Interactions of biofilm polysaccharides produced by human infective bacteria with molecules of the quorum sensing system. A microscopy and NMR study

Biofilms are the most common lifestyle adopted by bacterial communities where cells live embedded in a selfproduced hydrated matrix. Although polysaccharides are considered essential for matrix architecture, their possible functional roles are still rather unexplored. The primary structure of polysaccharides produced by Klebsiella pneumoniae and species of the Burkholderia cepacia Complex revealed a composition rich in rhamnose. The methyl group on carbon 6 of rhamnose units lowers the polymer hydrophilicity and can form low polarity regions on the polysaccharide chains. These regions promote chain-chain interactions that contribute to the biofilm matrix stability, but may also act as binding sites for low-polarity molecules, aiding their mobility through the hydrated matrix. In particular, quorum sensing system components crucial for the biofilm life cycle often display poor solubility in water. Therefore, cis-11-methyl-2-dodecenoic acid and L-homoserine-lactones were investigated by NMR spectroscopy for their possible interaction with polysaccharides. In addition, the macromolecular morphology of the polysaccharides was assessed using atomic force and electron microscopies to define the role of Rha residues on the three-dimensional conformation of the polymer. NMR data revealed that quorum sensing components interact with Rhamnose-rich polysaccharides, and the extent of interaction depends on the specific primary structure of each polysaccharide

Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type.

Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction

PEG hydration and conformation in aqueous solution: Hints to macromolecular crowding

The molecular structural dynamics of PEG in aqueous solution has been addressed with a series of experimental data correlating macroscopic solution properties of PEG with structural and hydration properties at molecular level, by using thermodynamic, UV Brillouin and Raman spectroscopy data. Water activity measured in PEG 600 solutions by a novel dynamical calorimetry approach shows that data reveal some non-equilibrium process for dilute solutions. Brillouin scattering data on aqueous solutions in the UV range made possible the measurement of the viscoelastic relaxation of the system with a characteristic temperature, TM, as precursor of the glass transition process at lower T, while at constant temperature, the addition of water to liquid PEG 600 first lead to a slight decrease and then to an increase in the solution viscoelastic relaxation consistent with the slowest polymer dynamics observed in the concentrated solution. Specific Raman bands corresponding to trans and gauche conformations of the C\u2013C and C\u2013O bonds and their sequences in the PEG chain have been identified and their relative intensities as a function of concentration evidence non-monotonous variations with a rather unusual concentration dependence for the frequency of C\u2013O bonds in the range of physiological temperatures. The heuristic result is that the extended time-space domains approach suggests an overall quasi-regular solution behavior of semidilute PEG, with opposite concentration dependence at low and at high water content, similar to discontinuities observed in excess thermodynamic properties and eutectic phase diagram. The cross-over of these distinct behaviors occurs at the concentration close to that usually employed to mimic the cellular crowding in biomolecular systems, highlighting the interplay of water molecules in solute-solute interactions

Determination of the capsular polysaccharide structure of the Klebsiella pneumoniae ST512 representative strain KPB-1 and assignments of the glycosyltransferases functions

Klebsiella pneumoniae strain KPB-1 was isolated in early 2011 from the pleural fluid of an inpatient admitted at an Italian hospital. It was characterized to produce the KPC-3 carbapenemase and to belong to sequence type 512, a derivative of sequence type 258 clade II characterized by the cps-2 gene cluster. The K-antigen of K. pneumoniae KPB-1 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives and 1D and 2D NMR spectroscopy of the native polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KPB-1 capsular polysaccharide:[Formula presented] The reactions catalysed by each glycosyltransferase in the cps-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens

Oligosaccharides derived from tramesan: Their structure and activity on mycotoxin inhibition in aspergillus flavus and Aspergillus carbonarius

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental prob-lems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, \u201cgreen\u201d, approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for myco-toxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no sig-nificant effect on mycotoxin inhibition; heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins

Polysaccharide solutions and gels: Isothermal dehydration study by dynamic calorimetric experiments with DSC

The isothermal dehydration of aqueous biosystems is a relevant topic in food, pharmaceutical and cosmetic industry and has been recently investigated for the assessment of a model calorimetric set-up and for the characterization of the parameters featuring the experimental calorimetric curve. In this study, the experimental Differential Scanning Calorimetry (DSC) data obtained under controlled conditions in isothermal mode have been collected on the dehydration of films consisting of solutions and gels of alginate, hydroxypropylmethylcellulose (HPMC), trehalose and mixtures thereof. Based on the proportionality between the calorimetric heat flow and water activity (aw) of solutions of known aw, the values calculated from calorimetry have been compared to those obtained with classic hygrometric measurements revealing a good consistency between the methods. Furthermore, the experimental data were mathematically turned into desorption isotherms, providing a continuous description of the water activity down to the low water activity limit. This experimental method represents an innovative approach to support other consolidated analytical techniques in the physico-chemical characterization of aqueous systems and, more importantly, a step forward in the determination of water activity as a continuous measurement in a timeframe far shorter than that necessary with other instruments (e.g., hygrometers)

Microwave generated solid dispersions containing ibuprofen

The purpose of this study was to apply the attractive technique of the microwaves irradiation (MW) for the preparation of solvent-free solid dispersions (SD). In particular, the microwave technology has been considered in order to prepare an enhanced release dosage form for the poorly soluble drug Ibuprofen (IBU), employing PVP/VA 60/40 (PVP/VA 64) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as hydrophilic carriers. Their physico-chemical characteristics and dissolution properties were compared to the corresponding physical mixtures and the drug alone. The results of physico-chemical characterization attested a correspondence of the solid state of the drug before and after irradiation treatment and that an amorphous form of the drug was obtained. This result, together with the presence of the hydrophilic polymers determined a remarkable enhancement of the in vitro dissolution rate of the drug suggesting that the microwave technique could be considered as a new and interesting method to prepare drug\u2013polymer systems

Proteomic studies of the biofilm matrix including outer membrane vesicles of burkholderia multivorans c1576, a strain of clinical importance for cystic fibrosis

Biofilms are aggregates of microbial cells encased in a highly hydrated matrix made up of self-produced extracellular polymeric substances (EPS) which consist of polysaccharides, proteins, nucleic acids, and lipids. While biofilm matrix polysaccharides are unraveled, there is still poor knowledge about the identity and function of matrix-associated proteins. With this work, we performed a comprehensive proteomic approach to disclose the identity of proteins associated with the matrix of biofilm-growing Burkholderia multivorans C1576 reference strain, a cystic fibrosis clinical isolate. Transmission electron microscopy showed that B. multivorans C1576 also releases outer membrane vesicles (OMVs) in the biofilm matrix, as already demonstrated for other Gram-negative species. The proteomic analysis revealed that cytoplasmic and membrane-bound proteins are widely represented in the matrix, while OMVs are highly enriched in outer membrane proteins and siderophores. Our data suggest that cell lysis and OMVs production are the most important sources of proteins for the B. multivorans C1576 biofilm matrix. Of note, some of the identified proteins are lytic enzymes, siderophores, and proteins involved in reactive oxygen species (ROS) scavenging. These proteins might help B. multivorans C1576 in host tissue invasion and defense towards immune system assaults