Thymidine-dependent Small-colony Variants Research Articles

Thymidine‐auxotrophic Staphylococcus aureus small‐colony variant bacteremia in a patient with cystic fibrosis

Small-colony variants (SCVs) are a morphologic subtype of Staphylococcus aureus that may occur through several mechanisms including auxotrophism for thymidine, hemin, or menadione. Auxotrophic SCV for thymidine fail to synthesize DNA specifically because of mutations in the thymidylate synthase gene. We isolated S. aureus thymidine-dependent SCVs (TD-SCV) from blood and respiratory samples of a pediatric patient with cystic fibrosis and pulmonary exacerbation. Nutritional dependence of SCVs on hemin, menadione, and thymidine was evaluated. Antimicrobial susceptibility testing was performed through broth microdilution. Polymerase chain reaction was carried out for mecA, ermA, ermB, ermC, msrA, and msrB resistance genes. DNA sequencing was used to determine mutations in thyA and the multilocus sequence typing to identify genetic relatedness. Methicillin-sensitive S. aureus with normal and TD-SCV phenotypes were isolated from respiratory samples and a TD-SCV phenotype was isolated from blood culture. Macrolides resistance was attributed to ermC and msrB genes. All isolates belonged to ST398. The thyA gene in S. aureus is 957 nucleotides in length and encodes a protein of 318 amino acids. The TD-SCV isolates carried a -2 nt frameshift mutation (delta 667GC668) in thyA, creating a stop codon at residue 222 close to the predicted binding site for deoxyuridine monophosphate. The pathogenesis of SCVs is complex and not fully elucidated. Factors inherent to the patient such as physiological conditions, recurrent infections, or coinfection should be considered. Although SCVs are considered less virulent, they showed the ability to invade and cause bacteremia in the patient.

Prevalence and clinical associations of Staphylococcus aureus small-colony variant respiratory infection in children with cystic fibrosis (SCVSA): a multicentre, observational study

Staphylococcus aureus is the bacterium cultured most often from respiratory secretions of people with cystic fibrosis. Both meticillin-susceptible S aureus and meticillin-resistant S aureus (MRSA) can adapt to form slow-growing, antibiotic-resistant isolates known as small-colony variants that are not routinely identified by clinical laboratories. We aimed to determine the prevalence and clinical significance of S aureus small-colony variants and their subtypes among children with cystic fibrosis. The Small Colony Variant Staphylococcus aureus (SCVSA) study was a 2-year longitudinal study of children aged 6-16 years at five US cystic fibrosis centres, using culture methods sensitive for small-colony variants. Children were eligible if they had a documented diagnosis of cystic fibrosis and a minimum of two cystic fibrosis clinic visits and two respiratory cultures in the previous 12 months at enrolment. Participants attended clinic visits quarterly, at which respiratory tract samples were taken and measures of lung function (percentage of predicted forced expiratory volume in 1 s [FEV1] and frequency of respiratory exacerbations) were recorded. We determined the prevalence of small-colony variants and their subtypes, and assessed their independent associations with lung function and respiratory exacerbations using linear mixed-effects and generalised estimating equation logistic regression models. Analyses included both univariate models (unadjusted) and multivariate models that adjusted for potential confounders, including age, sex, race, baseline microbiology, treatment with CFTR modulator, and CTFR genotype. Between July 1, 2014, and May 26, 2015, we enrolled 230 children. Participants were followed-up for 2 years, with a mean of 6·4 visits (SD 1·14) per participant (range 2-9 visits) and a mean interval between visits of 3·94 months (SD 1·77). Across the 2-year period, S aureus small-colony variants were detected in 64 (28%) participants. Most (103 [56%] of 185) of the small-colony variants detected in these participants were thymidine dependent. Children with small-colony variants had significantly lower mean percentage of predicted FEV1 at baseline than did children without small-colony variants (85·5 [SD 19] vs 92·4 [SD 18·6]; p=0·0145). Small-colony variants were associated with significantly lower percentage of predicted FEV1 throughout the study in regression models, both in univariate analyses (regression coefficient -7·07, 95% CI -12·20 to -1·95; p=0·0068) and in multivariate analyses adjusting for potential confounders (-5·50, -10·51 to -0·48; p=0·0316). Small colony variants of the thymidine-dependent subtype had the strongest association with lung function in multivariate regression models (regression coefficient -10·49, -17·25 to -3·73; p=0·0024). Compared with children without small-colony variants, those with small-colony variants had significantly increased odds of respiratory exacerbations in univariate analyses (odds ratio 1·73, 95% CI 1·19 to 2·52; p=0·0045). Children with thymidine-dependent small-colony variants had significantly increased odds of respiratory exacerbations (2·81, 1·69-4·67; p=0·0001), even after adjusting for age, sex, race, genotype, CFTR modulator, P aeruginosa culture status, and baseline percentage of predicted FEV1 (2·17, 1·33-3·57; p=0·0021), whereas those with non-thymidine-dependent small-colony variants did not. In multivariate models including small-colony variants and MRSA status, P aeruginosa was not independently associated with lung function (regression coefficient -4·77, 95% CI -10·36 to 0·83; p=0·10) and was associated with reduced odds of exacerbations (0·54, 0·36 to 0·81; p=0·0028). Only the small-colony variant form of MRSA was associated with reduced lung function (-8·44, -16·15 to -0·72; p=0·0318) and increased odds of exacerbations (2·15, 1·24 to 3·71; p=0·0061). Infection with small-colony variants, and particularly thymidine-dependent small-colony variants, was common in a multicentre paediatric population with cystic fibrosis and associated with reduced lung function and increased risk of respiratory exacerbations. The adoption of small-colony variant identification and subtyping methods by clinical laboratories, and the inclusion of small-colony variant prevalence data in cystic fibrosis registries, should be considered for ongoing surveillance and study. The Cystic Fibrosis Foundation and the National Institutes of Health.

Characterization of clinically isolated thymidine-dependent small-colony variants of Escherichia coli producing extended-spectrum β-lactamase.

Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without blaCTX-M-3, isolated from a patient. Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes. The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with blaCTX-M-3 failed to transfer blaCTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without blaCTX-M-3 experimentally received the plasmid encoding blaSHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2. Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

Addition of thymidine to culture media for accurate examination of thymidine-dependent small-colony variants of methicillin-resistant Staphylococcus aureus: A pilot study

Small-colony variants (SCVs) are slow-growing subpopulations of various auxotrophic bacterial strains. Thymidine-dependent SCVs (TD-SCVs) are unable to synthesize thymidine; hence, these variants fail to grow in a medium without thymidine. In this study, we used 10 TD-SCVs of Staphylococcus aureus, of which four strains possessed mecA. We compared the efficacy of a newly modified medium containing thymidine for the detection of TD-SCVs of methicillin-resistant S. aureus (MRSA) to the efficacy of routinely used laboratory media. We observed that none of the 10 TD-SCVs of S. aureus grew in Mueller–Hinton agar, and four TD-SCVs of MRSA failed to grow on all MRSA screening media, except for the ChromID™ MRSA medium. Laboratory tests conducted using medium with thymidine incorporated showed that thymidine did not affect the minimum inhibitory concentrations of oxacillin and cefoxitin for clinical isolates of S. aureus, and was able to detect MRSA, including TD-SCVs. These findings showed that thymidine-incorporated media are able to detect TD-SCVs of MRSA without altering the properties of other clinically isolated MRSA strains.

Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression.

ABSTRACTStaphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology.

Small colony variants (SCVs) of Staphylococcus aureus – A bacterial survival strategy

Small colony variants (SCVs) of Staphylococcus aureus have been implicated in chronic recurrent infections and have therefore gained renewed interest during the last decade. Moreover, SCVs have been shown to be part of the regular growth cycle, are highly dynamic or stable and can be selected during various harsh conditions. As such, the emergence of SCVs has been described not only in human, but also in veterinary medicine as well as in food microbiology. SCVs are characterized by impaired growth, down-regulation of genes for metabolism and virulence, while sigB and genes important for persistence and biofilm formation are up-regulated. Furthermore, SCVs are resistant to various antibiotics such as aminoglycosides, trimethoprim-sulfamethoxazole, fluorquinolones, fusidic acid or even to antiseptics such as triclosan. An underlying mechanism has been determined for hemin-, menadione- and thymidine-dependent SCVs as well as for SCVs which are impaired in their stress response. SCVs are optimized for persistence in the host. They are able to reverse and thereby constitute a highly dynamic subpopulation of S. aureus. Such phenotype switching constitutes an integral part of the infection process enabling the bacteria to hide inside the host cell without eliciting a strong host response.

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

We report the isolation of thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus from unusual infection sites of patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and septicemia. Furthermore, we provide evidence that the essential growth factor for TD-SCVs, i.e., thymidine, and its metabolite dTMP are present in various human specimens.