Staphylococcus 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.
Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA’T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA’T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid–auxotrophic staphylococcal SCVs.
Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) of clonal complex (CC) 398 has emerged in livestock across Europe over the past 10 years.Case presentation: Case 1 was a patient with a history of destructive chronic polyarthritis and immunosuppressive therapy who presented with dyspnoea and pain in the shoulders, back and hips. Microbiological analysis of tissue samples, punctures and blood cultures revealed MRSA. Echocardiography showed mitral valve endocarditis. The patient was treated with daptomycin and fosfomycin. Case 2 was a patient presenting with pneumonia after lung transplantation. Respiratory specimens and perianal swabs revealed MRSA. The patient was treated with teicoplanin and linezolid. The patients did not recover from their infections and died. The isolates belonged to spa types t2576 (case 1) and t011 (case 2), to sequence type 398 within CC398 as determined by multilocus sequence typing and to staphylococcal cassette chromosome mec type 5. An IdentiBAC Microarray revealed the absence of a bacteriophage integrating into the hlb gene indicative of the livestock origin of the isolates. In 2013, 170 of 534 MRSA cases (31.8 %) among inpatients of the University Hospital Mü nster, Germany, were caused by closely related spa types clustering in one spa-CC indicative of CC398. Two of 12 MRSA isolates from blood cultures (16.7 %) were caused by isolates associated with MRSA CC398.Conclusion: Livestock-associated MRSA CC398 is emerging as a cause of human infections. This observation is alarming and should inspire future efforts to control MRSA in livestock, forestall community spread and monitor changes of the occurrence of MRSA CC398 among cases of human infections.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.