Jpn. J. Infect. Dis., 57, 74-77, 2004
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Laboratory and Epidemiology Communications
Prevalence of Erythromycin-, Tetracycline-, and Aminoglycoside-Resistance Genes in Methicillin-Resistant Staphylococcus aureus in Hospitals in Tokyo and Kumamoto
Jun-ichiro Sekiguchi, Tomoko Fujino, Katsutoshi Saruta,
Hisami Konosaki, Haruo Nishimura, Akihiko Kawana, Koichiro Kudo,
Tatsuya Kondo, Yoshio Yazaki, Tadatoshi Kuratsuji, Hiroshi Yoshikura
International Medical Center of Japan, Tokyo 162-8655 and 1National Institute of Infectious Diseases, Tokyo 162-8640
Communicated by Hiroshi Yoshikura
(Accepted March 12, 2004)
*Corresponding author: Mailing address: International Medical Center of Japan, Toyama 1-21-1, Shinjuku-ku, Tokyo 162-8655, Japan. Fax: +81-3-3202-7364, E-mail: tkirikae@ri.imcj.go.jp
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of their emerging resistance to all current antibiotic classes. Investigating the spread of the drug-resistance genes in MRSA is important for the control of its dissemination (1).
In our previous papers (2-4), a total of 179 MRSA clinical isolates obtained in December 2000, October 2001, and October 2002 from a hospital with 24 wards and 925 beds in Tokyo were assessed using restriction fragment length polymorphism (RFLP) of genomic DNA using pulsed-field gel electrophoresis (PFGE). A band-based cluster analysis of the PFGE patterns of these isolates revealed that 111 of the 179 MRSA isolates formed a cluster of PFGE patterns, called cluster A.
Chromosomal DNA was typed by using a contour-clamped homogeneous electric field system (CHEF MapperTM, Bio-Rad Laboratories, Hercules, Calif., USA). Plasmid DNA was typed by using agarose gel electrophoresis. The antibiotic resistance of MRSA to tetracycline (TC) was analyzed using WalkAwayTM (Dade Behring, Deerfield, Ill., USA) and E-testTM (AB BIODISK, Dalvagen, Sweden). PCR was used to detect gentamicin (GM)-resistance genes [aac6'-aph2" and aph(3')-III], erythromycin (EM)-resistance genes (ermA, ermB, and ermC), and TC-resistance genes (tetK and tetM), while Southern blot used to detect aac6'-aph2", ermA, and tetM. Some of the PCR products were sequenced for confirmation. Based on these analyses, the isolates were classified into 33 types (Table 1).
Among the 111 MRSA isolates tested, all were resistant to EM, 13 were resistant to GM, and 102 were resistant to TC. The majority of the isolates (97 of 111) were resistant to EM and TC, but sensitive to GM. No isolates were sensitive to all three antibiotics (Table 1). PFGE of SmaI digests (Fig. 1A) revealed 23 different patterns. The most frequent pattern was A1, representing 31.5% of the total isolates (Table 1). The profiles of plasmid typing are shown in Fig. 2A. Plasmids of 27 different sizes, ranging from 2.4 kb to 300 kb, were detected. The isolates were classified into 26 plasmid patterns (Table 2). One-hundred-eight of 111 isolates had one or more different-sized plasmids. Three other isolates had no plasmids. Seventy-three isolates accounting for 68% of the total had plasmid pattern I, II, or III, and these isolates had both 50 kb and 35 kb plasmids (Table 2). Among the isolates with PFGE pattern A1, 34 had 50 kb plasmid. Among them, eight had plasmid pattern I, seven had plasmid pattern II, ten had plasmid pattern III, one had plasmid pattern X, and eight had plasmid pattern XXIV.
The results of PCR analysis are summarized in Table 1. Among the 111 MRSA isolates, 13 were PCR-positive for aac6'-aph2", all isolates were positive for ermA, and 103 were positive for tetM. No isolate was positive for aph(3')-III, ermB, ermC, or tetK. The majority of the isolates (103 of 111) were positive for the genes ermA and tetM, but negative for the others. Twelve isolates with type Nos. 10-12, 24, 28, and 33 were positive for aac6'-aph2", ermA, and tetM;. One isolate with No. 4 was positive for ermA and aac6'-aph2", and three with Nos. 3, 7, and 17 were positive for ermA.
Southern blotting detected aac6'-aph2" on 30 kb, 38 kb, 190 kb, or 200 kb plasmids carried by 6 isolates and on the chromosomes of 12 isolates. On the chromosomes, it was present in 110 kb and 220 kb SmaI fragments (2 isolates), in a 220 kb SmaI fragment (one isolate), and in a 500 kb SmaI fragment (five isolates) (Fig. 1 and Table 1). The ermA was found on the chromosomes of all the isolates, mostly on 220 kb and 580 kb SmaI fragments. The tetM was found on the chromosomes of 104 isolates, mostly in the 290 kb SmaI fragment.
The PCR analysis gave data consistent with resistance pattern of the bacteria in all the cases except three, which were types Nos. 2, 5, and 14. An isolate of type No. 2 was sensitive to GM, resistant to EM, and intermediately resistant to TC, while negative for aac6'-aph2", but positive for ermA and tetM in PCR. An isolate of type No. 5 was resistant to GM and EM, but sensitive to TC, while being PCR-negative for aac6'-aph2" and tetM, but positive for ermA. An isolate of type No. 14 was sensitive to GM and TC, but resistant to EM, while PCR-negative for aac6'-aph2", but positive for ermA and tetM. This discordance may probably be brought about by mutations in the coding or promoter region of the PCR-detected genes.
Among 111 MRSA isolates obtained from a hospital in Tokyo and whose PFGE patterns showed A clusters, 34 isolates showing PFGE pattern A1 were sensitive to GM, and resistant to EM and TC. They had ermA in 220 kb and 580 kb SmaI chromosomal digests and tetM in a 290 kb SmaI chromosomal digest, but they did not have plasmids harboring ermA, tetM, or any other of the drug-resistance genes tested. Previous studies (5,6) showed that MRSA isolates having the PFGE pattern A1 were wide spread in hospitals in Tokyo and in Kumamoto. Both the Kumamoto and the Tokyo isolates had ermA in 220 kb and 580 kb SmaI chromosomal fragments and tetM in 290 kb SmaI fragments. However, their antibiotic resistance patterns were different (1). Most Kumamoto isolates were resistant to GM, EM, and TC; they had a multi-drug resistant 40 kb plasmid harboring aac6'-aph2", ermA, and tetM, and 200 kb plasmid harboring aac6'-aph2". They also had aac6'-aph2" in a 110 kb SmaI chromosome fragment. The Tokyo isolates, meanwhile, were found to be GM-sensitive, and had no 40 kb or 200 kb plasmids and no aac6ﾕ-aph2ﾓ in theie chromosomes. In summary, there appears to have been a clonal expansion of closely related MRSAs in hospitals in Tokyo and in Kumamoto, but the MRSA in Kumamoto appeared to have recently acquired the GM-resistance gene, aac6'-aph2", which was not found in the Tokyo isolates.
REFERENCES
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