Spontaneously Occurring Small-Colony Variants of Staphylococcus aureus Show Enhanced Clearance by THP-1 Macrophages.

Simon M. Stoneham,Daire M. Cantillon,Simon J. Waddell,Martin J. Llewelyn

doi:10.3389/fmicb.2020.01300

Abstract

Staphylococcus aureus is a common cause of chronic and relapsing infection, especially when the ability of the immune system to sterilize a focus of infection is compromised (e.g., because of a foreign body or in the cystic fibrosis lung). Chronic infections are associated with slow-growing colony phenotypes of S. aureus on solid media termed small-colony variants (SCVs). Stable SCVs show characteristic mutations in the electron transport chain that convey resistance to antibiotics, particularly aminoglycosides. This can be used to identify SCVs from within mixed-colony phenotype populations of S. aureus. More recently, populations of SCVs that rapidly revert to a “wild-type” (WT) colony phenotype, in the absence of selection pressure, have also been described. In laboratory studies, SCVs accumulate through prolonged infection of non-professional phagocytes and may represent an adaptation to the intracellular environment. However, data from phagocytic cells are lacking. In this study, we mapped SCV and WT colony populations in axenic growth of multiple well-characterized methicillin-sensitive and methicillin-resistant S. aureus strains. We identified SCVs populations on solid media both in the presence and absence of gentamicin. We generated stable SCVs from Newman strain S. aureus, and infected human macrophages with WT S. aureus (Newman, 8325-4) and their SCV counterparts (SCV3, I10) to examine intracellular formation and survival of SCVs. We show that SCVs arise spontaneously during axenic growth, and that the ratio of SCV:WT morphology differs between strains. Exposure to the intracellular environment of human macrophages did not increase formation of SCVs over 5 days and macrophages were able to clear stable SCV bacteria more effectively than their WT counterparts.

Highlights

Staphylococcus aureus disease includes common acute skin and soft tissue infections, acute toxin-mediated processes, acute invasive infection such as bacteremia and chronic infections, e.g., of bone and prosthetic joints (Lowy, 1998)
To assess the proportion of small-colony variants (SCVs) and WT colonies detectable in axenic culture of S. aureus, stationary phase (24 h culture) NCTC 12493, 8532, 8325-4, Newman, and MRSA 252 strains of S. aureus were inoculated onto solid media containing gentamicin to select for gentamicin-resistant SCVs [Mueller–Hinton agar containing 2 μg/mL gentamicin (MHA+G)], and antibiotic-free media to enumerate WT colonies (LA) (Figure 1A)
To examine the dynamics of the SCV population over time, we extended culture of S. aureus strains 8325-4 and Newman up to 7 days with the hypothesis that nutrient depletion and acidification of the media would select for SCV formation by favoring slow-growing phenotypes

Summary

Introduction

Staphylococcus aureus disease includes common acute skin and soft tissue infections, acute toxin-mediated processes (food poisoning, toxic shock), acute invasive infection such as bacteremia and chronic infections, e.g., of bone and prosthetic joints (Lowy, 1998). Several studies have examined intracellular persistence of SCVs within nonprofessional phagocytes: endothelial cells (von Eiff et al, 1997; Rollin et al, 2017), epithelial cells (Tuchscherr et al, 2011; Leimer et al, 2016), and osteoblasts (Kalinka et al, 2014). This has led to the hypothesis that SCVs represent an adapted intracellular phenotype

Methods

Results

Conclusion