Cytotoxicity of staphylococcal toxins on human macrophages and mature osteoclasts.

<p>Human monocytes were differentiated for 6 days into macrophages with macrophage colony-stimulating factor (M-CSF) (A) or into mature osteoclasts in the presence of M-CSF and receptor activator of NFκ-B ligand (RANK-L) (B and C). Staphylococcal toxins were then added to the cell culture medium containing propidium iodide (PI) and cell death was quantified by monitoring PI incorporation over a 3 hours period (A, B and C). Fluorescence in each well was normalised to the fluorescence obtained with untreated cells. Results represent the mean cytotoxicity with 95% confidence interval, of 3 independent experiments performed in triplicate on 3 different donors (*p<0.05, ** p<0.01, ***p<0.001). Hla: α haemolysin, Hlb: β haemolysin, Hlg AB and Hlg BC: γ haemolysins AB and BC, Luk ED and Luk GH: leukocidins ED and GH, PVL: Panton Valentine Leukocidin, TSST-1: toxic shock syndrome toxin, SEA: Staphylococcal enterotoxin A.</p

TSST-1 stimulates bone resorption capacity of mature human osteoclasts.

<p>For bone resorption assay, mature osteoclasts were detached from plastic on day 6 and seeded at 2.10⁴ cells/well on mineralized matrix Osteo Assay Surface 96-well plates. TSST-1 was added to the cell culture medium. After 24 hours of culture, the osteocorning matrices were stained with 5% silver nitrate to measure the resorbed area (white area). The percentages of matrix that were resorbed by untreated (A) or TSST-1-treated osteoclasts (B, C) were measured using the Fiji software. Bars represent 100 μm. Results of resorption area quantification (D) represent the mean of resorbed area with 95% confidence interval, of 3 independent experiments realised in triplicate on 3 different donors (*p<0.05, ** p<0.01, ***p<0.001). TSST-1: Toxic shock syndrome toxin.</p

Genes significantly associated with animal and human isolates belonging to the ST398 lineage.

*<p>For each gene, isolates with ambiguous microarray result were excluded from the analysis and from P-value calculation. For this reason, percentages are not necessarily calculated relative to the total no. of isolates.</p>**<p>P-values were calculated for each gene using a two-tailed Fisher's exact test and corrected for multiple testing using the Holm-Bonferroni method. Inf, positive infinite.</p

Dendrogram (UPGMA method, circular representation) based on the results of DNA microarrays study (172 genes only) of the 105 MSSA CC398 strains and the 53 MRSA CC398 isolated in France.

<p>For each of the 172 genes, the average of the results of all the strains corresponding to the same <i>spa</i> type was calculated. Each group of strains is represented by the corresponding <i>spa</i> type. s<i>pa</i> types corresponding to MRSA strains are colored in red.</p

Dendrogram (UPGMA method, squared representation) based on the homology degree of the <i>spa-types</i> of the 105 MSSA CC398 strains and the 53 MRSA CC398 isolated in France.

<p>The homology between the repeats of the strains is of 76%. <i>spa</i>CC: <i>spa</i> clonal complex. s<i>pa types</i> corresponding to MRSA strains are colored in red.</p

Description of the studied populations, corresponding used methods, and objectives.

*<p>The 10 strains isolated from infective endocarditis are included in the 89 strains from infections.</p>†<p>The belonging of the strains to CC398 was confirmed by MLST in case of discrepancies between previous typing techniques. All strains were isolated in mainland France.</p

Nature and number of infections caused by 89 MSSA CC398 strains collected in mainland France between 1999 and 2011.

<p>Disseminated infections are defined by the presence of septic metastasis in at least 2 noncontiguous organs (by opposition to bloodstream infections). Infections classified as infective endocarditis correspond to bloodstream infections with infective endocarditis and without any other septic metastasis.</p

Phylogenic tree with 158 <i>S. aureus</i> CC398 strains (modified <i>Parsimony</i> method, circular representation), based on the analysis of 319 genes and alleles by DNA microarrays.

<p>Since the PCR analysis of the <i>agr</i> alleles confirmed that all the strains were <i>agr</i> 1, the analysis of the results of DNA microarrays concerned only 319 genes and alleles. MSSA group is subdivided in <i>bla−</i> and <i>bla+</i> clusters, and MRSA group in SCC<i>mec</i> IV and SCC<i>mec</i> V clusters.</p

Macroscopic findings after challenge with a high inoculum of CA-MRSA USA300.

<p>(<b>A</b>) Pulmonary hemorrhages demarcated by hyperemic regions in both lungs. (<b>B</b>) Abscesses in the left lung indicating disseminated infection. (<b>C</b>) White circled muscle abscess in the right leg. (<b>D</b>) Bone marrow filled with pus indicating osteomyelitis. (<b>E</b>) Splenomegaly with necrosis observed after disseminated sepsis.</p

Comparisons of osteomyelitis parameters observed in <i>Δpsmα</i> (●○)-, <i>Δpsmαβhld</i> ▲△)- and with LAC-WT (◆◇)–infected non-survivors (black) and D14 survivors (white).

<p>(<b>A</b>) Median bacterial bone densities, expressed in log₁₀ CFUs/g of bone. The <i>Δpsmα</i>- or <i>Δpsmαβhld</i>-infected non-survivors differed significantly from those infected with LAC WT. (<b>B</b>) Mean histological scores (0–4: none, minimal, mild, moderate, severe) for lung involvement. The bacterial densities in <i>Δpsmαβhld</i>-infected non-survivors differed significantly from those infected with LAC WT (*<i>P</i> = .017), whereas inflammation was more severe in <i>Δpsmαβhld</i>- and <i>Δpsmα</i>-infected D14 survivors than those infected with LAC WT (**<i>P</i> = .04 and ***.01, respectively).</p