Methods to identify enzymes that degrade the main extracellular polysaccharide component of Staphylococcus epidermidis biofilms

Abstract

The production of extracellular poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by Staphylococcus epidermidis is the principal determinant of biofilm formation on indwelling medical devices. Enzymes that degrade PNAG therefore provide an attractive strategy for biofilm removal and for the manufacture of biofilm-resistant coatings. Here we present methods that allow the identification of PNAG-degrading enzymes with the ability to detach biofilms. Our protocol includes the preparation of soluble PNAG from S. epidermidis cultures, the incubation of soluble PNAG with candidate enzymes and assays that detect the release of N-acetyl-d-glucosamine using high-pH anion-exchange chromatography (HPAEC) followed in parallel by pulsed amperometric detection (PAD) and online electrospray ionization mass spectrometry (ESI-MS). We validated our procedures using dispersin B, which is currently the only known PNAG-degrading enzyme.

Keywords: :

• Staphylococcus epidermidis
• biofilm
• exopolysaccharide
• high-pH anion-exchange chromatography
• poly-β-1,6-N-acetyl-d-glucosamine

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We wish to thank Wilma Ziebuhr for helpful discussions and for providing the S. epidermidis strain we used. The work was supported by the German federal state of Hessen as part of the LOEWE-Schwerpunkt Insektenbiotechnologie.