Direct identification of Staphylococcus aureus in blood cultures by detection of the gene encoding the thermostable nuclease or the gene product

Abstract

This study compares methods for direct identification of S. aureus in blood cultures by detection of the thermonuclease (TNase) of this bacterium or the nuc gene encoding it. The protein was detected by an enzyme diffusion test in o-toluidine blue DNA agar with a test time of at least 4 h, by a monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay (sELISA) with a test time of approximately 4 h, and by a MAb-based sandwich enzyme-linked immunofiltration assay (sELIFA) with a test time of 25-30 min (sample preparation included). The nuc gene was amplified by a polymerase chain reaction (PCR) with a test time (amplification plus detection) of approximately 3.5 h. The tests were optimized for direct examination of blood-containing cultures. All tests were positive with 67/67 blood cultures which grew S. aureus, negative with 35/35 cultures which grew coagulase-negative staphylococci, and negative with 37/37 cultures with various other bacteria. These results showed positive agreement with those of the commercial AccuProbe test but not with the StaphAurex agglutination kit. With an artificially seeded blood culture, minimum total times required (incubation plus testing) were as follows: nuc-PCR, 9.5 h; sELIFA, 12.5 h; enzymatic test, 16-36 h; AccuProbe, 14 h. Direct examination of both the nuc gene and the mecA gene encoding methicillin resistance demonstrated the mecA gene in all the coagulase-negative staphylococci (48.6%) which showed oxacillin resistance. The sELIFA had the particular advantage of its short test time, the PCR its high sensitivity and the possibility of simultaneous detection of the species-specific nuc gene and genes encoding other clinically important characters of the bacteria. These tests offer the prospect of direct application to a variety of clinical specimens for rapid diagnosis of S. aureus infection.