Recombinant S. aureus Penicillin Binding Protein 2a [His]

The genome of Staphylococcus aureus is constantly in a state of flux, acquiring genes that enable the bacterium to maintain resistance in the face of antibiotic pressure. The acquisition of the mecA gene from an unknown origin imparted S. aureus with broad resistance to beta-lactam antibiotics, with the resultant strain designated as methicillin-resistant S. aureus (MRSA). Epidemiological and genetic evidence suggests that the gene encoding PBP 2a of MRSA might have originated from Staphylococcus sciuri, an animal pathogen, where it exists as a silent gene of unknown function. We synthesized, cloned, and expressed the mecA gene of S. sciuri in Escherichia coli, and the protein product was purified to homogeneity. Biochemical characterization and comparison of the protein to PBP 2a of S. aureus revealed them to be highly similar. These characteristics start with sequence similarity but extend to biochemical behavior in inhibition by beta-lactam antibiotics, to the existence of an allosteric site for binding of bacterial peptidoglycan, to the issues of the sheltered active site, and to the need for conformational change in making the active site accessible to the substrate and the inhibitors. Altogether, the evidence strongly argues that the kinship between the two proteins is deep-rooted on the basis of many biochemical attributes quantified in this study.

Since its first identification in the early 1960s, Methicillin-Resistant Staphylococcus aureus (MRSA) has been recognized as a major human pathogen. The aim of this study was to identify the prevalence of MRSA in Alexandria Main University Hospital and to settle on a simple, rapid, accurate and cost-effective phenotypic test for the detection of MRSA from clinical specimens. One hundred S. aureus isolates, including 71 MRSA isolates, as confirmed by PCR for the presence or absence of the mecA gene as the gold standard, were isolated from patients from different departments at Alexandria Main University Hospital over a six month-period. They were tested for methicillin resistance by comparing five phenotypic tests (Mannitol salt agar-cefoxitin [MSA-FOX], oxacillin disc diffusion, cefoxitin disc diffusion, oxacillin MIC by broth microdilution and latex agglutination for PBP2a) to the gold standard genotypic test (detection of mecA gene by PCR). It was found that both oxacillin disc diffusion and latex agglutination showed 100% sensitivity, negative and positive predictive values of 100 and 97.3%, respectively. Both were found to be highly sensitive phenotypic tests for the detection of MRSA. However, the oxacillin disc diffusion test is much more cost-effective. The MSA-FOX, whose sensitivity was 95.8%, was found to be a highly sensitive, cost-effective screening medium for the detection of MRSA.