Imidazolium-based titanium substrates against bacterial colonization.

Nosocomial infections are often induced by the presence of pathogenic organisms on the surface of medical devices or hospital equipment. Chemical modifications of the surface are recognized as efficient strategies to prevent bacterial adhesion but they may have a negative impact on the material's interaction with living tissues. Here we have developed a photoactivated method for the modification of titanium substrates. A photoinduced technique employing a grafting-onto process has been successfully performed to covalently anchor an imidazolium-derivative siloxane onto titanium surfaces. Imidazolium surfaces showed higher bacteria-repellency performances than native titanium substrates, achieving more than 98% anti-adhesion efficiency against Escherichia coli after 24 h of incubation. In addition, these surfaces allowed for the adhesion and viability of osteoblasts cells without evidence of cytotoxicity.

Eosin-mediated synthesis of polymer coatings combining photodynamic inactivation and antimicrobial properties.

Polymer coatings exhibiting photodynamic bacterial inactivation properties have been successfully engineered. Such coatings were obtained by photoinduced crosslinking of a PEG-diacrylate monomer associated with the eosin Y dye which was used as both a radical photoinitiator and an antibacterial agent. A dual curing process was followed by combining compatible and solvent-free polymerization mechanisms, i.e. Aza-Michael reaction and free-radical polymerization in the presence of amines. The kinetics evolution of the photopolymerization process was followed using in situ Fourier transform infrared spectroscopy, allowing for the elucidation of the underlying mechanistic pathways. The influence of eosin Y and amines on the thermal and mechanical properties of the films was evidenced and discussed in terms of crosslinking chemistry. The antibacterial properties of the coatings against two different strains (Escherichia coli and Staphylococcus aureus) were evaluated on short and long terms, revealing that eosin confers both photodynamic inactivation and antimicrobial properties to the films. These coatings are therefore particularly promising for disposable medical devices.

Effect of glucose on glycerol metabolism by Clostridium butyricum DSM 5431.

The levels of 1,3-propanediol dehydrogenase and of the glycerol dehydrogenase in Clostridium butyricum grown on glucose-glycerol mixtures were similar to those found in extracts of cells grown on glycerol alone, which can explain the simultaneous glucose-glycerol consumption. On glycerol, 43% of glycerol was oxidized to organic acids to obtain energy for growth and 57% to produce 1,3-propanediol. With glucose-glycerol mixtures, glucose catabolism was used by the cells to produce energy through the acetate-butyrate production and NADH, whereas glycerol was used chiefly in the utilization of the reducing power since 92-93% of the glycerol flow was converted through the 1,3-propanediol pathway. The apparent K(m)s for the glycerol dehydrogenase was 16-fold higher for the glycerol than that for the glyceraldehyde in the case of the glyceraldehyde-3-phosphate dehydrogenase and fourfold higher for the NAD+, providing an explanation for the shift of the glycerol flow toward 1,3-propanediol when cells were grown on glucose-glycerol mixtures.

Properties of Allyl Alcohol-Resistant Mutants of Clostridium butyricum Grown on Glycerol.

Mutants of Clostridium butyricum E5 exhibiting resistance to allyl alcohol which produced the same quantities of 1,3-propanediol as the wild-type strain but more acetate than butyrate were isolated. The acetate-butyrate formation plays a major function in the regulation of the internal redox balance. Allyl alcohol resistance can be attributed not to the loss of 1,3-propanediol dehydrogenase but to a shift in the reductive properties of the enzyme. The data support the view that cellular regulation is modified to avoid intracellular accumulation of 3-hydroxypropionaldehyde.

Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose.

The metabolism of Clostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (q glucose = 11.1 mmol h-1 g-1) was around four times lower than the specific rate of glycerol catabolism (q glucose = 57.4 mmol h-1 g-1). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways.

Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids.

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain.

Isolation and characterization of exocellular polysaccharides produced by Bifidobacterium longum.

When grown anaerobically at pH values above 5.0, on ultrafiltered complex media containing excess lactose, Bifidobacterium longum formed up to 140 mg l-1 (glucose equiv.) exopolysaccharides. The highest yield was obtained when the cells were cultivated in a peptone/yeast extract medium with pH controlled by additions of NH4OH. Whatever the conditions under study, exopolysaccharides represented about 30% of the polysaccharides produced by B. longum after 48 h of culture. Crude pronase-treated exopolysaccharide preparations were adsorbed on ion-exchange chromatographic resin to yield an anionic heteropolysaccharide fraction. Two subfractions with apparent molecular masses of 1.2 MDa and 0.36 MDa respectively were subsequently recovered after gel filtration on Sepharose 4B. In both subfractions, glucose, galactose and small amounts of uronic acids and hexosamines were present in similar molar proportions, suggesting that the excreted polymers may be synthesized from the same base unit and may have a structure resulting from repeating subunits.