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.

Structure of a novel exopolysaccharide produced by Burkholderia vietnamiensis, a cystic fibrosis opportunistic pathogen.

Burkholderia vietnamiensis belongs to the Burkholderia cepacia complex and is an opportunistic pathogen for cystic fibrosis patients. As many other Burkholderia species, it has a mucoide phenotype, producing abundant exopolysaccharide. In general, polysaccharides contribute to bacterial survival in a hostile environment, are recognised as virulence factors and as important components in biofilm formation. The primary structure of the exopolysaccharide produced by B. vietnamiensis LMG 10929 was determined mainly by use of 1D and 2D NMR spectroscopy and ESI mass spectrometry. The polymer consists of the trisaccharidic backbone 3)-ß-D-Glcp-(1→4)-α-D-Glcp-(1→3)-α-L-Fucp-(1→ with the side chain α-D-Glcp-(1→4)-α-D-GlcAp-(1→3)-α-L-Fucp-(1→ linked to C-3 of the α-D-Glcp residue. The polysaccharide also bears acetyl substituents on about 20% of its repeating units and on at least two different positions. The presence of fucose residues is a novel structural feature among the exopolysaccharides produced by species of the B. cepacia complex.

Investigation of bacterial resistance to the immune system response: cepacian depolymerisation by reactive oxygen species.

Reactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS. Species belonging to the Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography, NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of some oligomers produced by NaClO oxidation is reported.

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.