DIRECT IDENTIFICATION OF STAPHYLOCOCCUS-AUREUS IN BLOOD CULTURES BY DETECTION OF THE GENE ENCODING THE THERMOSTABLE NUCLEASE OR THE GENE-PRODUCT

This study compares methods for direct identification of S. aureus in blood cultures by detection of the thermonuclease (TNase) of this bact erium or the nuc gene encoding it. The protein was detected by an enzy me 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 similar to 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 g ene was amplified by a polymerase chain reaction (PCR) with a test tim e (amplification plus detection) of similar to 3.5 h. The tests were o ptimized for direct examination of blood-containing cultures. All test s were positive with 67/67 blood cultures which grew S. aureus, negati ve with 35/35 cultures which grew coagulase-negative staphylococci, an d negative with 37/37 cultures with various other bacteria. These resu lts showed positive agreement with those of the commercial AccuProbe t est but not with the StaphAurex agglutination kit, With an artificiall y seeded blood culture, minimum total times required (incubation plus testing) were as follows: nuc-PCR, 9.5 h; sELIFA, 12.5 h; enzymatic te st, 16-36 h; AccuProbe, 14 h. Direct examination of both the nuc gene and the mecA gene encoding methicillin resistance demonstrated the mec A gene in all the coagulase-negative staphylococci (48.6%) which showe d 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 enco ding other clinically important characters of the bacteria. These test s offer the prospect of direct application to a variety of clinical sp ecimens for rapid diagnosis of S. aureus infection.