cloning of beta hemolysin gene from coagulase-negative staphylococci and testing of biological activity

Abstract

Coagulase-negative staphylococci (CoNS) are differentiated from the closely related but more virulent Staphylococcus aureus by their inability to produce free plasma coagulase. Coagulase-negative staphylococci are common etiologies of bacteremia that are resulted from intravascular device infections. They now represent one of the major nosocomial pathogens in clinical microbiology laboratories with S. epidermidis and S. haemolyticus being the significant species. Beta hemolysin (hlb) serves as an important virulence molecule involved in the pathogenesis of S. epidermidis. This cytotoxin has two distinct mechanisms of action; Sphingomyelinase activity and DNA bio-film ligase activity (i.e. the ability of β-toxin to cross-link itself in the presence of exogenous DNA). In this study, strains of S. epidermidis cultured from foot infections from diabetic patients were used. Primers with attached KpnI and EcoRI restriction sites were designed to amplify the hlb gene. The gene was subjected to cloning using pUC19 plasmid as a vector. After cloning, the amplicon obtained by M13 PCR was sent for confirmation by sequencing. From the sequence result, a percentage similarity rate of 98.95% of (Staphylococcus epidermidis strain Q47 sphingomyeline phosphodiesterase) was confirmed as expected. The recombinant protein was tested on a sheep blood agar which showed hemolysis of erythrocytes.