SY Park, et al agment of 372 bp were used8. For mecA gene detection, forward primer (5’-ATGAGATTAGGCATCGTTCC-3’) and reverse primer (5’-TGGATGACAGTACCTGAGCC-3’) with a fragment of 554 bp were used9. For mupA gene detec- tion, forward primer (5’-CCCATGGCTTACCAGTTGA-3’) and reverse primer (5’-CCATGGAGCACTATCCGAA-3’) with a fragment of 1.6 kb were used10. Prior to DNA extraction, frozen bacteria were subcultured twice onto Mueller-Hinton agar plates. For extraction, one to five bacterial colonies were suspended in 50μl of cell lysis buffer (Genotek Co., Daejeon, Korea) and this was heated at 100oC for 10 min. After centrifugation at 12,000 rpm for 10 min, 2μl of the supernatant was used for DNA extraction. The PCR reactions were performed using a Pri- mer Mix Kit (Genotek Co.). The thermal cycler (GeneAmp PCR system 9700, Perkin-Elmer Cetus, Foster City, CA, USA) was programmed with the following parameters: pre-denaturation at 95oC for 15 min, 30 cycles of denaturation at 94oC for 30 s, annealing at 68oC for 90 s, extension at 72oC for 90 s and a final extension at 72oC for 10 min. The amplified products were run on a 2% agarose gel for 20 min at 200 volts and visualized with ethidium bromide. Pulsed-field gel electrophoresis (PFGE) A total of 16 strains of mupirocin resistant MRSA (10 isola- tes with high-level resistance and 6 isolates with low-level resistance) were used for PFGE11. The high-level resistant strains were from (A) hospital. For the low-level strains, 2 strains were from (B) hospital, 3 strains were from (C) hospital and one strain was from (D) hospital. Each isolate was inoculated into 3 ml Luria-Vertani broth (Difco Laboratories) and incubated at 37oC overnight. The concentration of the cell suspensions were adjusted with saline using a spectrophotometer to an absorbance of 0.3 ∼0.25 at 610 nm. After adjustment, 1 ml of the cell suspension was centrifuged at 14,000 rpm for 5 min and the supernatant was aspirated. The pellet was washed once in 1 ml of Tris-EDTA (TE) buffer (10 mM Tris HCl, 1 mM EDTA [pH 8]) and centrifuged again. The washed cells were resuspended in 50μl of lysis II buffer (6 mM Tris-HCl, pH 8.0, 1 M NaCl, 100 mM EDTA, 0.5% Brij-58, 0.2% sodium deoxycholate, 0.5% sodium lauroyl- sarcosine) and equilibrated at 37oC for 15 min. Fifty micro- liters of 2% low-melting-pint agarose and 2μl of 100 units lysostaphin were added to the cell suspension, gently mixed and dispensed into a plug mold. The plugs were allowed to solidify in the refrigerator at 4oC for 5 min. The plugs were placed into a tube containing 250μl of lysis I buffer (50 mM Tris-HCl, pH 7.4, 100 unit lysostapin, lyso- zyme 1 mg/ml) and the cells in the plug were lysed for 1 h 34 Ann Dermatol at 37oC. Then the lysis I buffer were removed and repla- ced with 250μl of lysis II buffer at 37oC for 1 h. The lysis II buffer was removed and 250μl of proteinase K buffer (0.5 M EDTA, 25 unit/ml proteinase K) was added and this was incubated at 50oC overnight. The TE washings were repeated 3 more times. For electrophoresis, the plug was cut into small slices (2 by 5 mm) and these were placed in 125μl of a total restriction enzyme mixture that contained 20 U of SmaI (Sib Enzyme Ltd, Novosibirk, Russia). After 2 h incubation at 25oC with shaking at 140 rpm, the chromosomal restric- tion fragment patterns were analyzed by loading the trim- med slices of the plug into a well of SeaKem 1% agarose gel. The running gel was prepared in 0.5×TBE buffer (Bio- Rad Laboratories, Inc., Hercules, CA, USA). Electropho- resis was performed with a CHEP-DR III (Bio-Rad Labora- tories, Inc., Hercules, CA, USA). The running parameters were an initial pulse of 5 s, a final pulse of 40 s, 200 V, 20 hrs and 12∼14oC. After the electrophoresis run was com- pleted, the gel was stained with 0.5μg/ml ethidium bro- mide for 20 min and destained in fresh distilled water for 20 min. The gels were photographed and digitalized with a GelDoc (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The ima- ges were stored electronically as TIFF files. The restriction pattern was analyzed with the GelCompar II program (version 4.5; Applied Maths, Sint-Martens-Latem, Belgium) using the Dice coefficient, and cluster analysis of the similarity matrices was performed by the unweighted pair group method using the arithmetic average with 0.75% of tolerance12,13. The similarity cut-off value was 99%. RESULTS Prevalence of mupirocin resistant MRSA Of the total 193 clinical isolates of MRSA from the four university hospitals, 27 (14.1%) were mupirocin resistant (Table 1). Of these, 11 (5.7%) were found to have high- level resistance to mupirocin and all of them were from (A) hospital. The low-level strains were composed of 2, 13 and 1 isolates from (B), (C) and (D) hospitals, respectively. Antimicrobial susceptibility tests of the resistant MRSA mupirocin The antimicrobial susceptibility test results for the mupi- rocin resistant MRSA isolates are summarized in Table 2. The MICs of the high-level mupirocin resistant MRSA ranged from 512 to ≥1,024μg/ml, and the MIC50 and MIC90 were ≥1,024μg/ml, respectively. But for the low- level mupirocin resistant strains, the MICs ranged from 16 to 32, and the MIC50 and MIC90 was 16μg/ml in both