Genotype Analysis of Methicillin-Resistant Staphylococcus aureus With and Without Reduced Susceptibility to Vancomycin Using Pulsed-Field Gel Electrophoresis

To the Editor: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequently isolated bacterial pathogens in nosocomial infections. MRSA strains recently have emerged that show reduced susceptibility to vancomycin, including vancomycin-resistant S aureus (VRSA), vancomycin-intermediate S aureus (VISA), and S aureus heterogeneously resistant to vancomycin (heterogeneously resistant VRSA or heteroVRSA). Such strains, identified in Tokyo, Michigan, and New Jersey, are associated with failure of vancomycin therapy. While the frequency of isolation of these strains appears to be increasing, the strains have not been investigated previously at our university hospital. We therefore used pulsedfield gel electrophoresis (PFGE) to perform molecular epidemiological analysis of MRSA isolates with and without reduced susceptibility to vancomycin. Seventy-eight S aureus isolates that were isolated from many kinds of clinical samples at Sapporo Medical University Hospital from October to November 1998 were analyzed. All isolates were from different patients. MRSA was defined as S aureus showing a minimum inhibitory concentration (MIC) for oxacillin of 3=4 ug/mL using a Microscan Walkaway 96 system (Dade Behring, Tokyo, Japan) with a microdilution method (National Committee for Clinical Laboratory Standards methods). The N315 strain (MRSA clonotype II-A), the Mu3 strain (hetero-VRSA), and the Mu50 strain (VRSA) were generously provided by the Department of Bacteriology, Juntendo University, Tokyo, Japan. MU3 agar (BectonDickinson, Tokyo, Japan) was used for detection of MRSA with reduced susceptibility to vancomycin. The cultures were incubated at 37° C on sheep blood agar. Bacterial suspensions were adjusted to an optical density of 0.3 at 578 nm and smeared on MU3 agar on a base of brain-heart infusion agar containing vancomycin at 4.0 jig/mL and MU3 additive (Becton-Dickinson) at 1.0 mg/mL. To induce vancomycin resistance, three disks were placed on the MU3 agar prior to incubation at 35° C; one each contained cefminox, cefixime, and aztreonam (Sensidisk, BectonDickinson). Bacterial growth was inspected at 24 and 48 hours. S aureus was defined as vancomycin-sensitive if it revealed no growth on MU3 agar at either time point. Hetero-VRSA status also was assigned if the strain produced a subclone with a vancomycin MIC of 5=8 ug/mL upon selection with vancomycin, provided that the strain retained these properties after 9 days in drug-free medium. VSRA was identified by confluent growth on MU3 agar, confirmed by a vancomycin MIC of s=8 ug/mL. Coagulase type, staphylococcal enterotoxin type, and production of toxic shock syndrome toxin-1 (TSST-1) also were determined using an antiserum kit for detection of S aureus coagulase types, a SET-RPLA kit for S aureus enterotoxins, and a TST-RPLA kit for detection of S aureus TSST-1, respectively (Denkaseiken, Tokyo, Japan). PFGE was performed using the GenePath strain typing system (Bio-Rad, Tokyo, Japan). DNA from the isolates was prepared according to the manufacturer's protocol, and all reagents were supplied in the GenePath Reagents Kit 1. Samples of 150 uL from overnight cultures grown in 3 mL of brain-heart infusion broth were harvested by centrifugation at 12,000# for 1 minute, resuspended in 150 uL of cell suspension buffer, and