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Antimicrobial Agents and Chemotherapy, October 2008, p. 3755-3762, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.01613-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genomic Analysis Reveals a Point Mutation in the Two-Component Sensor Gene graS That Leads to Intermediate Vancomycin Resistance in Clinical Staphylococcus aureus{triangledown}

Benjamin P. Howden,1,2* Timothy P. Stinear,1 David L. Allen,1 Paul D. R. Johnson,2 Peter B. Ward,3 and John K. Davies1

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Wellington Rd., Clayton, Victoria, Australia,1 Infectious Diseases,2 Microbiology Departments, Austin Health, Heidelberg, Victoria, Australia3

Received 17 December 2007/ Returned for modification 15 February 2008/ Accepted 3 July 2008

Methicillin-resistant Staphylococcus aureus (MRSA), once restricted to hospitals, is spreading rapidly through the wider community. Resistance to vancomycin, the principal drug used to treat MRSA infections, has only recently emerged, is mainly low level, and characteristically appears during vancomycin therapy (vancomycin-intermediate S. aureus [VISA] and hetero-resistant VISA). This phenomenon suggests the adaptation of MRSA through mutation, although defining the mutations leading to resistance in clinical isolates has been difficult. We studied a vancomycin-susceptible clinical MRSA isolate (MIC of 1 µg/ml) and compared it with an isogenic blood culture isolate from the same patient, despite 42 days of vancomycin treatment (MIC of 4 µg/ml). A whole-genome sequencing approach allowed the nearly complete assembly of the genome sequences of the two isolates and revealed only six nucleotide substitutions in the VISA strain compared with the parent strain. One mutation occurred in graS, encoding a putative two-component regulatory sensor, leading to a change from a polar to a nonpolar amino acid (T136I) in the conserved histidine region of the predicted protein. Replacing the graS allele of the vancomycin-susceptible parent strain with the graS allele from the VISA derivative resulted in increased vancomycin resistance at a level between those of the vancomycin-susceptible S. aureus and VISA clinical isolates, confirming a role for graRS in VISA. Our study suggests that MRSA is able to develop clinically significant vancomycin resistance via a single point mutation, and the two-component regulatory system graRS is a key mediator of this resistance. However, additional mutations are likely required to express the full VISA phenotype.

* Corresponding author. Mailing address: Infectious Diseases Department, Austin Health, P.O. Box 5555, Heidelberg, Victoria 3084, Australia. Phone: 61 3 9496 6676. Fax: 61 3 9496 6677. E-mail: Benjamin.Howden{at}austin.org.au

{triangledown} Published ahead of print on 21 July 2008.

Antimicrobial Agents and Chemotherapy, October 2008, p. 3755-3762, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.01613-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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