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

Abstract

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.

Figure 1. Schematic for extraction and purification of EPS exopolysaccharides.

Depending on strain, yields range from 2–15 mg of purified polysaccharide per starting 1.2 L of sludge.

Figure 2. EPS staining of E. coli strain 43894 biofilms.

The FITC-labeled mannose-specific HHA lectin was used to stain exopolysaccharides (green) and Hoechst 33342 was used to stain the bacterial nucleic acids (blue). (A.) Extracellular green staining of the EPS by FITC-HHA can be seen on 1 day old biofilms of E. coli at 200X. Scale bar  = 5 µm. (B.) Confocal image of 3 day old E. coli biofilms at 63X. The large square panel is a plan view looking down on the biofilm. The top and right-side rectangular panels are vertical sections representing the XZ plane and YZ plane, respectively, at the positions indicated by the colored lines. The biofilm is 40 µm thick (i.e Z-axis).