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Antimicrobial Agents and Chemotherapy, June 2008, p. 2183-2189, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01395-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Thymidine-Dependent Small-Colony-Variant Phenotype Is Associated with Hypermutability and Antibiotic Resistance in Clinical Staphylococcus aureus Isolates

Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt am Main, Frankfurt am Main, Germany,¹ Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany²

Received 29 October 2007/ Returned for modification 20 January 2008/ Accepted 23 March 2008

Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate 10^–7) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.

Published ahead of print on 31 March 2008.