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

Maali Y, Martins-Simões P, Valour F, Bouvard D, Rasigade JP, Bes M, Haenni M, Ferry T, Laurent F, Trouillet-Assant S - Front Microbiol (2016)

Bottom Line: Compared to S. aureus, S. pseudintermedius was the only species that significantly adhered to human fibronectin.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.

View Article: PubMed Central - PubMed

Affiliation: Centre International de Recherche en Infectiologie, INSERM U1111, CNRS UMR5308, Université de Lyon 1, ENS de Lyon, Team "Pathogenesis of staphylococcal infections"Lyon, France; Department of Clinical Microbiology, Northern Hospital Group, Hospices Civils de LyonLyon, France.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: The capacity of each isolate to adhere to fibronectin in vitro was evaluated after staining with crystal violet and measuring the OD620, which is directly related to the amount of adherent bacteria. S. aureus 8325-4 and the DU5883 mutant were used to validate the protocol. The ability of the fnb-deleted DU5883 strain to adhere to fibronectin was reduced by 76% compared to the wild type strain (p < 0.001, Figure 1A). The results, which are expressed as percentages of the S. aureus 8325-4 adhesion rate (100%), showed that the adhesion rates of all of the tested SNA isolates were below the adhesion rate the negative control DU5883 (24.87 ± 2.90%). The only exception was S. pseudintermedius, which exhibited a significantly higher adhesion rate (100.25 ± 13.72%) than DU5883 (p < 0.001). Additionally, the adhesion rate of S. pseudintermedius was in the range of the adhesion rates observed for S. aureus 8325-4.


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

Maali Y, Martins-Simões P, Valour F, Bouvard D, Rasigade JP, Bes M, Haenni M, Ferry T, Laurent F, Trouillet-Assant S - Front Microbiol (2016)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940379&req=5

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).
Mentions: The capacity of each isolate to adhere to fibronectin in vitro was evaluated after staining with crystal violet and measuring the OD620, which is directly related to the amount of adherent bacteria. S. aureus 8325-4 and the DU5883 mutant were used to validate the protocol. The ability of the fnb-deleted DU5883 strain to adhere to fibronectin was reduced by 76% compared to the wild type strain (p < 0.001, Figure 1A). The results, which are expressed as percentages of the S. aureus 8325-4 adhesion rate (100%), showed that the adhesion rates of all of the tested SNA isolates were below the adhesion rate the negative control DU5883 (24.87 ± 2.90%). The only exception was S. pseudintermedius, which exhibited a significantly higher adhesion rate (100.25 ± 13.72%) than DU5883 (p < 0.001). Additionally, the adhesion rate of S. pseudintermedius was in the range of the adhesion rates observed for S. aureus 8325-4.

Bottom Line: Compared to S. aureus, S. pseudintermedius was the only species that significantly adhered to human fibronectin.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.

View Article: PubMed Central - PubMed

Affiliation: Centre International de Recherche en Infectiologie, INSERM U1111, CNRS UMR5308, Université de Lyon 1, ENS de Lyon, Team "Pathogenesis of staphylococcal infections"Lyon, France; Department of Clinical Microbiology, Northern Hospital Group, Hospices Civils de LyonLyon, France.

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.

No MeSH data available.


Related in: MedlinePlus