Rolf Bos

Electric double layer interactions in bacterial adhesion to surfaces

The DLVO (Derjaguin, Landau, Verwey, Overbeek) theory was originally developed to describe interactions between non-biological lyophobic colloids such as polystyrene particles, but is also used to describe bacterial adhesion to surfaces. Despite the differences between the surface of bacteria and that of non-biological particles, DLVO-descriptions of bacterial adhesion have nearly always treated bacteria as if they were non-biological particles and consequently in many cases these descriptions have failed to describe bacterial adhesion adequately. This review summarizes recent advances in colloid and surface science regarding the electrokinetic characterization of biological colloids, most notably bacteria, and their electric double layer interactions with surfaces

A reference guide to microbial cell surface hydrophobicity based on contact angles

Acid–base interactions form the origin of the hydrophobicity of microbial cell-surfaces and can be quantitated from contact angle measurements on microbial lawns with water, formamide, methyleneiodide and/or α-bromonaphthalene. This review provides a reference guide to microbial cell surface hydrophobicity based on contact angles with the above four diagnostic liquids and involves Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, actinomyces, Brevibacterium linens, various Candida species, Capnocytophaga gingivalis, Enterococci, Escherichia coli, lactobacilli, Leuconostoc mesenteroides, peptostreptococci, Porphyromonas gingivalis, Prevotella intermedia, pseudomonads, Serratia marcescens, staphylococci, and streptococci, adding up to a total of 142 isolates among which many ATCC and NCTC strains and two standard strains in hydrophobicity research. Comparison of the results of an acid-base analysis of the microbial cell surfaces on the basis of contact angles for the latter two strains and the results of the so-called MATH (microbial adhesion to hydrocarbons) assay for cell surface hydrophobicity, demonstrates that only contact angles can provide a real estimate of cell surface hydrophobicity. Furthermore, the compilation of contact angle data presented, makes clear that no generalizations concerning the physico-chemical surface properties of microorganisms may be made.

DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides.

'Soft-particle' analysis of the electrophoretic mobility of a fibrillated and non-fibrillated oral streptococcal strain : Streptococcus salivarius

The electrophoretic mobility of microbial cell surfaces can be analysed in terms of a so-called soft layer model, in which the electrophoretic mobility is described as originating from the potentials over the surface charge layer and the membrane fixed charges. Often, the polyelectrolyte layer deforms under the influence of ionic strength variations. In the soft layer analysis of electrophoretic mobilities this is expressed in the softness 1/lambda. Here, we determined the softness of two oral streptococcal strains, S. salivarius HB and HBC12 from particulate microelectrophoresis in KCl solutions of varying ionic strength. Electron microscopy of negatively-stained organisms and X-ray photoelectron spectroscopy showed that strain HB had several classes of proteinaceous fibrils with lengths up to 178 nm on its outermost surface, while variant HBC12 had a bald, peptidoglycan-rich outer surface. The fibrillated strain HB appeared as relatively soft (1/lambda equals 1.4 nm) from analysis of its electrophoretic mobility, while the bald variant HBC12 was hard (1/lambda equals 0.7 nm) due to its comparatively rigid, peptidoglycan-rich outer surface, characteristic to Gram-positive bacteria. The presence of proteinaceous fibrils on strain HB slightly shielded the membrane fixed charges on HBC12.

Controlled electrophoretic deposition of bacteria to surfaces for the design of biofilms

In this report, the formation of ordered clusters of both spherical and rod-shaped bacteria on an electrode during electrophoretic deposition is described. Inside clusters, adhering bacteria are regularly spaced with an interbacterial distance that can be controlled by adjusting the ionic strength of the suspending solution and the DC density used. Formed clusters can be immobilized on the surface by applying a sufficiently high current density. This method enables the design of bacterial biofilms for biotechnological and biomedical applications. When AC fields were used, rod-shaped bacteria adhering on the electrode were seen to align parallel to the applied field.

Measurement of charge transfer during bacterial adhesion to an indium tin oxide surface in a parallel plate flow chamber

An experimental method is described for the measurement of charge transfer during bacterial adhesion in situ to a transparent, semiconducting indium tin oxide (ITO) coated glass plate in a parallel plate flow chamber. Bacterial adhesion is measured simultaneously with either the electric potential or the capacitance of the surface. Initial bacterial adhesion was accompanied by a change in electric potential of the surface with no measurable change in capacitance. Consequently, it can be assumed that the change in electric potential of the surface is due to charge transfer between bacteria and the surface, and it can be calculated that, on average, a charge of about 10(-14) C per bacterium is exchanged during initial adhesion, which corresponds to only several percent of the total surface charge of a bacterium. Charge transfer could either be to or from the bacterial cell surface, dependent on the bacterial strain involved and the ionic strength used.

A QUANTITATIVE METHOD TO STUDY CO-ADHESION OF MICROORGANISMS IN A PARALLEL-PLATE FLOW CHAMBER - BASIC PRINCIPLES OF THE ANALYSIS

Intermicrobial aggregation is described as one of the factors contributing to dental plaque formation. Intermicrobial aggregation is usually measured by mixing potential partners suspended in a liquid phase (‘coaggregation’). However, even if aggregation in the liquid phase occurs, adhesion of microorganisms to partners already adhering to a substratum surface may also occur (‘co-adhesion’). Coaggregation assays have been performed in order to measure coaggregation and to model co-adhesion, although it is not yet clear which the two prevails in vivo. Apart from being semi-quantitative (scores run from 0 to 4) it is questionable whether coaggregation assays really mimic co-adhesion. This study was designed to develop a method to quantitative assess co-adhesion of microbial pairs in order to gain a better understanding of the mechanisms governing co-adhesion. Co-adhesion of coaggregating and non-coaggregating partners (S. oralis, S. sanguis and A. naeslundii) to glass has been studied in a parallel plate flow chamber using real time image analysis. The spatial arrangements of adhering bacteria were analyzed by radial pair distribution functions, revealing the relative density of adhering bacteria around adhering bacteria from the same (g22(r)) or a partner strain (g21(r)). Pair distribution functions g21(r) of coaggregating pairs clearly reveal a preference of coaggregating streptococci (S. oralis J22 and S. sanguis PK2951) to adhere around the actimomyces (A. naeslundii PK213 or T14V-J1), which were used to coat the bare glass substratum. Besides, the distribution function g21(r) showed differences in co-adhesion patterns for strains with the same coaggregation score. From the results presented in this paper it can be concluded that with a parallel plate flow chamber, co-adhesion can be quantified on a continuous scale under well controlled conditions, more closely resembling those occurring in vivo.

Charge transfer during staphylococcal adhesion to TiNOX® coatings with different specific resistivity

Adhesion of the bacterial strain Staphylococcus epidermidis 3399 to titanium-oxy-nitride (TiNOX) substrata with different specific resistivities was studied in a parallel plate flow chamber, while simultaneously measuring the electric potential of the substrata. During adhesion, bacteria either donated or accepted electrons to the substrata depending on the specific resistivity of the substratum and bacteria that had donated electrons to the substratum adhered more strongly than bacteria that had accepted electrons from the substratum. These results demonstrate that electron transfer plays a role in bacterial adhesion to conducting surfaces, which has hitherto been neglected.

The electrophoretic softness of the surface of Staphylococcus epidermidis cells grown in a liquid medium and on a solid agar

Many Staphylococcus epidermidis strains possess capsule or slime layers and consequently the staphylococcal cell surface should be regarded as a soft, polyelectrolyte layer allowing electrophoretic fluid flow through a layer of fixed charges. The presence of such a soft layer decreases the energy barrier due to electrostatic repulsion in the interaction of the organisms with negatively charged substrata [Morisaki, H., Nagai, S., Ohshima, H., Ikemoto, E. & Kogure, K. (1999), MICROBIOLOGY: 145, 2797-2802] and hence plays an important role in their adhesion. In this paper, the authors compare the electrophoretic softness and amount of fixed charge in the outer cell surface layers of 20 S. epidermidis strains, grown in a liquid medium or on a solid agar, as determined from the dependencies of their electrophoretic mobilities upon the ionic strength of a suspending fluid. Most of the staphylococcal cell surfaces were relatively soft, with a mean cell surface softness (1/lambda) for strains grown in liquid medium of 1.7+/-0.6 nm (standard deviation over all 20 strains) which is soft by comparison with a completely bald, peptidoglycan-rich streptococcal cell surface (1/lambda=0.7 nm). When the staphylococcal strains were grown on solid agar, the cell surface softness of 17 of the 20 strains increased, sometimes by a factor of two. On average for 20 strains, the cell surface softness increased significantly (P:<0.05, Students t-test) to 2.8+/-1.8 nm. The amount of fixed charge in the outer cell surface layer was -28+/-9 mM for bacteria grown in liquid medium and -24+/-12 mM for bacteria grown on agar. A soft, highly negatively charged polyelectrolyte layer was inferred by microelectrophoresis for all the staphylococcal cell surfaces, regardless of whether staining had indicated the presence of a capsule or slime layer.

Electrostatic interactions in the adhesion of an ion-penetrable and ion-impenetrable bacterial strain to glass

Deposition to glass of Streptococcus salivarius HB-C12 and Staphylococcus epidermidis 3399 in a parallel plate flow chamber has been studied as a function of ionic strength. Electrophoretic mobility measurements revealed that S. epidermidis 3399 possesses a thick ion-penetrable layer, probably associated with its encapsulation, while S. salivarius HB-C12 has an ion-impenetrable surface. Streaming potential measurements indicated that also the glass surface was covered with a relatively thin, ion-penetrable layer. Theoretical initial deposition rates of both strains to glass were obtained by numerically solving the convective-diffusion equation, while accounting for the ion-penetrability of the interacting surfaces. Experimentally, the initial deposition rate of the ion-penetrable strain S. epidermidis 3399 was found to be higher and less dependent on ionic strength than of the ion-impenetrable S. salivarius HB-C12, in accordance with theoretical expectations. Agreement between theoretical and experimental deposition rates could be obtained when glass was considered ion-penetrable when interacting with the ion-penetrable organism S. epidermidis 3399, while glass behaved as an ion-impenetrable surface when interacting with the ion-impenetrable S. salivarius HB-C12. Probably, interaction with an ion-impenetrable strain drives the diffuse double layer charges into the limited volume of the thin ion-penetrable layer on the glass, readily filling it up and making it appear ion-impenetrable. During interaction of glass with another ion-penetrable surface, as of S. epidermidis 3399, diffuse double layer charges move into both ion-penetrable surfaces, resulting in a much lower mobile charge density in the ion-penetrable layer on the glass which consequently continues to behave as ion-penetrable.