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Typing and antimicrobial susceptibilities of methicillin resistant Staphylococcus aureus (MRSA) strains isolated in a hospital in Korea.

Methicillin-resistant Staphylococcus aureus (MRSA) strains may cause serious nosocomial infections, including pneumonia and septicemia. The rate of methicillin-resistance among S. aureus isolates in Korea is over 50%. In this study, 90 MRSA isolates from Kyung Hee University Hospital were characterized employing bacteriophage typing, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. Eighty percent of the strains could be phage-typed. The largest group or 40% of the strains belonged to lyso group III, followed by 32% of the isolates which produced a reaction with regional additional phages. Phage type 83A was most frequently encountered, followed by phage type D11. PFGE patterns confirmed the presence of two major clusters, which comprise the isolates belonging to lyso group III and the strains that were typable with regional additional phages. The latter group also contained a number of strains that were nontypable with bacteriophages. The resistance rates to ciprofloxacin, erythromycin, tetracycline, gentamicin and clindamycin were over 94%. Strains with intermediate resistance to vancomycin strains or resistance to mupirocin were not found. In conclusion, this study demonstrates that the results of phage typing are confirmed and supplemented by PFGE data.

out by testing production of free coagulase, DNase, and growth on mannitol salt agar: all isolates were coagulase positive and DNase positive.

Pulsed-field gel electrophoresis
Chromosomal DNA for the SmaI restriction digest was purified from 31 strains as described previously (8). PFGE was performed with a Bio-Rad CHEF DR III system (Bio-Rad, M nich, Germany) employing Pulsed Field Certified Agarose (1%) (Bio-Rad). Run conditions were 6 V/cm, a field angle of 120°, and with switching times of 5-15 sec for 7 hr to 15-60 sec for further 19 hr. A chromosomal DNA digest of S. aureus strain NCTC 8325 served as a mass standard.

Antimicrobial susceptibility tests
Antimicrobial susceptibility tests were performed according to the protocols recommended by the National Committee for Clinical Laboratory Standards employing the disk diffusion method. Screening for occurrence of heterogeneous intermediate resistance to vancomycin was performed as recommended (9). Vancomycin minimal inhibitory concentrations were determined by the microbroth dilution method in brain heart infusion broth (9) and read after 24 hr of incubation at 37℃.

Phage typing
The distribution of clinical samples from which the isolates were derived is shown in Table 1 and 2. Seventy-two (80%) of 90 MRSA isolates were identified using bacteriophages of the IPS or regional additional phages, with 40% of the strains belonging to lyso group III. Eighteen isolates (20%) were nontypable and 32% reacted only with regional additional phages ( Table 3). None of the strains reacted with phages of group II.
Pulsed-field gel electrophoresis PFGE patterns of 11 MRSA strains representing the major types encountered are shown in Fig. 1. Lanes 2 to 7 show examples of strains that were typable only with regional additional phages or nontypable. Lanes 10 to 12 display the PFGE patterns of strains that belong to lyso group III. The strain in lane 13 was the single isolate that was found to belong to group I. The isolate in lane 14 was nontypable and showed an unrelated PFGE pattern. Corresponding to the results of the phage typing, the isolates in lanes 2-7 and lanes 10-13 formed groups of related strains.   The first group displayed a similarity to the banding pattern of the Japanese vancomycin intermediately resistant isolate Mu50 (lane 9) which is also nontypable by phages of the IPS. The second group is distinguishable from the first group by its PFGE pattern, but some similarities remain, indicating that the two groups may be related.

Antimicrobial susceptibility
The resistance rates to ciprofloxacin, erythromycin, tetracycline, and gentamicin were higher than 94% (Table 4). All strains were screened for the occurrence of subpopulations with intermediate resistance to vancomycin, that is, with an minimal inhibitory concentration (MIC) greater than or equal to 8 mg/L vancomycin on agar plates containing 4 g/mL vancomycin. No vancomycin-intermediate S. aureus (VISA) strains were detected. Two isolates which were able to form colonies on the selection agar harbored less sensitive subpopulations with an MIC of only 4 g/mL vancomycin and therefore were considered as sensitive. One of these strains (PFGE of in lane 12 in Fig. 1) belonged to the largest group of isolates giving a reaction with group III phages, and the other strain (lane 14 in Fig. 1) was nontypable and displayed an unrelated PFGE pattern. The antibiotic resistance phenotypes and phage types are given in Table 5, showing that the antibiograms of the strains are heterogeneous and vary within a single phage type.

DISCUSSION
MRSA is one of the most important nosocomial pathogens, causing surgical wound infections, infections at indwelling catheters, abscesses related to injections, etc. The most serious infections include pneumonia and septicemia. The infections are usually acquired during the stay in hospital by direct or indirect contact or airborne transmission. In order to control the transmission of MRSA in a hospital environment, special precautions have to be taken. Typing of the isolates is necessary for epidemiologic monitoring and especially, identification of outbreak strains, which possess a high virulence and often seem to defy infection control measures that successfully inhibit transmission of ordinary S. aureus strains.
Phage typing, which has been the classical typing method of S. aureus, divided the isolates of this study into three groups, namely, those belonging to lyso group III, those typable with additional phages, and nontypable strains. The 19 isolates that reacted with phage 83A and 12 strains that were typable with phage D11 displayed identical resistance profiles, which indicates at least two clones in the hospital. The group III strains represented the largest proportion of the isolates, and their PFGE patterns also confirmed the close relatedness among the strains. Of notes, lysotype 83A that is rarely encountered in Germany predominated in this group.
Cho et al. (10) and Lee et al. (11) have previously reported on the isolation of lyso group II and lyso group III strains in Korea. Compared with these strains, which were isolated from the skin, the MRSA strains characterized in this paper formed a clearly distinct cluster. Fifty-two percent of the isolates were nontypable with the IPS, and by employing additional regional phages, this proportion could be reduced to 20%. The fact that a high percentage of MRSA strains are nontypable with the IPS is well known and has been reported previously (12). The additional phages that were    employed for typing have been selected because of their ability to lyse the local German epidemic strains, among which the Southern German epidemic strain gives a reaction with the regional additional phages D11, 16, and 192. However, the PFGE pattern of this strain did not show similarity to the Korean isolates (data not shown) and rather resembled the Japanese isolate Mu50. S. aureus Mu50 belongs to the clonotype IIa MRSA which are prevalent in Japan and U.S.A. (9). PFGE revealed that many of the strains that were nontypable with the IPS and the strains that reacted with the regional additional phages are closely related to each other.
In conclusion, this study demonstrates that the results of the phage typing are confirmed and supplemented by PFGE data. Phage typing is still be used as an inexpensive screening method in an epidemiological context especially for mass typing. In the meantime, the number of strains nontypable with IPS has been reduced by the introduction of new MRSA phages. PFGE is essential for the examination of nontypable strains, identification and characterization of epidemic strains or confirmation of ambiguous results, although it is more time-consuming, cumbersome and requires expensive equipment.