Pentadecanoic acid against Candida albicans-Klebsiella pneumoniae biofilm: towards the development of an anti-biofilm coating to prevent polymicrobial infections.

The ability to form biofilms is a common feature of microorganisms, which can colonize a variety of surfaces, such as host tissues and medical devices, resulting in infections highly resistant to conventional drugs. This aspect is particularly critical in polymicrobial biofilms involving both fungi and bacteria, therefore, to eradicate such severe infections, new and effective anti-biofilm strategies are needed. The efficacy of pentadecanal and pentadecanoic acid as anti-biofilm agents has been recently reported against different bacterial strains. Their chemical similarity with diffusible signal factors (DSFs), plus the already known ability of fatty acids to act as anti-biofilm agents, suggested to explore their use against Candida albicans and Klebsiella pneumoniae mixed biofilm. In this work, we demonstrated the ability of both molecules to prevent the formation and destabilize the structure of the dual-species biofilm. Moreover, the pentadecanoic acid anti-biofilm coating, previously developed through the adsorption of the fatty acid on polydimethylsiloxane (PDMS), was proved to prevent the polymicrobial biofilm formation in dynamic conditions by confocal laser scanning microscopy analysis. Finally, the evaluation of the expression levels of some biofilm-related genes of C. albicans and K. pneumoniae treated with pentadecanoic acid provided some insights into the molecular mechanisms underpinning its anti-biofilm effect.

Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.

Structure of Lipid A from Pseudomonas corrugata by electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

The use of the electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOFMS) technique for the structural determination of Lipid A from Pseudomonas corrugata is described. This technique appears to be more sensitive with respect to other commonly used tandem mass spectrometric approaches, and was very valuable in the structural determination of the highly heterogeneous Lipid A fractions. The Lipid A fraction consists mainly of a pentaacyl component in which 3-hydroxydecanoyl [10:0(3-OH)] and 3-hydroxydodecanoyl [12:0(3-OH)] are linked as primary acyl substituents to the classical bisphosphorylated beta-(1' --> 6)-linked D-glucosamine disaccharide. Secondary substitution of N-acyl fatty acids with dodecanoyl residues [12:0] and/or its 2-OH derivatives was also observed.

Determination of phosphorylation sites in lipooligosaccharides from Pseudoalteromonas haloplanktis TAC 125 grown at 15 degrees C and 25 degrees C by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Lipooligosaccharides (LOSs) are macromolecules present on the external cellular membrane of Gram-negative bacteria, structurally made of two distinct regions, lipid A and Core. By varying their growth temperature, bacteria such as psychrophiles change the phosphorylation distribution of the LOSs produced. The level of phosphorylation and the phosphate group positions in LOSs produced by the extremophile psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125, grown at 15 degrees C and 25 degrees C, were investigated by nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) and tandem mass spectrometry (MS/MS). The samples, obtained by phenol/chloroform/petroleum ether (PCP) extraction of dried cells, were treated with hydrazine at 37 degrees C in order to reduce the heterogeneity by removal of the ester-linked fatty acid moieties. The molecular ion distributions in these LOS fractions were investigated in negative ion mode. Based on these data it was postulated that the sample grown at 25 degrees C contained four phosphate groups while that at 15 degrees C contained three. In order to determine phosphorylation sites in sugar chains, the samples were submitted to low collision energy MS/MS for sequencing. In the sample with three phosphates, one was found to be linked to the tetrasaccharide Core region, more precisely to position C-4 of the Kdo unit. The two remaining phosphate groups were both linked to the 2-acylamide-2-deoxy-D-glucopyranose of the lipid A moiety, and two possible distributions could be postulated on the basis of the fragmentation pattern obtained; in the first case both phosphate groups are linked as a pyrophosphate moiety to position C-1 of the proximal glucosamine (reducing residue), while in the second case one phosphate is linked to position C-1 of the proximal glucosamine and the other to position C-4' of the distal glucosamine (non-reducing residue). This distribution was also found in the lipid A moiety of the tetraphosphorylated sample grown at 25 degrees C, which bears two phosphate groups on the Core region, one on position C-4 of the Kdo and the other on position C-7 or C-8 of the same residue. The phosphate locations were derived from the intra-ring cleavage ions of sugar moieties in the LOSs obtained by an optimized CID procedure using negative ion QTOF-MS/MS.

Structural investigation on the lipooligosaccharide fraction of psychrophilic Pseudoalteromonas haloplanktis TAC 125 bacterium.

The core structure of the cell-wall lipooligosaccharide (LOS) fraction of an Antarctic Gram-negative bacterium, Pseudoalteromonas haloplanktis TAC 125 strain, was determined to be deacetylated alditols. These were obtained from native LOS fraction by O-deacylation, dephosphorylation, reduction and finally N-deacylation. Two novel structures were detected, the more highly represented molecule consisting of the following hexasaccharide chain: alpha-D-ManpNH(2)-(1-->3)-beta-D-Galp-(1-->4)-alpha-L-glycero-D-manno-Hepp-(1-->5)-alpha-D-Kdo-(2-->6)-beta-D-GlcpNH(2)-(1-->6)-D-GlcNH(2)(ol) while the corresponding pentasaccharide, lacking the ManpNH(2) residue, was less abundant. To the best of our knowledge, the structural investigation presented here, mainly performed by NMR and MS methods, is the first report of the lipopolysaccharide fraction of a psychrophilic bacterium.

Structural determination of the phytotoxic mannan exopolysaccharide from Pseudomonas syringae pv. ciccaronei.

The structural determination was performed of a mannan exopolysaccharide from the gram negative bacterium Pseudomonas syringae pv. ciccaronei, which is the pathogenic agent responsible for the leaf spots of carob plants. The structure, obtained by chemical, enzymatic and spectroscopic methods, consisted of a backbone of alpha-(1-->6)-linked mannopyranose units with 80% substituted at C-2 by mono-, di- and trisaccharide side chains. In addition, terminal glucose units and phosphate groups were found to be present. This is, to the best of our knowledge, the first report of a mannan exopolysaccharide structure from a phytopathogenic bacterium. The pure polysaccharide showed phytotoxic effects, i.e., chlorosis and necrosis on tobacco leaves.

Structural characterization of a xylanase from psychrophilic yeast by mass spectrometry.

The complete structural characterization of the xylanase, a glycoprotein constituted of 338 amino acids, from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. The verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites and the investigation of glycosylation pattern were performed. This analysis revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures. The data obtained on both the N- and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophylic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophylic and thermophylic cells. The cysteine pairings were eventually identified as Cys173-Cys205 and Cys272-Cys278, with Cys89 showing a free thiol group. These data suggest that a common folding motif might occur within the entire xylanase family in which the second Cys is linked to the third one with the fourth and fifth joined together.

Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp.

An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp. was found to contain D-galactopyranuronic acid (GalpA), 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (QuipNAc4NAc), 2-acetamido-2-deoxy D-mannopyranuronic acid (ManpNAcA) and one uncommon unit of D-galactopyranuronic acid with the carboxyl group amide-linked to glycine [GalpA(Gly)]. The polysaccharide was studied by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis. The following linear structure of the repeating unit was established: -->3)-alpha-D-GalpA(Gly)-(1-->4)-beta-D-ManpNAcA-(1-->4)-alp ha-D-Galp A-(1-->3)-alpha-D-QuipNAc4NAc-(1-->. A preliminary phylogenetic assignment for the bacterium is also reported.

Chemical structure of two phytotoxic exopolysaccharides produced by Phomopsis foeniculi.

The two main exocellular polysaccharides produced in vitro by Phomopsis foeniculi, a fungal pathogen of fennel, were isolated and characterized by chemical and spectroscopic methods as a galactan with the known structure [-->6)-beta-D-Galf-(1-->5)-beta-D- Galf-(1-->5)-beta-D-Galf-(1-->]n and a mannan. The latter consists of a backbone of alpha-(1-->6)-linked mannopyranose units. Almost all of these are branched at the 2 position with arms containing 2- and 3-linked mannopyranose units. The crude polysaccharide fraction and its components, galactan and mannan, showed phytotoxic effects, i.e. chlorosis, necrosis and/or wilting, on fennel and on two non-host plants, tobacco and tomato.

Structural determination of the O-deacetylated O-chain of lipopolysaccharide from Burkholderia (Pseudomonas) cepacia strain PVFi-5A.

On the basis of chemical degradation methods and one-and two-dimensional 1H and 13C NMR experiments the novel following structure was established for the O-deacetylated repeating unit of the O-chain of the main Burkholderia (Pseudomonas) cepacia (strain PVFi-5A) lipopolysaccharide: -->4)-beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->6)-alpha-D-GlcpNAc-(1-->.

Lipopolysaccharides from three phytopathogenic pseudomonads.

Analysis of the polysaccharide and lipid moieties of the lipopolysaccharides (LPSs) of the phytopathogenic bacteria, Pseudomonas amygdali and P. syringae pv. ciccaronei has demonstrated that for both bacteria, the O-chain consists of a tetrasaccharide repeating unit of three alpha-L-Rhap and one terminal nonreducing alpha-D-Fucp3NAc. Two of the rhamnosyl residues are 3-linked, the third one 2,3-linked. This structure had been previously found for the O-chains of three phytopathogenic strains of P. syringae subsp. savastanoi, but this is the first report on its occurrence in P. amygdali and P. syringae pv. ciccaronei. The results of the LPS lipid residue analysis made it possible to make some chemotaxonomic considerations and therefore classify P. amygdali as a chemotype, which is different from that of the other two bacteria examined.

Structural investigation of the polysaccharide fraction from the mucilage of Diceroaryum zanguebaricum Merr.

The polysaccharide fraction from the mucilage of Dicerocaryum zanguebaricum (Pedaliaceae) appears to be mainly constituted of a chemically homogeneous polysaccharide. By NMR and chemical degradative methods its structure appeared to consist of alternate-->4)-beta-D-GlcpA-(1--> and -->2)-alpha-D-Man p-(1-->units. Single branch units of beta-D-Xyl p and alpha-D-Gal p are linked to the O-3 positions of Man p and a significant number of Glc pA residues.

Polysaccharides from seeds of Strychnos species.

The chemical composition of polysaccharide fractions from Strychnos nux-vomica and S. innocua seeds and comparison with those from S. potatorum seeds are reported. The structural features of the galactomannans from the three Strychnos species are also discussed.

Composition of the coagulant polysaccharide fraction from Strychnos potatorum seeds.

The composition of the coagulant polysaccharide fraction from Strychnos potatorum seeds is described. This fraction comprises a 1:1.7 mixture of a galactomannan and a galactan. The structure of these polysaccharides is also discussed. In addition, the coagulant properties of the polysaccharide fractions of two other Strychnos species, innocua and nux-vomica, have been assayed.

Cycloartane glucosides from Juncus effusus.

Five new cycloartane glucosides, juncosides I-V, have been isolated from Juncus effusus. The structures have been determined on the basis of chemical and spectroscopic studies.

Structural investigation of Ceratozamia spinosa mucilage.

The polysaccharide fraction from Ceratozamia spinosa appears to be made up mainly by a chemically homogeneous polysaccharide but with a wide range of molecular weight. By NMR and chemical degradative methods, it is shown to consist essentially of a backbone of alternate-->4)-beta-D-GlcpA-(1-->and-->2)-alpha-D-Manp-(1--> units. On the 4 position of the latter, beta-D-GlcpA residues are linked. End units of alpha-L-Araf, beta-D-Xylp, alpha-L-Rhap, and alpha-L-3-OMe-Rhap are linked to C-3 and/or C-4 positions of beta-D-GlcpA residues.

Triterpenoid oligoglycosides from Chionodoxa luciliae.

Four minor novel oligoglycosides of triterpenes having a lanostane-type skeleton were isolated from the bulbs of Chionodoxa luciliae. The C30 skeleton of the aglycones has been found for the first time in Liliaceae and can be considered the metabolic precursor of the C29 eucosterol skeleton.

Nortriterpenoid oligoglycosides from Chionodoxa luciliae.

Six new oligoglycosides of eucosterol derivatives were isolated from the bulbs of Chionodoxa luciliae Bossis. A chemotaxonomical relationship based on glycoside and homoisoflavanone content is revealed for some Muscari, Bellevalia, Chionodoxa and Scilla species.