Korea, but there are only a few studies about mupirocin resistance in MRSA4-6. Therefore, we measured the preva- lence, genotype and antimicrobial susceptibility of mupiro- cin-resistant MRSA from four Korean university hospitals. MATERIALS AND METHODS Bacterial isolates A total of 193 MRSA isolates were collected from the cli- nical cultures taken from patients at four Korean university hospitals (A∼D) from April 2008 to July 2009. The speci- mens originated from the skin, pus, blood, central venous catheter tip, sputum, tracheal tip, urine, and wound. Of these, 103 strains were from (A) hospital and 30 strains were from the other 3 hospitals, respectively. All the MRSA isolates were randomly collected from each of the hospitals. Antimicrobial susceptibility test The susceptibility to antimicrobial agents was determined by agar dilution tests, according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI). Eight antimicrobial agents were tested: ciprofloxacin, clin- damycin, erythromycin, fusidic acid, mupirocin, rifampin, tetracycline and vancomycin. S. aureus ATCC 33591 was used as a control strain. High- and Low-level Mupirocin Resistant MRSA Briefly, serial two-fold dilutions of the antibiotics were prepared in Muller-Hinton agar (Difco Laboratories, Det- roit, MI, USA). The strains were subcultured on tryptic soy agar (Difco Laboratories), suspended in tryptic soy broth (Difco Laboratories) and adjusted to the turbidity of a 0.5 McFarland standard. Next, the suspension was diluted 1： 10 and inoculated on each plate coated with antibiotic containing media by a Steer replicator. The inoculated plates were incubated at 35oC for 24 hr. The lowest concentration of antibiotic that inhibits the visible growth of an organism was regarded as the MIC, and growth of one isolate was ignored. The MIC50 and MIC90 were the MICs that were required to inhibit 50% and 90% of the organisms, respectively. The MIC determination was evaluated according to the CLSI guidelines7. Detection of the femA, mecA and mupA genes by polymerase chain reaction (PCR) All the isolates were confirmed to be S. aurues and MRSA by detection of the femA and mecA genes using a PCR assay. The high level mupirocin resistant strains were also confirmed by detection of the mupA gene (Fig. 1∼4). Three primer pairs were used for the detection of the femA, mecA and mupA genes. For femA gene detection, forward primer (5’-CATGATGGCGAGATTACAGG-3’) and reverse primer (5’-CGCTAAAGGTACTAACACACGG-3’) with a fr- Fig. 1. Agarose gel electrophoresis of the amplified 372-bp DNA fragments of the femA gene. The samples are lane M: a 100 bp size marker, lanes 1-9: mupirocin resistant MRSA, PC: femA- positive control (S. aureus ATCC 33591) and NC: femA-negative control (S. epidermidis ATCC 2228). MRSA: methicillin-resistant Staphylococcus aureus. Fig. 3. Agarose gel electrophoresis of the amplified 1.6 kb DNA fragments of the mupA gene. The samples are lane M: a 1-kb size marker, lanes 1-2, 4-5, 9-11: high-level mupirocin resistant MRSA, PC: mupA-positive control (KCKC 6129), and NC: mupA- negative control (ATCC 25923). MRSA: methicillin-resistant Sta- phylococcus aureus. Fig. 2. Agarose gel electrophoresis of the amplified 554-bp DNA fragments of the mecA gene. The samples are lane M: a 100 bp size marker, lanes 1-9: mupirocin resistant MRSA, PC: mecA- positive control (ATCC 33591) and NC: mecA-negative control (ATCC 25923). MRSA: methicillin-resistant Staphylococcus aureus. Fig. 4. Agarose gel electrophoresis of the amplified 1.6 kb DNA fragments of the mupA gene. The samples are lane M: a 1-kb size marker, lanes 15, 21-26: low-level mupirocin resistant MRSA, PC: mupA-positive control (KCKC 6129) and NC: mupA-negative control (ATCC 25923). MRSA: methicillin-resistant Staphylococ- cus aureus. Vol. 24, No. 1, 2012 33