Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1-35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

An antarctic psychrotrophic bacterium Halomonas sp. ANT-3b, growing on n-hexadecane, produces a new emulsyfying glycolipid.

A bacterial strain ANT-3b was isolated at the sea-ice seawater interface from Terra Nova Bay station, Ross Sea, Antarctica. It was isolated on mineral medium supplemented with 2% diesel fuel as a sole carbon and energy source and grown routinely on 2% n-hexadecane. Analysis of 16S rRNA gene sequence indicates that the strain has 99.8% sequence similarity with Halomonas neptunia. The strain ANT-3b was grown in mineral medium supplemented with n-hexadecane between 4 and 20 degrees C, but not at 30 degrees C. The maximum degradation rate of the n-alkane was measured at 15 degrees C, with 5.6+/-1.7 mg O2 microg(-1) protein d(-1). The strain ANT-3b produced emulsifying compounds when grown on n-hexadecane, but not on mineral medium supplemented with D-fructose. A preliminary characterisation of the emulsifier was carried out. The lipid moiety contained a mixture of fatty acids with a following composition in molar ratio: caprylic acid 18.85, myristic acid 1.0, palmitic acid 9.68, palmitoleic acid 5.69 and oleic acid 1.26. The polysaccharide moiety also contained a mixture of sugars with the following molar ratio: mannose 1.71, galactose 1.00 and glucose 2.96. The molecular weight of the glycolipid component determined by gel permeation chromatography was in the 18 kDa range and contained smaller fragments, possibly oligomeric contaminants. Transmission electron microscopy showed contact between the glycolipid secreted by the strain and n-hexadecane broken down to nanodroplets at the water interface, to form a material with mesophase (liquid crystal) organisation.

Versatility of the Burkholderia cepacia complex for the biosynthesis of exopolysaccharides: a comparative structural investigation.

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable.

A novel highly charged exopolysaccharide produced by two strains of Stenotrophomonas maltophilia recovered from patients with cystic fibrosis.

Stenotrophomonas maltophilia is a non-fermenting Gram-negative microorganism capable of causing chronic pulmonary infection in cystic fibrosis patients and its ability to form biofilms on polystyrene and glass surfaces, as well as on cystic fibrosis-derived bronchial epithelial IB3-I cells was recently demonstrated. The latter evidence might explain the power of S. maltophilia to produce persistent lung infections, despite intensive antibiotic treatment. In addition to being important components of the extracellular biofilm matrix, polysaccharides are involved in virulence, as they contribute to bacterial survival in a hostile environment. With the aim of contributing to the elucidation of S. maltophilia virulence factors, the exopolysaccharides produced by two mucoid clinical isolates of S. maltophilia obtained from two cystic fibrosis patients were completely characterised, mainly by means of ESI-MS and NMR spectroscopy. The results showed that, although the two isolates were recovered from two different patients living in different countries (Italy and France), the exopolysaccharides produced have an identical primary structure, with the following repeating unit: The exopolysaccharide is highly negatively charged for the presence of three uronic acids on four residues in the repeating unit. Moreover, an ether-linked d-lactate substituent is located on C-3 and one O-acetyl group on C-4 of the galacturonic acid side chain. Another O-acetyl group substitutes C-2 of the galacturonic acid in the backbone, making this primary structure unique.