Purification of Staphylococcus aureus type 5 capsular polysaccharide by DEAE sephacel and sephacryl chromatography

Abstract

Staphylococcus aureus (S. aureus) type 5 and 8 are an important cause of both nosocomial and community acquired infections. Out of the 40 million patients that undergo surgery, approximately 5% of these patients acquire S. aureus infections. Community acquired S. aureus bacteremia is considered to be even more serious than hospitalized infections comprising approximately forty percent of all S. aureus infections. Resistant strains toward recent antibiotics have been increasing in number and many antibiotics are ineffective. The development of new forms of treatment are needed for these resistant forms of bacteria. A resistant capsular polysaccharide (CP) layer located on the outer cell wall, aides the bacteria in pathogenesis. The purpose of these studies was to purify CP from S. aureus type 5. The bacteria was treated with DNAse, RNAse, protease, and lysostaphin to release the CP from the cell wall Purification of lysates was accomplished by means of a DEAE charge separation and size exclusion chromatography. Collected fraction samples from chromatography purification were tested for the presence of organic molecules by an absorbance of 206 nm. The presence of reducing sugars by a red tetrazolium test, and contamination by teichoic acid was evaluated by a phosphate test. Anti-type 5 S. aureus monoclonal antibodies were used in an ELISA to test for specificity of purified CP. Monoclonal antibodies to type 8 S. aureus were developed to test preparations of purified type 8 CP. Purified CP will be used in future studies to determine the specificity of antibodies and phage display libraries used to develop immunotherapies for S. aureus infections.