Pathophysiological Mechanisms of Staphylococcus Non-aureus Bone and Joint Infection: Interspecies Homogeneity and Specific Behavior of S. pseudintermedius

Abstract

Implicated in more than 60% of bone and joint infections (BJIs), Staphylococci have a particular tropism for osteoarticular tissue and lead to difficult-to-treat clinical infections. To date, Staphylococcus aureus internalization in non-professional phagocytic cells (NPPCs) is a well-explored virulence mechanism involved in BJI chronicity. Conversely, the pathophysiological pathways associated with Staphylococcus non-aureus (SNA) BJIs have scarcely been studied despite their high prevalence. In this study, 15 reference strains from 15 different SNA species were compared in terms of (i) adhesion to human fibronectin based on adhesion microplate assays and (ii) internalization ability, intracellular persistence and cytotoxicity based on an in vitro infection model using human osteoblasts. Compared to S. aureus, S. pseudintermedius was the only species that significantly adhered to human fibronectin. This species was also associated with high (even superior to S. aureus) internalization ability, intracellular persistence and cytotoxicity. These findings were confirmed using a panel of 17 different S. pseudintermedius isolates. Additionally, S. pseudintermedius internalization by osteoblasts was completely abolished in β1 integrin-deficient murine osteoblasts. These results suggest the involvement of β1 integrin in the invasion process, although this mechanism was previously restricted to S. aureus. In summary, our results suggest that internalization into NPPCs is not a classical pathophysiologic mechanism of SNA BJIs. S. pseudintermedius appears to be an exception, and its ability to invade and subsequently induce cytotoxicity in NPPCs could explain its severe and necrotic forms of infection, notably in dogs, which exhibit a high prevalence of S. pseudintermedius infection.

Keywords: Staphylococcus non-aureus; Staphylococcus pseudintermedius; bone and joint infection (BJI); fibronectin; integrin α5β1; invasion.

FIGURE 1

Evaluation of Staphylococcus spp. in terms of their adherence to human fibronectin, their internalization and their persistence in MG63 cells. (A) Quantification of the fibronectin adhesion capacity of 15 Staphylococcus spp. reference strains. All of the results are expressed as percentages of the values obtained for the S. aureus 8325-4 strain. The horizontal bars denote the means derived from three independent experiments performed in quadruplicate. The fibronectin adhesion capacity of the SNA strains was compared to that of Δfnb S. aureus using the one-tailed Mann–Whitney test with an α risk of 0.05. (^∗∗∗p < 0.001; SNA: Staphylococcus non-aureus). (B) MG63 cells were infected for 2 h at 37°C with staphylococci at a multiplicity of infection (MOI) of 100:1 for all strains. The invasion and persistence capacities were assessed by quantifying the viable intracellular bacterial loads at 3 h and 24 h post-infection after gentamicin treatment. Bars represent means ± standard deviations derived from three experiments performed in triplicate, and the results are expressed as the percentages of initial inoculum internalized. (C) Quantifications of LDH release, reflecting cytotoxicity, were performed on culture supernatants at 24 h post-infection. All of the results are expressed as the percentages of the values obtained for the control “uninfected cells” (100%), represented by the red line. Bars represent means ± standard deviations derived from three experiments performed in triplicate. The increase in the LDH concentration in the cells infected with an SNA strain compared to the uninfected control cells was evaluated using a one-tailed Mann–Whitney test with an α risk of 0.05 (^∗∗∗p < 0.001; LDH: lactate dehydrogenase; SNA: Staphylococcus non-aureus; pi: post-infection).

FIGURE 2

Fibronectin adhesion and internalization of 17 S. pseudintermedius clinical isolates into MG63 osteoblasts. (A) Adhesion of the isolates to human fibronectin was evaluated by measuring the absorbance at 620 nm normalized to the reference strain S. aureus. Plots and means were derived from three experiments performed in triplicate. The difference in the capacity of the SNA strains to adhere to fibronectin compared to DU5883 Δfnb S. aureus was assessed using the Mann–Whitney one-tailed test with an α risk of 0.05 (^∗∗p < 0.01, ^∗∗∗p < 0.001; OD: optical density). (B) Capacity of internalization of S. pseudintermedius clinical isolates into MG63 osteoblasts. Bars represent means ± standard deviation derived from two experiments performed in triplicate, and the results are expressed as percentages relative to the initial inoculum († = 0.01 ± 0.01%).

FIGURE 3

Determination of the involvement of β1 integrin in the S. pseudintermedius internalization process using murine osteoblast cell lines (OB-β₁^fl/fl and OB-β₁^-/-) with functional and non-functional β₁ subunits, respectively. Internalization capacity of S. aureus 8325-4 (positive control), DU5883 S. aureus Δfnb (negative control) and the reference strain S. pseudintermedius LMG 22219 in OB-β₁^fl/fl and OB-β₁^-/- osteoblasts measured 3 h post-infection derived from three experiments performed in triplicate. The bilateral Mann–Whitney test was used to compare the data obtained for the OB-β₁^fl/fl and OB-β₁^-/- osteoblasts at an α risk of 0.05 (^∗∗∗p < 0.001).