Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient.

David A Goulding,Francesca L Short,Julian Parkhill,Matthew J Dorman,Theresa Feltwell

doi:10.3791/58679

Abstract

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

Highlights

Many bacterial species produce a polysaccharide capsule, which protects the bacterial cell from various physical stresses and from recognition and killing by the immune system
The exact result to expect will depend on the bacterial species, the set-up of the density gradients, and whether the user is examining a single strain or a pool of mutants
We have demonstrated a robust method for capsule-based separation of bacterial populations, with multiple potential applications in conjunction with different upstream or downstream protocols

Summary

Introduction

Many bacterial species produce a polysaccharide capsule, which protects the bacterial cell from various physical stresses and from recognition and killing by the immune system. For Klebsiella species, these include the string test[5], in which a toothpick touched to a colony is pulled upwards and the length of the string produced measured, and the mucoviscosity assay[6], which involves the slow centrifugation of a culture followed by measuring the optical density of the supernatant. These methods are simple and quick, but lack sensitivity when used on classical Klebsiella strains rather than capsule overproducing strains. Only microscopy allows the user to observe different capsulation states within a single population, and none of these methods enables the physical separation of capsulated and non-capsulated bacteria

Methods

Results

Conclusion