Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus

Thymidine-Dependent Staphylococcus aureus Small-Colony Variants: Human Pathogens That Are Relevant Not Only in Cases of Cystic Fibrosis Lung Disease

“…The experiment was performed in triplicate and showed that four of the five TD-SCV isolates were able to revert to the NCV phenotype during subcultivation, depending on the extent of the alterations in thyA (Table 2). In this context, sequence analysis revealed that, in line with TD-SCVs isolated from CF respiratory samples, TD-SCVs isolated from unusual infection sites harbored various mutations in thyA, comprising individual nucleotide substitutions that resulted in premature stop codons and amino acid substitutions as well as a 9-bp, in-frame deletion (1,4). Furthermore, it became evident that an SCV-inducing nonsense mutation can be compensated for by different missense mutations in the same codon.…”

“…While SCVs dependent on hemin or menadione can be isolated from patients with osteomyelitis, persistent soft tissue infection, and device-related infections (15,17,20,21), thymidine-dependent SCVs (TD-SCVs) are well-known in the context of cystic fibrosis (CF) lung disease, especially after prior use of trimethoprimsulfamethoxazole (SXT) (2,7,9). It has been shown recently that random mutations in the thymidylate synthase-encoding thyA gene which lead to an intracellular lack of dTMP are responsible for the formation of the TD-SCV phenotype (1,4,24) and that hypermutability due to a defect DNA mismatch repair system favors the acquisition of these mutations (3). As TD-SCVs are apparently able to use thymidine and/or the metabolite dTMP from the environment, these variants are able to bypass the antibiotic effect of folic acid antagonists and consequently are resistant to SXT (7,13,24).…”

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

“…For the disc method, commercially available 10% hemin discs (Sigma-Aldrich, Saint Louis, MO) or discs impregnated with thymidine (100 g/ml) or menadione (25 g/ml) were placed on Mueller-Hinton agar (MHA) plates inoculated with a 0.5 McFarland standard of the isolate. The isolate was considered auxotrophic if it showed normal colonies or increased growth surrounding the impregnated disc compared to the periphery following 24 h of incubation in air at 37°C (5,17). For the agar method, following growth overnight on SBA, 3 to 4 similar colonies were inoculated onto MHA with and without supplementation with 125 g/ml thymidine, 25 g/ml menadione, or 10 g/ml hemin.…”

“…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).…”

“…The ideal approach to assessing the antimicrobial susceptibility of thymidine-dependent S. aureus SCVs is unclear. We assessed susceptibility using MHA supplemented with 5% defibrinated sheep blood, as described by Besier et al (5,6). The addition of defibrinated sheep blood is not recommended by the Clinical and Laboratory Standards Institute or the manufacturer of Etest strips, as it may supply constituents that bypass the effects of TMP-SMX, potentially causing TMP-SMXsusceptible strains to appear resistant to TMP-SMX (13).…”