Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections

Abstract

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of β-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections.

Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.

Introduction

Total joint prostheses and fracture fixation devices, are essential for the operative treatment of orthopaedic patients. However, these implants are prone to colonization of pathogenic bacteria and these bacteria can cause implant-associated bone infections (IABI), which might result in significant morbidity, amputation or even increased mortality (Montanaro et al., 2011; Osmon et al., n.d; Zimmerli et al., 2004). It is widely known that miscellaneous Gram-negative and Gram-positive bacteria like Enterobacteriaceae and staphylococci can cause these IABI, respectively. Recently, it has been shown that 35% of total IABI were associated with S. aureus, which also could play a role in polymicrobial infections (Montanaro et al., 2011).

S. aureus is a versatile bacterium causing a broad array of infections, such as skin infections to life-threatening diseases such as endocarditis, septicemia, and osteomyelitis. S. aureus as commensal bacterium is present in nares of around 30% human population and actively turn into pathogenic when host immune system is breached (Fitzgerald and Holden, 2016). Several recent studies suggest that certain mutations in the araC like regulators lead to the conversion of nasal carriage S. aureus into life-threatening bacteremia (Das et al., 2016; Laabei et al., 2015). The main cause of IABI was reported to be acute infections with highly virulent bacterial pathogens. The low virulent isolates are mainly detected in the chronic IABI due to lack of early clinical symptoms (Moran et al., 2010; Tande and Patel, 2014; Trampuz and Zimmerli, 2005).

During pre-, peri-, or post-operative stages of surgery, S. aureus initiate IABI by adhering to the implant devices with the help of their surface proteins and form biofilms which subsequently lead to the disintegration of implants. This biofilm formation is regulated by the quorum sensing (agr locus) system, which consists of two divergent transcriptional units (agrBDCA and RNAIII) and also regulates a wide variety of virulence factors of staphylococcal infections (Bronesky et al., 2016). In addition, S. aureus also invades osteoblasts. After invading osteoblasts, S. aureus enters the dormant stage with a major decrease in metabolic activity and can appear as small colony variants (SCV) that cause persistent infections (Mohamed et al., 2014; Wright and Nair, 2010). Gentamicin has been largely used with polymethylmethacrylate (PMMA) as PMMA gentamicin beads or gentamicin PMMA spacers in orthopaedic surgery for the treatment of osteomyelitis and implant-associated infections since the initial discoveries of Buchholz and Engelbrecht in the seventies of the last century (Buchholz and Engelbrecht, 1970; Klemm, 1979). Staphylococcus aureus has been shown to develop SCVs under the influence of low concentrations of gentamicin and other antibiotics that could be promote chronic infections (Tuchscherr et al., 2016). Despite vast literature that has described multi-faceted virulence of this facultative pathogen, the virulence regulation and molecular mechanism of its pathogenicity at various conditions remain unclear. Moreover, with its capability of acquiring mobile genetic elements and a high mutation rate, S. aureus is able to modify its genome organization quickly, to evade host immune system as well as to acquire resistance to prophylactic antibiotics (Harris et al., 2010).

We aimed at the identification of potential virulence properties of S. aureus strains isolated from acute and chronic IABI by means of their in vivo lethality, invasion of osteoblasts-like cell line in vitro, as well as proliferation and biofilm formation. These clinical isolates demonstrated low, intermediate and high virulence phenotypes in the alternative insect infection model G. mellonella. Furthermore, comparative genomics was performed between very high and very low virulent strains to identify potential genetic factors involved in acute and chronic infections in patients. The virulence properties of these strains displayed a high correlation to their biofilm formation and osteoblast invasion capabilities. Comparative genomics of selected low and high virulent strains revealed crucial insights in virulence and pathogenicity that reflect severity and chronicity of infection in patients. We thus demonstrate that novel insights into the genetic factors related to virulence mechanisms of S. aureus in deep invasive IABI can be obtained through comparative genomics.