) films , 2000 .

[10]  J. Block,et al.  Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances , 1998 .

[11]  Edward J. Bouwer,et al.  Reversibility and mechanism of bacterial adhesion , 1995 .

[12]  Nicholas D. Spencer,et al.  The influence of pH on friction between oxide surfacesin electrolytes, studied with lateral force microscopy:application as a nanochemical imaging technique , 1997 .

[13]  H. C. van der Mei,et al.  Bacterial adhesion to surface hydrophilic and hydrophobic contact lenses. , 2001, Biomaterials.

[14]  Materials Characterization by Scanned Probe Analysis , 2003 .

[15]  Detection of coccoid forms of Sulfitobacter mediterraneus using atomic force microscopy. , 2002, FEMS microbiology letters.

[16]  I. Beech,et al.  Microbial polysaccharides and corrosion , 1991 .

[17]  R. Neumann,et al.  The role of topography and friction for the image contrast in lateral force microscopy , 1993 .

[18]  Ralph Mitchell,et al.  Mechanism of the Initial Events in the Sorption of Marine Bacteria to Surfaces , 1970 .

[19]  H. Busscher,et al.  X-ray photoelectron spectroscopy for the study of microbial cell surfaces , 2000 .

[20]  J. Wimpenny,et al.  Heterogeneity in bio¢lms , 2000 .

[21]  G. Whitesides,et al.  Scanning Force Microscopies Can Image Patterned Self-Assembled Monolayers , 1995 .

[22]  L. Kotra,et al.  Dynamics of the Lipopolysaccharide Assembly on the Surface of Escherichia coli , 1999 .

[23]  M. Asther,et al.  Stretching cell surface macromolecules by atomic force microscopy , 2001 .

[24]  G. Leggett,et al.  Scanning force microscopy of poly(ethylene terephthalate) surfaces: comparison of SEM with SFM topographical, lateral force and force modulation data , 1997 .

[25]  P. Lawson,et al.  Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., alpha-3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho Lake. , 2000, International journal of systematic and evolutionary microbiology.

[26]  W. Norde,et al.  Protein adsorption and bacterial adhesion to solid surfaces: a colloid-chemical approach. , 1989 .

[27]  Jost Wingender,et al.  Microbial extracellular polymeric substances : characterization, structure, and function , 1999 .

[28]  J. Vanderleyden,et al.  Direct evidence for the involvement of extracellular proteins in the adhesion of Azospirillum brasilense. , 1996, Microbiology.

[29]  I. Beech,et al.  Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel , 1991, Applied Microbiology and Biotechnology.

[30]  Y. Dufrêne.  Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells. , 2001, Micron.

[31]  Charles M. Lieber,et al.  Chemical Force Microscopy: Exploiting Chemically-Modified Tips To Quantify Adhesion, Friction, and Functional Group Distributions in Molecular Assemblies , 1995 .

[32]  J. T. Staley.  Prosthecomicrobium and Ancalomicrobium: New Prosthecate Freshwater Bacteria , 1968, Journal of bacteriology.