Genotypic variations between wild-type and small colony variant of Staphylococcus aureus in prosthetic valve infectious endocarditis: a comparative genomic and transcriptomic analysis

Highlights

• A S. aureus small colony variant (SCV) strain grew more slowly than the isogenic wild-type strain.

• Virulence of the SCV strain was decreased.

• Genes harbouring mutations in the SCV were involved in metabolism, virulence and DNA repair.

• Most differentially expressed genes in the SCV were involved in metabolism.

Abstract

Staphylococcus aureus small colony variants (SCVs) can cause persistent infections. However, the genomes and transcriptomes of S. aureus SCVs remain poorly understood. A pair of isogenic wild-type and SCV methicillin-resistant S. aureus (MRSA) strains (IE1 and IE2, respectively) were isolated from a patient with prosthetic valve infectious endocarditis. The SCV strain IE2 grew more slowly than the wild-type strain, and serum killing and mouse lethality assays revealed that the virulence of SCV strain IE2 was decreased. Whole-genome sequencing of the SCV and wild-type strains revealed 15 mutations in nine genes associated with metabolism, virulence and DNA repair, including serine/threonine-protein kinase PrkC (prkC), glycerol-3-phosphate acyltransferase (plsY), 2-deoxyribose-5-phosphate aldolase (deoC), extracellular adherence protein (eap), iron compound ABC uptake transporter substrate-binding protein (sstD), RecU Holliday junction resolvase (recU), excinuclease ABC subunit B (uvrB), type I restriction–modification system, M subunit (hsdM) and smooth muscle caldesmon. Sequencing of RNA transcripts revealed that expression levels of 321 genes were upregulated and 582 genes were downregulated in SCV strain IE2 compared with IE1. Most of the differentially expressed genes were involved in metabolism. Expression levels of several genes involved in the pathways to which plsY, deoC, eap and sstD belonged were changed, associated with the metabolism and virulence of S. aureus. In conclusion, the reduced growth rate and decreased virulence of MRSA SCV strains may be related to mutations in and downregulation of genes associated with metabolism and virulence, especially plsY, deoC, eap and sstD.

Introduction

Small colony variants (SCVs) of Staphylococcus aureus are usually isolated from chronic infectious diseases such as cystic fibrosis, osteomyelitis, infective endocarditis and device-related infections [1], [2], [3], [4]. Phenotypically, SCV isolates have a slower growth rate, smaller colony size, decreased pigmentation, decreased haemolytic and coagulase activities, increased resistance to aminoglycosides and decreased toxin production [5]. Staphylococcus aureus SCVs show different auxotrophic phenotypes, including isolates defective in electron transport, thymidine biosynthesis, CO₂ auxotrophy, F0F1-ATPase and cytochromes. Several genetic mutations (menD, hemB and ctaA) have been associated with the electron transport-defective phenotype in SCVs [5], and random mutations in the thymidylate synthase gene (thyA) were found in thymidine biosynthesis-defective SCV strains [5].

Staphylococcus aureus SCV strains can cause recurrent and persistent infections. Although several studies have indicated that S. aureus SCV strains are less virulent than wild-type strains [6], [7], they persist for longer in eukaryotic cells than the corresponding normal phenotype strains [8]. A better understanding of the phenotype of SCV strains might help to explain their improved survival in the host. Treatment of S. aureus SCVs currently presents a clinical challenge and no standard guidelines are available [9]. Rifampicin, fluoroquinolones and quinupristin/dalfopristin might be effective against S. aureus SCV strains, but aminoglycosides are not recommended [10].

In the current study, a S. aureus SCV strain responsible for several recurrent infections was isolated from a patient with subacute infective endocarditis. Few studies to date have explored the genome and transcriptome of S. aureus SCVs [11]. We therefore explored the potential molecular mechanisms responsible for the persistence of this methicillin-resistant S. aureus (MRSA) SCV strain by examining growth curves and conducting serum killing assays, animal experiments, and genome and transcriptome sequencing.