Integration of Raman microscopy, differential interference contrast microscopy, and attenuated total reflection Fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in Candida albicans biofilms.

Two spectroscopic techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and Raman microscopy (RM), were used to characterize transport of chlorhexidine digluconate (CHG) in Candida albicans (CA) biofilms. Different (volumetric) regions of the biofilm are sampled by these two vibrational spectroscopies making them complementary techniques. Simple mathematical models were developed to analyze ATR-FTIR and RM data to obtain an effective diffusion coefficient describing transport through CA biofilms. CA biofilms were composed primarily of yeast and hyphal forms, with some pseudohyphae. Upper regions of biofilms that had become confluent, (i.e., biofilms that completely covered the germanium (Ge) substratum) were composed primarily of a tangled mass of hyphae with openings between germtubes about 10 to 50 microm across. Quantitative analysis of ATR-FTIR kinetic data curves indicated that the effective diffusion coefficient for transport of CHG through confluent biofilms about 200-microm thick was reduced 0.1 to 0.3 times compared to the diffusion coefficient for CHG in water. Effective diffusion coefficients obtained from analysis of RM data were consistently higher than those indicated by ATR-FTIR data suggesting that transport is more hindered in regions near the base of the biofilm than in the outer layers. Analysis of both ATR-FTIR and RM data obtained from thicker films indicated that adsorption of CHG to biofilm components was responsible for a substantial portion of the transport limitation imposed by the biofilm. Comparison of ATR-FTIR and RM data for both types of biofilms indicated that sites of CHG adsorption were more concentrated in the interfacial region than in the bulk biofilm. Comparison of results for ATR-FTIR and RM measurements suggests that these relatively thick CA biofilms can be modeled, for purposes of predicting transport, approximately as a homogeneous thin planar sheet. Thus, these biofilms offer a relatively tractable model system for initial investigations of the relation between antimicrobial transport and kinetics of antimicrobial action.

Comparison of adsorption behavior of two Mytilus edulis foot proteins on three surfaces.

The sea mussel Mytilus edulis fabricates a hold-fast (an adhesive plaque) from a proteinaceous mixture that it extrudes into a cavity formed by an organ called the 'foot'. A family of four proteins in the mixture known as M. edulis foot proteins (Mefp 1-4) have been purified to homogeneity. Mefp-1 and 2 are the most well-characterized and most easily purified members of the Mefp family. They constitute about 5 and 25% of the content of the plaque, respectively. It has been proposed that Mefp-1 mediates bonding to the intended substratum while Mefp-2 serves more as a structural component. In order to provide data relevant to this hypothesis, the adsorption behavior of Mefp-1 and 2 was compared on three surfaces using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Surfaces were germanium (Ge), polystyrene (PS) or poly(octadecyl)methacrylate (POMA). Polymer surfaces were prepared by spin casting onto the flat face of Ge trapezoidal internal reflection elements (IRE). Adsorption behavior was characterized by analyzing the kinetics of adsorption using a double exponential fit. The data indicate that the adsorption behavior of Mefp-1 and 2 is similar on the three surfaces both in terms of rate of adsorption and surface coverage attained over a short (<60 min) time period.

Characterization of extracellular chitinolytic activity in biofilms.

Extracellular enzymes produced by bacterial biofilms tend to become an integral, permanent part of the biofilm/substratum system. Thus, characterizing extracellular enzyme activity is an essential component of understanding biofilm ecology. Methods have been presented for characterizing three aspects of extracellular enzyme activity in biofilms: promoter activity of the structural gene, local catalytic activity, and kinetics of collective substrate degradation. The abundance of intracellular transcript derived from a structural gene is only indirectly related to the magnitude of catalytic activity of the corresponding enzyme. This relationship may be particularly tenuous in the case of extracellular enzymes, which must be transported out of the cell in order to become active. Fluorogenic substrates that allow direct detection of an increasingly greater variety of enzyme activities are becoming available. There are technical problems, originating from surface roughness and intrinsic fluorescence, associated with microscopic examination of biofilms on natural materials. Thin films provide one option for acquiring data about biofilms colonizing relevant materials.

Investigation of interactions between antimicrobial agents and bacterial biofilms using attenuated total reflection Fourier transform infrared spectroscopy.

Biomaterial-centred infections are often difficult to treat. An impaired immune response, acute inflammatory reactions and the presence of recalcitrant attached microorganisms are all contributing factors. A brief review of the role of attached bacteria in biomaterial-centred infections is presented. Two major hypotheses which may explain the recalcitrance of biofilms to antimicrobial agents are discussed. The analytical capabilities of attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy for providing information on both transport of an antimicrobial agent to bacteria embedded in the biofilm and interactions between an antimicrobial agent and biofilm components are described.

Comparison of recalcitrance to ciprofloxacin and levofloxacin exhibited by Pseudomonas aeruginosa bofilms displaying rapid-transport characteristics.

Attenuated total reflection Fourier transform infrared spectroscopy was used to measure transport of the fluoroquinolones (FQs) ciprofloxacin and levofloxacin into Pseudomonas aeruginosa biofilms. Biofilms were exposed to each FQ at dose levels of 100, 250, and 500 microg/ml for 30 min. A mathematical transport model was used to extract the diffusion coefficient, binding site density, and adsorption and desorption rates for each experiment. Recalcitrance of the biofilms toward each FQ was evaluated by comparison of efficacies with planktonic bacteria. By this criterion, biofilms were found to exhibit more recalcitrance toward levofloxacin than ciprofloxacin under the experimental conditions. These results cannot be explained by the more hindered transport of levofloxacin, implicating the domination of physiological factors.

Combined light microscopy and attenuated total reflection fourier transform infrared spectroscopy for integration of biofilm structure, distribution, and chemistry at solid-liquid interfaces.

Reflected differential interference contrast microscopy and attenuated total reflection Fourier transform infrared spectroscopy were used to obtain complementary data on the structural and chemical properties of a biofilm. This information was obtained nondestructively, quasisimultaneously, and in real time, thereby permitting the verification of time-dependent relationships between the biofilm's population structure, distribution, and interfacial chemistry. The approach offers opportunities to examine these relationships on a variety of substrata in the presence of a bulk aqueous phase under controlled hydrodynamic conditions.

Influence of protein conditioning films on binding of a bacterial polysaccharide adhesin from Hyphomonas MHS-3.

A putative polysaccharide adhesin which mediates non-specific attachment of Hyphomonas MHS-3 (MHS-3) to hydrophilic substrata has been isolated and partially characterized. A polysaccharide-enriched portion of the extracellular polymeric substance (EPS(P)) from MHS-3 was separated into four fractions using high performance size exclusion chromatography (HPSEC). Comparison of chromatograms of EPS(P) from MHS-3 and a reduced adhesion strain (MHS-3 rad) suggested that one EPS(P) fraction, which consisted of carbohydrate, served as an adhesin. Adsorption of this fraction to germanium (Ge) was investigated using attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry. Binding curves indicated that the isolated fraction had a relatively high affinity for Ge when ranked against an adhesive protein from Mytilis edulis, mussel adhesive protein (MAP) and an acidic polysaccharide (alginate from Macrocystis pyrifera). Spectral features were used to identify the fraction as a polysaccharide previously reported to adsorb preferentially out of the EPS(P) mixture. Conditioning the Ge substratum with either bovine serum albumin (BSA) or MAP decreased the adsorption of the adhesive polysaccharide significantly. Conditioning Ge with these proteins also decreased adhesion of whole cells.

Adhesion of biofilms to inert surfaces: A molecular level approach directed at the marine environment.

Protein/ligand interactions involved in mediating adhesion between microorganisms and biological surfaces have been well-characterized in some cases (e.g. pathogen/host interactions). The strategies microorganisms employ for attachment to inert surfaces have not been so clearly elucidated. An experimental approach is presented which addresses the issues from the point of view of molecular interactions occurring at the interface.

Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms.

Bacterial infections associated with indwelling medical devices often demonstrate an intrinsic resistance to antimicrobial therapies. In order to explore the possibility of transport limitation to biofilm bacteria as a contributing factor, the penetration of a fluoroquinolone antibiotic, ciprofloxacin, through Pseudomonas aeruginosa biofilms was investigated. Attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry was employed to monitor bacterial colonization of a germanium substratum, transport of ciprofloxacin to the biofilm-substratum interface, and interaction of biofilm components with the antibiotic in a flowing system. Transport of the antibiotic to the biofilm-substratum interface during the 21-min exposure to 100 micrograms/ml was found to be significantly impeded by the biofilm. Significant changes in IR bands of the biofilm in regions of the spectrum associated with RNA and DNA vibrational modes appeared following exposure to the antibiotic, indicating chemical modification of biofilm components. These results suggest that transport limitations may be an important factor in the antimicrobial resistance of biofilm bacteria and that ATR/FT-IR spectrometry may be used to follow the time course of antimicrobial action in biofilms in situ.

Investigation of specific binding of antifluorescyl antibody and Fab to fluorescein lipids in Langmuir-Blodgett deposited films using quartz crystal microbalance methodology.

Antifluorescyl IgG antibody and Fab binding to two fluorescein-conjugated lipids was measured using the quartz crystal microbalance methodology. By use of the Langmuir-Blodgett technique, the fluorescein lipids, which were diluted to 5% in a L-alpha-dipalmitoyl phosphatidylethanolamine (DPPE) matrix, were deposited directly onto one gold electrode of the quartz crystal. Binding to films containing the fluorescein hapten was significantly enhanced compared to films of the pure DPPE matrix lipid, indicating that binding occurred primarily through a specific interaction. Association constants were 40-300 times less than for binding to haptens free in solution. Binding of IgG to the lipid in which the hydrocarbon chains and the fluorescein hapten were linked via a hydrophilic spacer was approximately 7 times as great as to the lipid containing no spacer. IgG binding to the lipid containing the spacer was increased 1.5-4.4 times compared to Fab binding for the same lipid. Equilibrium binding curves and kinetic measurements are analyzed quantitatively and compared.