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Author Notes:

Address correspondence to Timothy D. Read, tread@emory.edu.

K.D.P. and S.W.S. contributed equally to this article.

We acknowledge Jean Patel and Brandi Limbago at the CDC for strain acquisition, Eric Skaar and Laura Anzaldi at Vanderbilt University for plasmids and discussions, Anjana Varadarajan for computer assistance, Kellie Vinal and Max Schroeder for help with molecular biology, Chad Haase, Alex Chan, and Ryan Weil in the Georgia Research Alliance (GRA) Emory Genomics Core, Hong Yi at the Robert P. Apkarian Integrated Electron Microscopy Core at Emory University, and Ewelina Lyzskowicz and Monica Farley at the Atlanta VA hospital.

We also thank Mark Driscoll, Brian Desany, and Roche-454 for generously providing sequencing support and Eric Skaar, Tony Richardson, and Bill Shafer for help with complementation experiments.


Research Funding:

This work was funded by NIH grant 1R56AI091827-01A1.

This work made use of the Emory Genomics Center.

Funding for the equipment and maintenance of the Emory Genomics Center (EGC) comes from the Emory University School of Medicine, Georgia Research Alliance, and Atlanta Clinical & Translational Sciences Institute.

A Mutation in the PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin


Journal Title:

Antimicrobial Agents and Chemotherapy


Volume 56, Number 10


, Pages 5212-5223

Type of Work:

Article | Final Publisher PDF


Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediate S. aureus (VISA). In this study, we characterized two isogenic USA300 S. aureus isolates collected sequentially from a single patient with endocarditis where the S. aureus isolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein of S. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII, sarT, and saeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquired in vivo. Creation of de novo mutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype.

Copyright information:

© 2012, American Society for Microbiology. All Rights Reserved.

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