Discovery of potent 1,1-diarylthiogalactoside glycomimetic inhibitors of Pseudomonas aeruginosa LecA with antibiofilm properties.

In this work, ß-thiogalactoside mimetics bearing 1,1-diarylmethylene or benzophenone aglycons have been prepared and assayed for their affinity towards LecA, a lectin and virulence factor from Pseudomonas aeruginosa involved in bacterial adhesion and biofilm formation. The hit compound presents higher efficiency than previously described monovalent inhibitors and the crystal structure confirmed the occurrence of additional contacts between the aglycone and the protein surface. The highest affinity (160 nM) was obtained for a divalent ligand containing two galactosides. The monovalent high affinity compound (Kd = 1 µM) obtained through structure-activity relationship (SAR) showed efficient antibiofilm activity with no associated bactericidal activity.

Impairment of Pseudomonas aeruginosa Biofilm Resistance to Antibiotics by Combining the Drugs with a New Quorum-Sensing Inhibitor.

Pseudomonas aeruginosa plays an important role in chronic lung infections among patients with cystic fibrosis (CF) through its ability to form antibiotic-resistant biofilms. In P. aeruginosa, biofilm development and the production of several virulence factors are mainly regulated by the rhl and las quorum-sensing (QS) systems, which are controlled by two N-acyl-homoserine lactone signal molecules. In a previous study, we discovered an original QS inhibitor, N-(2-pyrimidyl)butanamide, called C11, based on the structure of C4-homoserine lactone, and found that it is able to significantly inhibit P. aeruginosa biofilm formation. However, recent data indicate that P. aeruginosa grows under anaerobic conditions and forms biofilms in the lungs of CF patients that are denser and more robust than those formed under aerobic conditions. Our confocal microscopy observations of P. aeruginosa biofilms developed under aerobic and anaerobic conditions confirmed that the biofilms formed under these two conditions have radically different architectures. C11 showed significant dose-dependent antibiofilm activity on biofilms grown under both aerobic and anaerobic conditions, with a greater inhibitory effect being seen under conditions of anaerobiosis. Gene expression analyses performed by quantitative reverse transcriptase PCR showed that C11 led to the significant downregulation of rhl QS regulatory genes but also to the downregulation of both las QS regulatory genes and QS system-regulated virulence genes, rhlA and lasB. Furthermore, the activity of C11 in combination with antibiotics against P. aeruginosa biofilms was tested, and synergistic antibiofilm activity between C11 and ciprofloxacin, tobramycin, and colistin was obtained under both aerobic and anaerobic conditions. This study demonstrates that C11 may increase the efficacy of treatments for P. aeruginosa infections by increasing the susceptibility of biofilms to antibiotics and by attenuating the pathogenicity of the bacterium.

Antibacterial, antifungal and antileishmanial activities of indolone-N-oxide derivatives.

An alarming increase in microbial resistance to traditional drugs and classical pharmacophores has spurred the search for new antimicrobial compounds. Indolone-N-oxides (INODs) possess a redox pharmacophore with promising, recently established, antimalarial activities. In this study, the anti-infectious properties of a series of INODs were investigated. The antibacterial activity was evaluated against five bacterial strains Gram-positive (Staphylococcus aureus, Enterococcus hirae), Gram-negative (Pseudomonas aeruginosa, Escherichia coli) and acid-fast (Mycobacterium tuberculosis). The antifungal activity was assessed using two fungal strains (Aspergillus niger, Candida albicans). The antileishmanial activity was tested against two leishmanial strains, axenically-cultured amastigote (Leishmania infantum, Leishmania amazonensis). The pharmacological activities are discussed as a function of structural and lipophilic characteristics. The Gram-positive bacterial strain E. hirae was found to be the most sensitive strain, whereas the Gram-negative E. coli was resistant to this family of compounds. One compound (64) was more potent than nalidixic acid against E. hirae, whereas another one (52) was equipotent as clotrimazole against C. albicans. INODs were microbe -cidal rather than -static. INODs showed good antitubercular activity in the low micromolar range (similar to ciprofloxacin). In addition, INOD-antiprotozoal potencies were confirmed against the leishmania parasite. INODs showed a broad spectrum of antimicrobial activity and offer a promising anti-infectious prototype worthy of being developed.

Effects of ionic strength on bacteriophage MS2 behavior and their implications for the assessment of virus retention by ultrafiltration membranes.

Bacteriophage MS2 is widely used as a surrogate to estimate pathogenic virus elimination by membrane filtration processes used in water treatment. Given that this water technology may be conducted with different types of waters, we focused on investigating the effects of ionic strength on MS2 behavior. For this, MS2 was analyzed while suspended in solutions of various ionic strengths, first in a batch experiment and second during membrane ultrafiltration, and quantified using (i) quantitative reverse transcriptase PCR (qRT-PCR), which detects the total number of viral genomes, (ii) qRT-PCR without the RNA extraction step, which reflects only particles with a broken capsid (free RNA), and (iii) the PFU method, which detects only infectious viruses. At the beginning of the batch experiments using solutions containing small amounts of salts, losses of MS2 infectivity (90%) and broken particles (20%) were observed; these proportions did not change during filtration. In contrast, in high-ionic-strength solutions, bacteriophage kept its biological activity under static conditions, but it quickly lost its infectivity during the filtration process. Increasing the ionic strength decreased both the inactivation and the capsid breakup in the feed suspension and increased the loss of infectivity in the filtration retentate, while the numbers of MS2 genomes were identical in both experiments. In conclusion, the effects of ionic strength on MS2 behavior may significantly distort the results of membrane filtration processes, and therefore, the combination of classical and molecular methods used here is useful for an effective validation of the retention efficiency of ultrafiltration membranes.

Growth inhibition of adherent Pseudomonas aeruginosa by an N-butanoyl-L-homoserine lactone analog.

The discovery of quorum sensing (QS) communication systems regulating bacterial virulence has afforded a novel opportunity for controlling infectious bacteria by interfering with QS. Pseudomonas aeruginosa is an example of an opportunistic human pathogen for which N-acyl homoserine lactone (AHL)-related compounds have been described as potent inhibitors of biofilm formation and virulence factors, given their similarity to the natural QS autoinducers (AHLs). Our purpose was to design potent analogs of N-butanoyl-L-homoserine lactone (C4-HSL) and to screen them for biological activity. Eleven original compounds characterized by the modification of the lactone moiety were screened for their ability to impair biofilm formation. Among them, compound 11 was able to modify the growth kinetics and to restrict the number of adherent cells when added from the early stages of biofilm formation (i.e., adhesion and microcolony formation) in a dose-dependent manner. To demonstrate antagonism with C4-HSL, we showed that the inhibition of biofilm formation by compound 11 was impaired when C4-HSL was added. Structure-activity relationships are discussed with respect to the results obtained.

Effect of xylitol on an in vitro model of oral biofilm.

PURPOSE: The aim of the present study was to examine whether xylitol, at different concentrations, inhibits the formation of an experimental model of oral biofilm. MATERIALS AND METHODS: Biofilms of six bacterial species (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus rhamnosus, Actinomyces viscosus, Porphyromonas gingivalis and Fusobacterium nucleatum) were prepared on hydroxyapatite (HA) discs according to the Zürich Biofilm Model. Xylitol was tested at two concentrations, 1% and 3%. At the end of their designated incubation times, some HA discs were destined for confocal laser scanning microscopy (CLSM) and the others were harvested using a sterile surgical instrument. Aliquots of harvested biofilms were diluted and plated onto specific media. After a 48-h anaerobic incubation at 37 degrees C, the colony-forming units (CFUs) were counted. RESULTS: CLSM images showed that only a small amount of isolated bacteria was observed on the surface of HA discs. Culture of harvested biofilms showed an inhibition in the growth of different species included in the biofilms. CONCLUSIONS: Xylitol has a clear inhibitory effect on the formation of the experimental biofilms. This study shows that xylitol is not only efficient in inhibiting the acid production of cariogenic bacteria, but also in preventing the formation of a multispecies biofilm; it confirms the relevance of the use of this polyol for the prevention of oral diseases caused by dental plaque.

Hopeaphenol: the first resveratrol tetramer in wines from North Africa.

Grapes and wines are now known to constitute a rich source of phenolics such as stilbenes and flavonoids. These compounds have been shown to have cancer chemopreventive activity and potential beneficial effects on cardiovascular diseases thanks to their antioxidant and antiplatelet properties. However, because little is known about African wines and their phenolic compositions, we investigated wine samples from North Africa. A three-step method was used for the fractionation of the Merlot variety wine: column chromatography followed by centrifugal partition chromatography and reversed-phase semipreparative high-performance liquid chromatography (HPLC). Six polyphenolic compounds of the Merlot variety (from Algeria) were isolated and identified by NMR spectroscopy, five of which are known (trans-resveratrol, trans-piceid, trans-epsilon-viniferin, pallidol, and astilbin) and one that is reported for the first time in wine, (+)-hopeaphenol, a stilbene tetramer. Furthermore, these molecules were quantified in 10 commercial wines from North Africa by means of an analytical HPLC system coupled with diode array detection. Differences in concentrations were found ranging in mg/L from 4.6 to 45 (trans-piceid), 0.66 to 3.45 (trans-resveratrol), 0.2 to 1.2 (trans-epsilon-viniferin), 0.2 to 9.2 (pallidol), 0.3 to 3.8 (hopeaphenol), and 10.8 to 24.22 (astilbin). Such a high level of pallidol and astilbin has never been recorded in wine. North African wines may contribute to a significant proportion of dietary intake of stilbene and astilbin, which may have health benefits.

The influence of storage conditions on delta amino levulinic acid induced toxicity and phototoxicity in vitro.

Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent advances in PDT. ALA itself or its derivatives are marketed for a variety of clinical indications. Despite the development of clinical applications, experimental ALA results are very heterogeneous and experimentally used parameters are still not standardized. This suggests that some problems remain unsolved that are likely to impair experiments to be performed but also that clinical results obtained could be greatly improved. Frequently unmentioned or imprecise data concern solvents, pH of ALA solutions, storage time, ALA degradation or ALA efficacy. In addition, diversity of experimental model is huge while capabilities of ALA transformation into PpIX are known to vary from one cell to the other. Thus, the aim of the present paper was to quantify the level of ALA degradation or changes in ALA efficacy using one single cell line without presuming of the mechanisms and determine the conditions of storage inducing the best transformation into PpIX and/or cell phototoxicity. We added ALA diluted in water, PBS or RPMI to C6 cells, a murine brain tumour cell line that can be used in vivo as an orthotopic graft. We measured in cells used as tools for final bio efficacy estimation, both the induced fluorescence and phototoxicity in various conditions of storage before use chosen to be as close as possible to the real lab conditions. Water had been found to better preserve ALA than, respectively, PBS and RPMI and this for any temperature or storage durations. The lowest temperature and the shortest duration for storage used had also been shown to better preserve ALA-induced fluorescence and phototoxicity. The fact that these properties were found to be better preserved in 7.4 buffered solvent could be in relationship with a fast ALA condensation occurring at neutral or lightly acidic pH modifying its availability for an optimal transformation into PpIX.