Purification and Characterization of Biofilm-Associated EPS Exopolysaccharides from ESKAPE Organisms and Other Pathogens

In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kD...

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Bibliographic Details
Published in:	PloS one Vol. 8; no. 6; p. e67950
Main Authors:	Bales, Patrick M, Renke, Emilija Miljkovic, May, Sarah L, Shen, Yang, Nelson, Daniel C
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 21-06-2013 Public Library of Science (PLoS)
Subjects:	Acids Acinetobacter - physiology Acinetobacter baumannii Alginic acid Analysis Antimicrobial agents Arabinose Bacteria Biofilms Biology Biopolymers Biotechnology Carbohydrates Chromatography Drug resistance E coli Escherichia coli Escherichia coli - physiology Exopolysaccharides Fucose Galactose Gas Chromatography-Mass Spectrometry Glucosamine Glucose Klebsiella Klebsiella - physiology Klebsiella pneumoniae Linkage analysis Lipids Mannose Mannose - analysis Microscopy, Fluorescence Molecular weight N-Acetylglucosamine Nucleic acids Pathogenesis Pathogenic microorganisms Pathogens Pneumonia Polysaccharides, Bacterial - analysis Polysaccharides, Bacterial - chemistry Polysaccharides, Bacterial - isolation & purification Polysaccharides, Bacterial - metabolism Proteins Pseudomonas aeruginosa - physiology Rhamnose Slime Sludge Staphylococcus - physiology Staphylococcus epidermidis Strains (organisms) Xylose United States > US Maryland
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Summary:	In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kDa) exopolysaccharides from biofilms of eight human pathogens, including species of Staphylcococcus, Klebsiella, Acinetobacter, Pseudomonas, and a toxigenic strain of Escherichia coli O157:H7. Glycosyl composition analysis indicated a high total mannose content across all strains with P. aeruginosa and A. baumannii exopolysaccharides comprised of 80-90% mannose, K. pneumoniae and S. epidermidis strains containing 40-50% mannose, and E. coli with ∼10% mannose. Galactose and glucose were also present in all eight strains, usually as the second and third most abundant carbohydrates. N-acetyl-glucosamine and galacturonic acid were found in 6 of 8 strains, while arabinose, fucose, rhamnose, and xylose were found in 5 of 8 strains. For linkage analysis, 33 distinct residue-linkage combinations were detected with the most abundant being mannose-linked moieties, in line with the composition analysis. The exopolysaccharides of two P. aeruginosa strains analyzed were consistent with the Psl carbohydrate, but not Pel or alginate. The S. epidermidis strain had a composition rich in mannose and glucose, which is consistent with the previously described slime associated antigen (SAA) and the extracellular slime substance (ESS), respectively, but no polysaccharide intracellular adhesion (PIA) was detected. The high molecular weight exopolysaccharides from E. coli, K. pneumoniae, and A. baumannii appear to be novel, based on composition and/or ratio analysis of carbohydrates.
Bibliography:	Conceived and designed the experiments: PMB DCN. Performed the experiments: PMB EMR SLM YS. Analyzed the data: PMB DCN. Wrote the paper: PBM DCN. Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0067950