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Antimicrobial Agents and Chemotherapy, August 2009, p. 3190-3196, Vol. 53, No. 8
0066-4804/09/$08.00+0     doi:10.1128/AAC.00834-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Selection of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus by Imipenem{triangledown}

Yuki Katayama,* Hiroko Murakami-Kuroda, Longzhu Cui, and Keiichi Hiramatsu

Department of Bacteriology, Faculty of Medicine, Juntendo University, Tokyo, Japan

Received 24 June 2008/ Returned for modification 2 August 2008/ Accepted 9 May 2009

Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain {Delta}IP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraSH14) that caused an amino acid substitution (S329->L). This mutation is different from the VraS mutation (N5->I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of {Delta}IP with the mutated vraSH14 gene confirmed that the S329->L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N5->I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of {Delta}IP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus.

* Corresponding author. Mailing address: Department of Bacteriology, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan. Phone: 81-3-5802-1041. Fax: 81-3-5684-7830. E-mail: yukk{at}juntendo.ac.jp

{triangledown} Published ahead of print on 18 May 2009.

Antimicrobial Agents and Chemotherapy, August 2009, p. 3190-3196, Vol. 53, No. 8
0066-4804/09/$08.00+0     doi:10.1128/AAC.00834-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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