A panel of 24 methicillin-resistant Staphylococcus aureus strains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.

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.

Mupirocin resistance in