The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates

Abstract

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 > or = 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.

FIG. 1.

Mutation frequencies of S. aureus isolates. CF-derived TD-SCVs, NCVs, and non-CF NCVs cultured from nasal swabs or blood cultures were analyzed. Each dot represents the mean mutation frequency plus standard deviation calculated from three independent experiments for one isolate. CF isolates (TD-SCVs and NCVs) showed significantly higher mutation frequencies than non-CF isolates (P < 10⁻⁴, Kruskal-Wallis test). The TD-SCVs harbored significantly more strong hypermutators (mutation rate ≥ 10⁻⁷) than the CF and non-CF NCVs (P = 0.047, Fisher's exact test). Culture conditions are defined in Materials and Methods.

FIG. 2.

Antimicrobial resistance of S. aureus CF-derived TD-SCVs (n = 20), CF NCVs (n = 20), and non-CF NCVs (n = 20).

FIG. 3.

Pulsed-field gel electrophoresis patterns of SmaI digests of total DNA of the clinical CF isolates S33, S68, and S231 (TD-SCVs) and S187 (NCV), which all showed a premature termination of the mutL coding sequence at the same amino acid position. Lane M, DNA size marker. Molecular sizes of the marker fragments (in kilobases) are given on the left.