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.

Bacteria coordinate their behavior using quorum sensing (QS), whereby cells secrete diffusible signals that generate phenotypic responses associated with group living. The canonical model of QS is one of extracellular signaling, where signal molecules bind to cognate receptors and cause a coordinated response across many cells. Here we study the link between QS input (signaling) and QS output (response) in the ComQXPA QS system of Bacillus subtilis by characterizing the phenotype and fitness of comQ null mutants. These lack the enzyme to produce the ComX signal and do not activate the ComQXPA QS system in other cells. In addition to the activation effect of the signal, however, we find evidence of a second, repressive effect of signal production on the QS system. Unlike activation, which can affect other cells, repression acts privately: the de-repression of QS in comQ cells is intracellular and only affects mutant cells lacking ComQ. As a result, the QS signal mutants have an overly responsive QS system and overproduce the secondary metabolite surfactin in the presence of the signal. This surfactin overproduction is associated with a strong fitness cost, as resources are diverted away from primary metabolism. Therefore, by acting as a private QS repressor, ComQ may be protected against evolutionary competition from loss-of-function mutations. Additionally, we find that surfactin participates in a social selection mechanism that targets signal null mutants in coculture with signal producers. Our study shows that by pleiotropically combining intracellular and extracellular signaling, bacteria may generate evolutionarily stable QS systems.social interactions | social evolution B acteria secrete and share quorum-sensing (QS) signaling molecules that bind to specific receptors, and upon reaching critical concentration induce cell density-dependent adaptive responses within the population (1). In Gram-positive bacteria small peptide QS signals are typically produced from oligopeptide precursors that are modified by a signal-processing enzyme before they are secreted from the cell. It is assumed that these bacteria secrete QS signals during growth, and then in the stationary phase when the signal threshold concentration is reached, the signals activate specific histidine kinase receptors. These activate specific response regulators through phosphotransfer, which then initiate a QS response (2). The QS response often involves expression of adaptive extracellular factors (such as food-degrading enzymes, virulence factors, antibiotics, or biosurfactants) that are considered public goods (3, 4), as they can be shared within the population. Recently, however, it was shown that the QS of Gramnegative bacteria may also regulate adaptive metabolic pathways that produce molecules that remain private as they are not secreted by the responsive cells (5).The ComQXPA QS system of Bacillus subtilis is a typical QS system of Gram-positive bacteria that controls expression of nearly 200 genes, including both extracellular and private...

Extracellular polymeric substances (EPS) play an important role in cell aggregation, cell adhesion, and biofilm formation, and protect cells from a hostile environment. The EPS was isolated by trichloroacetic acid/ethanol extraction from broth culture of a marine bacterium isolate. The EPS was composed of glucose and galactose as determined by HPLC and TLC; the protein content was on average 15 +/- 5% of EPS dry mass. The solution structure of EPS at different values of pH was revealed by small-angle x-ray scattering. Scattering curves of EPS solutions (0.4%, w/v) consistently showed two nearly linear log-log regions with slopes a and b in the q-ranges from 0.06 nm(-1) to 0.26 nm(-1), and from 0.27 nm(-1) to 0.88 nm(-1), respectively. Slope a was sensitive to pH changes whereas slope b was not. The observed sensitivity to pH was not a consequence of ionic strength variation with pH, as checked by salt addition. The pH variation causes major rearrangements of EPS structure mainly at length scales above 24 nm. To get a better understanding of the pH effect on EPS structure, the original model proposed by Geissler was refined into a mathematical model that enabled fitting of the experimental scattering curves in the pH range from 0.7 to 11.0. The model describes EPS structure as a network of randomly coiled polymeric chains with denser domains of polymeric chains. The results obtained from the model indicate that dense domains increase in average size from 19 nm at pH 11.0 to 52 nm at pH 0.7. The average distance between the polysaccharide chains at pH 0.7 was 2.3 nm, which indicates a compact EPS structure. Swelling was found to be at a maximum around pH = 8.8, where the average distance between the chains was 4.8 nm.