Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant Staphylococcus spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia.

The incidence of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continues to increase in many countries worldwide. Rapid identification of MRSA from clinical specimens and screening of high-risk patients for MRSA colonization have been found to be cost-effective measures for limiting the spread of the organism in hospitals (1, 6). Several screening media for the enhanced recovery of methicillin-resistant organisms and for the differentiation of S. aureus from coagulase-negative staphylococci have been developed (2,4,5,7,8). Recently, a novel medium, oxacillin resistance screening agar base (ORSAB; Oxoid Limited, Basingstoke, England), has been developed for the detection of MRSA in clinical specimens. The medium uses aniline blue to demonstrate mannitol fermentation in staphylococci. The dual antibiotic supplement (oxacillin, 2.0 g/ml; polymixin B, 50,000 IU/l) and the presence of 5.5% NaCl have the potential to reduce the growth of nonstaphylococcal organisms and to select for the growth of MRSA. We evaluated the performance of ORSAB as compared to that of mannitol salt agar supplemented with 2.0 g/ml oxacillin (MSA) for the recovery of MRSA from clinical specimens.A total of 455 specimens were obtained for MRSA culture, consisting of nares (132 specimens), perineum (109 specimens), skin and soft tissue (206 specimens), sputum (5 specimens), and urine (3 specimens). In order to increase the yield of positive cultures, specimens were obtained from patients known to have had MRSA colonization in the past, from hospital contacts of these patients, and from other patients thought to be at high risk for MRSA colonization. Each specimen was cultured on ORSAB and MSA media, with incubation for 48 h in ambient air at 35°C. The plates were examined at 24 and 48 h for the presence of mannitol-fermenting colonies, which were blue on ORSAB and yellow on MSA. Each morphotype of a mannitol-fermenting colony was subcultured onto sheep blood agar and identified by conventional tests, including Gram staining and tests for catalase activity, tube coagulase activity, and latex agglutination (Pastorex Staph Plus, Bio-Rad, Mississauga, Ontario, Canada). The MRSAScreen test (Denka-Seiken, Tokyo, Japan) was used for the detection of PBP2a associated with oxacillin resistance in staphylococci (3). Identification as MRSA was subsequently confirmed by multiplex PCR for the nucA and mecA genes (3).MRSA was isolated from 104 specimens. Four MRSA isolates were detected only on the ORSAB medium, and two were recovered only from the MSA. After 24 h of incubation, 79 (76%) of the MRSA isolates were evident on the ORSAB medium, and 67 (64%) were evident on the MSA. After 48 h of incubation the yield of MRSA increased for both ORSAB and MSA to 98% (102 of 104 specimens) and 96% (100 of 104 specimens) respectively (Table 1). A greater number of specimens yielded mannitol-fermenting colonies on the MSA (177 specimens) as compared to the ORSAB (138 specimens). However, a total of 77 (44%) of mannitol-fermenting colonies gr...

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84 mecA-positive strains and 116 mecA-negative strains) consisting of 108 Staphylococcus epidermidis, 37 S. saprophyticus, 15 S. haemolyticus, 11 S. hominis, 10 S. capitis, 10 S. warneri, and 3 S. lugdunensis species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 g/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the mecA gene was used as the "gold standard." The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.Coagulase-negative staphylococci (CoNS) are recognized as important causes of nosocomial infection, especially in neonates, those who are immunocompromised, and patients with indwelling prosthetic devices (6). The rate of resistance to oxacillin among CoNS has been increasing (2), and the empirical treatment of choice for infections caused by these organisms is often vancomycin. The rapid and accurate identification of oxacillin resistance is essential in order to determine the most appropriate antimicrobial therapy. The major mechanism of oxacillin resistance in staphylococci is mediated by the production of a modified penicillin-binding protein, PBP 2a, specified by the mecA gene. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) lowered the breakpoint for determination of oxacillin resistance in CoNS from Ն4 to Ն0.5 g/ml (12) in order to more accurately reflect mecA-mediated resistance. In addition, the use of the oxacillin agar screen plate (Mueller-Hinton agar with 6 g of oxacillin per ml supplemented with 4% NaCl) was no longer recommended by NCCLS for the detection of oxacillin resistance in CoNS, as reports in the literature had indicated that this method may not be able to detect mecA-mediated oxacillin resistance in these strains (15,17).A rapid latex agglutination test, the MRSA-Screen (DenkaSeiken, Tokyo, Japan), has recently been developed in order to detect the presence of oxacillin resistance mediated by PBP 2a. Although several studies have evaluated the use of this test for the detection of oxacillin resistance in Staphylococcus aureus (8,18,19), its accuracy for the detection of oxacillin resistance in CoNS has not been extensively assessed (5, 9). In the study described here, we evaluated the MRSA-Screen for its ability to detect PBP 2a in clinical strains of CoNS. In ...