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Antimicrobial Agents and Chemotherapy, November 2008, p. 4017-4022, Vol. 52, No. 11
0066-4804/08/$08.00+0 doi:10.1128/AAC.00668-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Identification of the Genetic Basis for Clinical Menadione-Auxotrophic Small-Colony Variant Isolates of Staphylococcus aureus
Jonas Lannergård,1,
Christof von Eiff,2,
Gunnar Sander,2
Tina Cordes,2
Jochen Seggewiβ,2
Georg Peters,2
Richard A. Proctor,3
Karsten Becker,2 and
Diarmaid Hughes1*
Microbiology Program, Department of Cell and Molecular Biology, Box 596, The Biomedical Center, Uppsala University, Uppsala S-75124, Sweden,1 Institute of Medical Microbiology, University Hospital of Münster, Domagkstrasse 10, Münster 48149, Germany,2 Department of Medical Microbiology & Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin3
Received 21 May 2008/ Returned for modification 27 May 2008/ Accepted 1 September 2008
Small-colony variants (SCVs) of Staphylococcus aureus are associated with persistent infections and may be selectively enriched during antibiotic therapy. Three pairs of clonally related S. aureus isolates were recovered from patients receiving systemic antibiotic therapy. Each pair consisted of an isolate with a normal phenotype and an isolate with an SCV phenotype. These SCVs were characterized by reduced susceptibility to gentamicin, reduced hemolytic activity, slow growth, and menadione auxotrophy. Sequencing of the genes involved in menadione biosynthesis revealed mutations in menB, the gene encoding naphthoate synthase, in all three strains with the SCV phenotype. The menB mutations were (i) a 9-bp deletion from nucleotides 55 to 63, (ii) a frameshift mutation that resulted in a premature stop codon at position 230, and (iii) a point mutation that caused the amino acid substitution Gly to Val at codon 233. Fluctuation tests showed that growth-compensated mutants arose in the SCV population of one strain, strain OM1b, at a rate of 1.8 x 10–8 per cell per generation. Sequence analyses of 23 independently isolated growth-compensated mutants of this strain revealed alterations in the menB sequence in every case. These alterations included reversions to the wild-type sequence and intragenic second-site mutations. Each of the growth-compensated mutants showed a restoration of normal growth and a loss of menadione auxotrophy, increased susceptibility to gentamicin, and restored hemolytic activity. These data show that mutations in menB cause the SCV phenotype in these clinical isolates. This is the first report on the genetic basis of menadione-auxotrophic SCVs determined in clinical S. aureus isolates.
* Corresponding author. Mailing address: Microbiology Program, Department of Cell and Molecular Biology, Box 596, The Biomedical Center, Uppsala University, Uppsala S-75124, Sweden. Phone: 46-18-4714354. Fax: 46-18-530396. E-mail: diarmaid.hughes{at}icm.uu.se
Published ahead of print on 8 September 2008.
These two authors contributed equally to the manuscript.
Antimicrobial Agents and Chemotherapy, November 2008, p. 4017-4022, Vol. 52, No. 11
0066-4804/08/$08.00+0 doi:10.1128/AAC.00668-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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