For direct in situ quantification of attached bacteria on various biomaterial surfaces or on other surfaces exposed to biofouling, a novel fluorescence-based bacterial overlay technique was developed. The detection of bacteria by this method, which was based on biotinylation of bacteria and subsequent detection by fluorescence-tagged avidin, was methodically simple to perform and allowed the simultaneous measurement of a large number of different small material specimens. This method was tested in an established model system based on silanized silicon wafers with and without prior protein coating by using exemplary bacterial strains exhibiting different adhesion properties. With this novel method, bacterial attachment was found to depend on a variety of parameters, including surface wettability, surface roughness, strain of bacteria employed, and prior protein adsorption.
Bacterial overlay methods are well-established tools for examination of bacterial adhesin-mediated binding to immobilized host glycolipid (18
) or glycoprotein (24
) receptors separated by either thin-layer chromatography or gel electrophoresis and subsequent blotting but have so far not been employed for quantification of bacteria attached to material surfaces. When the newly established overlay technique was compared to the very simple and direct agar replica technique or to visualization by SEM, similar qualitative adhesion patterns on the differently modified wafer surfaces could be verified. Certain quantitative deviations between these methods might be caused by inherent technical shortcomings. The agar imprint method requires viable bacteria and reflects only the number of bacteria that are transferable to the agar medium, whereas an uncertainty regarding the number of firmly adherent residual bacteria on the material surface remains. SEM, on the other hand, shows only the firmly attached bacteria remaining on the material surface, whereas uncertainty regarding organisms lost during sample preparation exists. Alternative methods for quantification, such as radiolabeling, crystal violet-staining, quantification by metabolites, and enumeration by use of a Coulter Counter or flow cytometry, require large surface areas to obtain reliable results. Such large surface areas are sometimes difficult to realize with certain materials. Similar limitations might apply also to other techniques that have been described in the past to quantify surface-attached bacteria (1
). Thus, the technique of choice depends on the particular problem that one is interested in as well as on the type of material examined. The most obvious advantage of the fluorescence-based overlay method described in the present study is the possibility for screening of in situ bacterial attachment on many parallel small samples within one experimental setting.
Despite the inherent limitations of the different techniques for enumeration of bacteria attached to material surfaces, certain common principles regarding the influence of surface properties on bacterial adhesion can be deduced from the literature. There is a general tendency for hydrophilic materials to be more resistant to bacterial or fungal adhesion than hydrophobic ones (2
). This is reflected in the present investigation by the low numbers of bacteria found on oxidized wafers compared to those on OTS or HFS-modified surfaces. This is also in agreement with previous reports investigating the adhesion of a variety of bacteria, including S. aureus
, to self-assembled monolayers (8
) and with SEM studies using various materials within a wide range of surface free energy (41
). In the present investigation, also in agreement with these reports, the lowest numbers of attached organisms for each of the three bacterial strains tested were recorded for PEG-modified surfaces. This may be explained by the fact that PEG chains, depending on their lengths (31
), provide a template for water nucleation resulting in the formation of an interfacial water layer, whereby these PEG brushes prevent direct contact between bacteria and the surface (17
Higher roughness on the chemically modified wafer surfaces was found in the present study to increase bacterial attachment but did not alter the relative adhesion patterns found for smooth wafer surfaces. Other studies also found that roughening of, e.g., polymeric surfaces, promoted bacterial adhesion and biofilm deposition, whereas smooth surfaces did not (27
). An accepted explanation for this behavior is that rough surfaces have greater surface areas and that the depressions in the roughened surfaces provide more favorable sites for colonization (2
), an observation that was also observed by SEM visualization (images not shown).
Exposure of the modified wafers to protein solutions before incubation with the bacterial strains resulted in significantly altered adhesion patterns that were to a great degree dependent on the type of protein or protein mixture and on the particular bacterial strain examined. Differences in wettability between the chemically modified wafer surfaces were moderated to a certain extent by adsorption of a protein layer, whereby hydrophobic surfaces became more hydrophilic and vice versa. However, preadsorption of the proteins did not completely mask the influence of the original surface chemistry. Preadsorption of wafers with HSA reduced bacterial attachment in most cases, which is in agreement with prior studies using different material surfaces (23
). The hypothesis that this reduction of bacterial adhesion by albumin might be caused by changing substratum surface hydrophobicity (2
) was supported by the combined results for the water contact angle measurements and bacterial adhesion experiments of the present study. Preadsorption of wafers with serum, similar to that with HSA, reduced bacterial attachment in most instances, which might in fact be due to the high albumin content in serum, as suggested previously (23
). However, for S. gordonii
DL1, an increase in bacterial numbers attached to serum-precoated surfaces was observed. This exceptional adhesion behavior of S. gordonii
DL1 could be explained by the expression of an adhesin activity recognizing putative receptor molecules in this complex body fluid. This was further supported by the present dot blot experiments, showing preferential binding of this strain to serum proteins immobilized on nitrocellulose (Fig. ). Precoating of wafers with fibronectin, in certain instances, resulted in greater numbers of attached bacteria. This could be explained by expression of fibronectin-binding adhesin activities in these bacteria (7
) and was also reflected by bacterial binding to fibronectin in the dot blot experiment (Fig. ). For saliva precoating, it is not surprising that particularly the adhesion of S. gordonii
DL1 was enhanced in all situations because multiple adhesin activities that interact with salivary proteins have been described for this bacterium (33
), including a lectin-like sialic acid binding adhesin (38
) that might in part also be responsible for the observed binding to serum proteins. This was supported in the present investigation by strong binding of S. gordonii
DL1 to mixed whole saliva in the dot blot experiment. S. mitis
, although considered one of the earliest colonizers of the tooth surface (12
), did not exhibit stronger binding to saliva-precoated wafers and was found to bind only weakly to whole saliva immobilized on nitrocellulose. Thus, it appears that adsorbed proteins do not mask the original surface properties but rather decorate the surface in a fashion whereby their presence adds additional biochemical properties to the already present physicochemical properties. It is, however, still the original surface chemistry that determines the amount, the composition, the conformation, and ultimately the biological activity of these adsorbed proteins.
For many applications, there is a need for materials that resist bacterial colonization (15
). The new method described in the present study not only allows quantification of surface-attached bacteria but also more closely imitates the real situation by considering the conditioning influence of the surrounding macromolecule-containing fluid environment. For each particular application, e.g., when evaluating implant materials, blood-contacting transcutaneous or extracorporal devices, contact lenses, dental restorative materials, or even surfaces exposed to a marine environment (9
), it is crucial to select those bacteria that are of relevance in each given particular situation. It would be advantageous to establish model strains for each test system together with a set of adhesin-deficient mutants (26
) for discrimination between general physicochemical influences and the more specific biochemical sterical recognition mechanisms.