This study examines bacterial adhesion on titanium-substrates used for bone implants. Adhesion is the most critical phase of bacterial colonization on medical devices. The surface of titanium was modified by hydrothermal treatment (HT) to synthesize nanostructured TiO₂-anatase coatings, which were previously proven to improve corrosion resistance, affect the plasma protein adsorption, and enhance osteogenesis. The affinity of the anatase coatings toward bacterial attachment was studied by using a green fluorescent protein-expressing Escherichia coli (gfp-E. coli) strain in connection with surface photoactivation by UV irradiation. We also analyzed the effects of surface topography, roughness, charge, and wettability. The results suggested the dominant effects of the macroscopic surface topography, as well as microasperity at the surface roughness scale, which were produced during titanium machining, HT treatment, or both. Macroscopic grooves provided a preferential site for bacteria deposit within the valleys, while the microscopic roughness of the valleys determined the actual interaction surface between bacterium and substrate, resulting in an “interlocking” effect and undesired high bacterial adhesion on nontreated titanium. In the case of TiO₂-coated samples, the nanocrystals reduced the width between the microasperities and thus added nanoroughness features. These factors decreased the contact area between the bacterium and the coating, with consequent lower bacterial adhesion (up to 50% less) in comparison to the nontreated titanium. On the other hand, the pronounced hydrophilicity of one of the HT-coated discs after pre-irradiation seemed to enhance the attachment of bacteria, although the increase was not statistically significant (p > 0.05). This observation may be explained by the acquired similar degree of wetting between gfp-E. coli and the coating. No correlation was found between the bacterial adhesion and the ζ-values of the samples in PBS, so the effect of surface charge was considered negligible in this study.

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.

Significance: In a social process called “quorum sensing” (QS) bacteria secrete and share signaling molecules that bind to specific receptors and induce adaptive responses within the population. We use the ComQXP QS system of Bacillus subtilis to study the intracellular codependence of two essential QS functions: signal production and signal response. We demonstrate that the QS signal-deficient mutants have an overly responsive QS system, a disturbed balance between primary and secondary metabolism, and an overproduction of the secondary metabolite surfactin in the presence of exogenously provided signal. Such mutants fail to compete with the socially active signal producers due to surfactin-related mechanisms that discriminate the two populations. We believe that a constraint on signal production preserves QS functionality in the natural microbial populations.

Liposomes are widely applied in research, diagnostics, medicine and in industry. In this study we show for the first time the effect of hydrodynamic cavitation on liposome stability and compare it to the effect of well described chemical, physical and mechanical treatments. Fluorescein loaded giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles were treated with hydrodynamic cavitation as promising method in inactivation of biological samples. Hydrodynamic treatment was compared to various chemical, physical and mechanical stressors such as ionic strength and osmolarity agents (glucose, Na+, Ca2+, and Fe3+), free radicals, shear stresses (pipetting, vortex mixing, rotational shear stress), high pressure, electroporation, centrifugation, surface active agents (Triton X-100, ethanol), microwave irradiation, heating, freezing-thawing, ultrasound (ultrasonic bath, sonotrode). The fluorescence intensity of individual fluorescein loaded lipid vesicles was measured with confocal laser microscopy. The distribution of lipid vesicle size, vesicle fluorescence intensity, and the number of fluorescein loaded vesicles was determined before and after treatment with different stressors. The different environmental stressors were ranked in order of their relative effect on liposome fluorescein release. Of all tested chemical, physical and mechanical treatments for stability of lipid vesicles, the most detrimental effect on vesicles stability had hydrodynamic cavitation, vortex mixing with glass beads and ultrasound. Here we showed, for the first time that hydrodynamic cavitation was among the most effective physico-chemical treatments in destroying lipid vesicles. This work provides a benchmark for lipid vesicle robustness to a variety of different physico-chemical and mechanical parameters important in lipid vesicle preparation and application.