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 treatmentresistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthaseencoding 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.

Infection Prevention and Control Guideline for Cystic Fibrosis: 2013 Update

“…S. aureus persists in the airways of people with CF for years and can develop a hypermutator phenotype that promotes adaptive changes, including SCVs, thought to facilitate survival of this organism within the CF airway. 84,85 SCVs are detected visually on laboratory agar plates and exhibit slower growth rates due to metabolic defects (eg, thymidine biosynthesis deficiency 86 ), and they therefore require special susceptibility testing. However, the testing method has not been standardized by the Clinical and Laboratory Standards Institute.…”

“…SCV strains of S. aureus are thought to have increased resistance to the innate immune system and increased resistance to antibiotics. 85,87,88 In vitro, SCVs have an increased ability to infect normal and CF airway epithelial cells. 89 In non-CF patients, SCV S. aureus are associated with chronic or recurrent infections, such as endocarditis and osteomyelitis.…”

Bacterial hypermutation: clinical implications

“…For example, thymidine-dependent smallcolony variants of Staphylococcus aureus, which are frequently isolated in CF patients and are implicated in persistent and recurrent infections, are hypermutators (Besier et al, 2008). Inactivation of the MMR system favours long-term persistence of oropharyngeal colonization by P. aeruginosa in CF mice .…”

Thymidine Auxotrophic Staphylococcus aureus Small-Colony Variant Endocarditis and Left Ventricular Assist Device Infection

“…Antimicrobial susceptibility was tested by Etest (bioMérieux, Marcy l'Etoile, France) by following the manufacturer's instructions (except that 5% defibrinated sheep blood was added to the medium, as previously described [5,6]) for all tested antimicrobics except gentamicin, amikacin, tobramycin, and kanamycin, which were tested by agar dilution following Clinical and Laboratory Standards Institutes guidelines (10) (except that 5% defibrinated sheep blood was added to the media, as previously described [5,6]). All testing was simultaneously performed on control strain S. aureus ATCC 29213 and was within the quality control range specified by Clinical and Laboratory Standards Institutes guidelines (11).…”

“…Thymidine auxotrophs are thought to arise due to antibiotic pressure after prolonged exposure to TMP-SMX (6,7). This has been best described in isolates from the airways of cystic fibrosis patients on chronic TMP-SMX therapy (15,17), but long-term TMP-SMX has also been associated with thymidine auxotrophy in S. aureus isolates from patients without cystic fibrosis (7).…”