This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ishino, K.
Right arrow Articles by Hotta, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishino, K.
Right arrow Articles by Hotta, K.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, February 2007, p. 607-609, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.02099-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Usefulness of PCR-Restriction Fragment Length Polymorphism Typing of the Coagulase Gene To Discriminate Arbekacin-Resistant Methicillin-Resistant Staphylococcus aureus Strains{triangledown}

Keiko Ishino,1* Naofumi Tsuchizaki,1,2 Jun Ishikawa,1 and Kunimoto Hotta1

Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan,1 Japan Microbiological Clinic, Co., Ltd., 5-17-1, Okada, Atsugi, Kanagawa 243-0021, Japan2

Received 12 October 2006/ Accepted 5 December 2006


arrow
ABSTRACT
 
We compared the results of two typing methods for 678 strains of methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. PCR-restriction fragment length polymorphism typing of the coagulase gene was a more reliable method than coagulase serotyping from the viewpoint of arbekacin resistance.


arrow
TEXT
 
Molecular typing plays an important role in epidemiological studies of nosocomial infection, such as methicillin-resistant Staphylococcus aureus (MRSA) infection. Pulsed-field gel electrophoresis and multilocus sequence typing are considered the most discriminatory and reliable methods of typing, although they are technically complex, time consuming, and expensive. Coagulase serotyping is widely used in Japan in addition to conventional and genetic methods for distinguishing S. aureus strains. On the other hand, it has been reported that PCR-restriction fragment length polymorphism (RFLP) typing of the coagulase gene (coa) can be used to discriminate S. aureus strains on the basis of sequence variation within the 3' end coding region of the gene (1, 2, 8). Arbekacin is effective, even in gentamicin-resistant MRSA strains, and has been used extensively in Japan since its approval as an anti-MRSA agent in 1990 (9, 10). We have studied arbekacin resistance in MRSA and recently reported the Japanese trend of arbekacin-resistant MRSA strains in the last two decades by using coa-RFLP typing (10). The results showed that arbekacin-resistant MRSA strains were distributed over only a few coa-RFLP types and remained at a low level. In this study, we assessed the clinical usefulness of coa-RFLP typing, especially from the viewpoint of arbekacin resistance, by comparing coagulase serotyping in both clinically isolated MRSA and clinically isolated methicillin-susceptible S. aureus (MSSA) in Japan.

Coagulase serotyping was performed with a coagulase-typing reagent kit (Denka Seiken Co. Ltd., Tokyo, Japan) according to the manufacturer's instructions. Based on the results, a total of 678 isolates, including 206 MSSA and 472 MRSA strains from unrelated clinical sources in Japan between 1979 and 2000 (4, 6, 7), were classified into nine serotypes, I to VIII and "other" (Table 1). MRSA strains were divided into serotypes I, II, III, IV, and VII, and most (82%) of the MRSA strains belonged to serotype II. A similar trend was previously observed in MRSA isolated in Japan (3). The serotype distribution of MSSA strains differed from that of MRSA strains. MSSA strains contained all serotypes. However, the majority of the MSSA strains were classified into serotypes II (28%) and VII (35%).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Correlation between the coagulase serotype and the coa-RFLP type in MRSA and MSSAa

We next carried out coa-RFLP typing according to the method of Hookey et al., with some modifications (2). coa gene fragments were amplified from colonies grown on agar plates with the primers coaF (ATAGAGATGCTGGTACAGG) and coaR (GCTTCCGATTGTTCGATGC). As shown in Fig. 1A, PCR products of eight sizes were obtained from 678 isolates and named XS, SS, S, M, L, LL, XL, and XXL according to their sizes. The sizes of the products ranged from 350 to 917 bp in increments of 81 bp, reflecting the number of 81-bp repeat units contained in the coa gene. After digestion with AluI, 31 coa-RFLP types were detected and numbered to allow them to be distinguished from each other (Fig. 1B). The majority (77%) of MRSA strains belonged to type L21, indicating the spread of a specific type of MRSA in Japan. In contrast, there was no such tendency in MSSA. This result was in agreement with the results of serotyping.


Figure 1
View larger version (53K):
[in this window]
[in a new window]

  		FIG. 1. Agarose gel electrophoresis pattern. (A) PCR amplification of coa genes. (B) Schematic representation of PCR-amplified coa genes digested with AluI.

A comparison of the results of the two typing methods may illuminate the advantages of coa-RFLP typing from the viewpoint of arbekacin resistance. Serotype II contains 11 coa-RFLP types, including type L31. Previously, we reported strain PRC104 as a highly arbekacin-resistant strain (128 µg/ml) (5). Although the typing data for this strain were not incorporated in Table 1 because of its history of isolation, the strain belongs to type L31 (unpublished data). This implies that this important coa-RFLP type cannot be recognized by coagulase serotyping alone. Types M22 and M31 are also important due to their high incidence of arbekacin-resistant strains (10). The coa-RFLP typing clearly distinguished type M22 or M31 isolates from other strains belonging to serotype VII or III, respectively. Thus, our results demonstrated that coa-RFLP typing has higher discriminatory power than coagulase serotyping and may be useful for discriminating groups that may be potential reservoirs of arbekacin-resistant MRSA.


arrow
ACKNOWLEDGMENTS
 
We thank T. Okubo, S. Kondo, K. Kikuchi, Y. Arakawa, and A. Wada for MRSA strains and Y. Uehara for helpful discussion.

This work was supported by a grant from the Ministry of Health, Labor, and Welfare of Japan (Molecular Analysis of Drug-Resistant Bacteria and Establishment Rapid Identification Methods, no. H12-Shinkou-19).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81 3 5285 1111. Fax: 81 3 5285 1272. E-mail: ishino{at}nih.go.jp. Back

{triangledown} Published ahead of print on 13 December 2006. Back


arrow
REFERENCES
 
    1
  1. Goh, S.-H., S. K. Byrne, J. L. Zhang, and A. W. Chow. 1992. Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. J. Clin. Microbiol. 30:1642-1645.[Abstract/Free Full Text]
    2. 2
  2. Hookey, J. V., J. F. Richardson, and B. D. Cookson. 1998. Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol. 36:1083-1089.[Abstract/Free Full Text]
    3. 3
  3. Ida, T., R. Okamoto, C. Shimauchi, T. Okubo, A. Kuga, and M. Inoue. 2001. Identification of aminoglycoside-modifying enzymes by susceptibility testing: epidemiology of methicillin-resistant Staphylococcus aureus in Japan. J. Clin. Microbiol. 39:3115-3121.[Abstract/Free Full Text]
    4. 4
  4. Ike, Y., Y. Arakawa, X. Ma, K. Tatewaki, M. Nagasawa, H. Tomita, K. Tanimoto, and S. Fujimoto. 2001. Nationwide survey shows that methicillin-resistant Staphylococcus aureus strains heterogeneously and intermediately resistant to vancomycin are not disseminated throughout Japanese hospitals. J. Clin. Microbiol. 39:4445-4451.[Abstract/Free Full Text]
    5. 5
  5. Ishino, K., J. Ishikawa, Y. Ikeda, and K. Hotta. 2004. Characterization of a bifunctional aminoglycoside-modifying enzyme with novel substrate specificity and its gene from a clinical isolate of methicillin-resistant Staphylococcus aureus with high arbekacin resistance. J. Antibiot. 57:679-686.[Medline]
    6. 6
  6. Kondo, S., Y. Ikeda, S. Hattori, M. Hamada, and T. Takeuchi. 1991. Susceptibility of methicillin-resistant Staphylococcus aureus to various antibiotics. Classification by aminoglycoside-modifying enzymes and antibiotics active against MRSA. Jpn. J. Antibiot. 44:1211-1215.[Medline]
    7. 7
  7. Piao, C., T. Karasawa, K. Totsuka, T. Uchiyama, and K. Kikuchi. 2005. Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan. Microbiol. Immunol. 49:959-970.[Medline]
    8. 8
  8. Schwarzkopf, A., and H. Karch. 1994. Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker. J. Clin. Microbiol. 32:2407-2412.[Abstract/Free Full Text]
    9. 9
  9. Tabata, M., M. Shimizu, M. Araake, and H. Ogawa. 2003. Relationship between arbekacin-susceptibility and aminoglycoside-resistant gene of methicillin-resistant Staphylococcus aureus (MRSA). Jpn. J. Antibiot. 56:36-43.
    10. 10
  10. Tsuchizaki, N., K. Ishino, F. Saito, J. Ishikawa, M. Nakajima, and K. Hotta. 2006. Trends of arbekacin-resistant MRSA strains in Japanese hospitals (1979 to 2000). J. Antibiot. 59:229-233.[Medline]

Journal of Clinical Microbiology, February 2007, p. 607-609, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.02099-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ishino, K.
Right arrow Articles by Hotta, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishino, K.
Right arrow Articles by Hotta, K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»