In Vivo Mutations of Thymidylate Synthase (Encoded by thyA ) Are Responsible for Thymidine Dependency in Clinical Small-Colony Variants of Staphylococcus aureus

Abstract

Trimethoprim-sulfamethoxazole (SXT)-resistant

Characterization of Clinical Enterococcus faecalis Small-Colony Variants

“…The endocarditis was finally cured after replacement of the prosthetic aortic valve and appropriate antimicrobial therapy for 6 weeks. Among gram-positive bacteria, S. aureus SCVs have been most extensively investigated, and much is known about the biochemical and pathophysiological alterations in SCVs relative to their normal phenotypes (3,4,15,17,23). Like S. aureus SCVs and those of other bacterial species, the Enterococcus SCVs described here formed smaller colonies than the clonally related normal strain on universal culture media and failed to grow on simple media such as Mueller-Hinton-agar and broth without the addition of blood.…”

“…Reduced susceptibility to antimicrobial substances relative to that of their normal counterparts (26), which are often isolated simultaneously, restrict the therapeutic options for SCV infections. While SCVs of staphylococci, especially those of S. aureus, are intensively studied for their morphological, ultrastructural, metabolic, and growth characteristics (3,4,(15)(16)(17)22), little is known about the SCVs of Enterococcus species. Their occurrence in a patient with endocarditis (13) and in chickens with amyloid arthropathy (20) has only been briefly mentioned, without further characterization.…”

“…Generally, SCVs display a small colony size, a low growth rate, and atypical colony morphology (16). Biochemical characteristics of SCVs include a deficiency in electron transport or in thymidine biosynthesis (4,23). SCVs are specialized for intracellular persistence in host cells and have been shown to be associated with persistent and recurrent infections, such as osteomyelitis, arthritis, abscesses, and respiratory infections, in humans (23).…”

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).…”