Jpn. J. Infect. Dis., 56, 129-132, 2003

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Laboratory and Epidemiology Communications

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Kumamoto Hospital in 2002

Fumio Kawano*, Hisayoshi Miyazaki, Jun-ichi Takami, Tomoko Fujinol, Jun-ichiro Sekiguchil, Katsutoshi Sarutal, Tadatoshi Kuratsujil and Teruo Kirikael

Kumamoto National Hospital, Kumamoto 860-0008 and 1International Medical Center of Japan, Tokyo 162-8655

Communicated by Hiroshi Yoshikura

(Accepted July 22, 2003)

*Corresponding author: Mailing address: Kumamoto National Hospital, Ninomaru 1-5, Kumamoto 860-0008, Japan. Fax: +81-96-325-2519, E-mail:

Methicillin-resistant Staphylococcus aureus (MRSA) is a prevalent nosocomial pathogen in healthcare facilities. Epidemiological analysis of MRSA isolates present in a hospital, based, for example, on the restriction fragment length polymorphisms of genomic DNA determined using pulsed-field gel electrophoresis (PFGE), is essential for assessment of hospital infection control (1,2).

Fifty-six MRSA isolates were obtained from 24 inpatients during October 2002 in a hospital with 11 wards and 550 beds in Kumamoto Prefecture. Of these isolates, 24 isolates, each derived from a single patient, were analyzed for chromosomal DNA typing by using a contour-clamped homogeneous electric field system (CHEF MapperTM: Bio-Rad Laboratories, Hercules, Calif., USA), plasmid DNA typing by use of agarose gel electrophoresis, antibiotic resistance (VITEKTM: bioMerieux, Marcy-l'Etoile, France), enterotoxin serotyping (SET-RPLA: Denka Seiken Co., Tokyo), toxic shock syndrome toxin-1 (TSST-1) production (TST-RPLA: Denka Seiken), and coagulase serotyping (Denka Seiken).

Thirteen different PFGE patterns of SmaI DNA digests were detected (Fig. 1). A band-based cluster analysis of these patterns (Molecular AnalystTM: Bio-Rad) revealed nine clusters (clusters A, AH, AI, AE, AM, AL, AN, AJ, and AK) (a cluster was defined as a group of patterns with more than 70% similarity (Fig. 2A). The frequency distribution of MRSA isolates based on PFGE patterns is shown in Fig. 2B. The most frequent pattern (A1) represented 45.8% of the total isolates. PFGE pattern A25 was detected in two isolates. The other 11 PFGE patterns were detected in only one isolate.

The profiles of plasmid typing are shown in Fig. 3. All MRSA isolates had plasmids. Twelve different sizes of plasmids, ranging from 2.4 kb to 280 kb, were detected. Each isolate had two to five different sized plasmids. Thirteen plasmids different in size were detected (Table 1). The frequency distribution of MRSA isolates based on plasmid patterns is shown in Table 1. All isolates had the 40 kb plasmid. Isolates with patterns a, b, and g accounted for 50% of the total, and these isolates had both 200 kb and 40 kb plasmids.

The geographic distribution of MRSA isolates in the hospital is shown in Table 2. The two, two, two, one, three, and one isolates from intensive care unit and wards e2, w3, a2, a5, and a6, respectively, belonged to the same cluster, A1, suggesting a clonal spread of MRSA in the hospital. Among these isolates, one from ward a2 and two from ward a5 belonged to the same plasmid pattern a; and one from ward e2, one from ward a5 and one from ward a6 belonged to the pattern b, indicating that transmission of a clone of MRSA with PFGE pattern A1 and plasmid pattern a occurred in the ward a5. Two isolates belonging to PFGE pattern A25, but having different respective plasmid patterns (z and h) were isolated from wards a3 and a5, respectively. Isolates with other PFGE and plasmid patterns appeared to be limited to wards w3, e2, w2, a3, a5, e1, a3, w1, e1, a3, and w2.

Sensitivity to antibiotics is shown in Table 3. The MRSA isolates were resistant to 9 - 12 of 15 tested drugs. Those isolates had a spectrum of drug-resistance showing eight different patterns. The spectra of drug-resistance were similar to each other; e.g., a difference in susceptibility was found against only one drug when isolates with pattern c were compared with those with pattern a, b, d, e, f, or g. One isolate was resistant to arbekacin (pattern b). Those isolates were resistant to 9 - 12 of 15 tested drugs. (One isolate with PFGE pattern AH2.) All the isolates were sensitive to vancomycin or teicoplanin. No correlation was found between the antibiotic resistance patterns and PFGE patterns (Table 4).

Among 24 MRSA isolates, 23 isolates produced coagulase type II, and the remaining isolate produced coagulase type III (Table 4). Twenty-two isolates produced enterotoxin type C; the twenty-third produced isolate enterotoxin types B and C; and the last isolated produced neither enterotoxin types A, B, C, or D. Twenty-three isolates produced TSST-1; the twenty-fourth did not. Collectively, among 24 MRSA isolates, 21 isolates produced coagulase type II, enterotoxin type C, and TSST-1; i.e., most of the isolates shared common characteristics regarding these parameters.

PFGE-based MRSA surveillance was conducted in the same hospital in October 2001 (1) and in a hospital in Tokyo in October 2002 (2). In these surveillance studies, a total of 42 PFGE patterns were detected (Fig. 4). PFGE patterns A1, A6, A25(M5), and AI2(O5) were common in Kumamoto in 2001 and 2002, whereas the other patterns were unique to each year (Fig. 4)(1), indicating the co-existence of persistence and rapid turnover of MRSA in a hospital. Patterns A1, A2(M1), A16, AE, detected in the hospital in Kumamoto in either 2001 or 2002, were also detected in a hospital in Tokyo in 2002 (Fig. 4)(2). Among these patterns, pattern A1 was most frequently detected in both hospitals (Fig. 2B)(2). The data indicate the clonal expansion of MRSA not only within hospitals but also nationwide.


  1. Kawano, F., Miyazaki, H., Takami, J., Fujino, T., Saruta, K., and Kirikae T. (2002): Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a Kumamoto hospital in 2001. Jpn. J. Infect. Dis., 55, 29-30.
  2. Fujino, T., Sekiguchi, J., Kawana, A., Konosaki, H., Nishimura, H., Saruta, K., Kudo, K., Kobori, O., Yazaki, Y. Kuratsuji, T. and Kirikae, T. (2002): Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a Tokyo hospital in 2002. Jpn. J. Infect. Dis., 55, 210-213.

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