Development of an antibody-based assay for methicillin resistant Staphylococcus aureus using synthetic peptidoglycan precursors

Methicillin-resistant Staphylococcus aureus (MRSA) are isolates of the bacterium Staphylococcus aureus that have acquired genes encoding antibiotic resistance to all penicillins, including methicillin and other narrow-spectrum β-lactamase-resistant penicillin antibiotics. Outbreaks of MRSA occur quite frequently as there is no quick screening test for the presence of MRSA. The aim of this project is to try and develop an antibody based detection test for rapid detection of MRSA, which could help in the prevention of outbreaks. An antigen (UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys(ε-NH2-Gly)5-D-Ala-D-Ala) that resembles the outer surface of the Staphylococcus aureus (contains a Gly5 moiety that is specific for S. aureus) cell wall peptidoglycan has been prepared, and attached to a carrier protein. Sheep antibodies raised against this antigen were screened using ELISA assays. The results showed that antibodies did have specificity for the antigen. Cell walls were prepared from several different bacteria, including two MRSA and one methicillin-sensitive Staphylococcus aureus (MSSA) strain. ELISA assays using these cell walls showed that the antibodies had specificity for cell walls containing (Gly)5 in the order of S aureus (Gly5) > S. simulans (less Gly5) > S. pneumoniae (no Glyn) > E. coli (no Glyn, Gram-negative strain). A particularly high response was observed for one of the two MRSA strains, detectable at 0.1 μg of cell wall. An HPLC-based UV-Vis assay was developed to monitor the activity of peptidoglycan polymerisation enzymes from S. aureus, while preparing polymeric peptidoglycan as an antigen for immunisation. Several intermediates in peptidoglycan polymerisation were detected using S. aureus monofunctional glycosyltransferase (MGT), which allowed us to propose a new hypothesis for the early steps of peptidoglycan transglycosylation