Characterization of Klebsiella pneumoniae Extracellular Polysaccharides

Abstract

Klebsiella pneumoniae is a clinically significant, Gram-negative pathogen in which the production of extracellular polysaccharides is a key virulence factor. Extracellular polysaccharides such as the capsule and its mucoviscosity play a significant role in K. pneumoniae infection. In this article, we explain several standard protocols used to characterize the extracellular polysaccharides of K. pneumoniae. Several of these protocols are adapted specifically for K. pneumoniae and describe methods to purify and quantify the extracellular polysaccharide of K. pneumoniae. We also present a standardized protocol to quantify K. pneumoniae mucoviscosity, a unique feature of K. pneumoniae extracellular polysaccharide. These protocols will help create uniformity in standard protocols used in K. pneumoniae extracellular polysaccharide studies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extracellular polysaccharide isolation and purification. Basic Protocol 2: Large-scale isolation and purification of extracellular polysaccharide. Basic Protocol 3: Uronic acid quantification of extracellular polysaccharide. Basic Protocol 4: Extracellular polysaccharide visualization by SDS-PAGE. Basic Protocol 5: Klebsiella pneumoniae mucoviscosity measurement by sedimentation resistance assay. Alternate Protocol 5: 96-well plate-based Klebsiella pneumoniae sedimentation resistance assay. Support Protocol 5: Determination of plate to cuvette conversion factor.