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

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

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

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

Frequency of antibiotic resistance of 105 MSSA CC398 strains of human origin isolated between 1999 and 2011 in mainland France.

<p>Frequency of antibiotic resistance of 105 MSSA CC398 strains of human origin isolated between 1999 and 2011 in mainland France.</p

Data_Sheet_3_Understanding the Virulence of Staphylococcus pseudintermedius: A Major Role of Pore-Forming Toxins.DOCX

<p>Staphylococcus pseudintermedius is responsible for severe and necrotizing infections in humans and dogs. Contrary to S. aureus, the pathophysiological mechanisms involved in this virulence are incompletely understood. We previously showed the intracellular cytotoxicity induced after internalization of S. pseudintermedius. Herein, we aimed to identify the virulence factors responsible for this cytotoxic activity. After addition of filtered S. pseudintermedius supernatants in culture cell media, MG63 cells, used as representative of non-professional phagocytic cells (NPPc), released a high level of LDH, indicating that the cytotoxicity was mainly mediated by secreted factors. Accordingly, we focused our attention on S. pseudintermedius toxins. In silico analysis found the presence of two PSMs (δ-toxin and PSMε) as well as Luk-I leukotoxin, the presence of which was confirmed by PCR in all clinical strains tested (n = 17). Recombinant Luk-I leukotoxin had no cytotoxic activity on NPPc but the ectopic expression of the CXCR2 receptor in U937 cells conferred cytotoxity to Luk-I. This is in agreement with the lack of Luk-I effect on NPPc and the previous report of Luk-I cytoxic activity on immune cells. Contrary to Luk-I, synthetic δ-toxin and PSMε had a strong cytotoxic activity on NPPc. The secretion of δ-toxin and PSMε at cytotoxic concentrations by S. pseudintermedius in culture supernatant was confirmed by HPLC-MS. In addition, the supplementation of such supernatants with human serum, known to inhibit PSM, induced a complete abolition of cytotoxicity which indicates that PSMs are the key players in the cytotoxic phenotype of NPPc. The results suggest that the severity of S. pseudintermedius infections is, at least in part, explained by a combined action of Luk-I that specifically targets immune cells expressing the CXCR2 receptor, and PSMs that disrupt cell membranes whatever the cell types. The present study strengthens the key role of PSMs in virulence of the different species belonging to Staphylococcus genus.</p

Data_Sheet_1_Understanding the Virulence of Staphylococcus pseudintermedius: A Major Role of Pore-Forming Toxins.DOCX

<p>Staphylococcus pseudintermedius is responsible for severe and necrotizing infections in humans and dogs. Contrary to S. aureus, the pathophysiological mechanisms involved in this virulence are incompletely understood. We previously showed the intracellular cytotoxicity induced after internalization of S. pseudintermedius. Herein, we aimed to identify the virulence factors responsible for this cytotoxic activity. After addition of filtered S. pseudintermedius supernatants in culture cell media, MG63 cells, used as representative of non-professional phagocytic cells (NPPc), released a high level of LDH, indicating that the cytotoxicity was mainly mediated by secreted factors. Accordingly, we focused our attention on S. pseudintermedius toxins. In silico analysis found the presence of two PSMs (δ-toxin and PSMε) as well as Luk-I leukotoxin, the presence of which was confirmed by PCR in all clinical strains tested (n = 17). Recombinant Luk-I leukotoxin had no cytotoxic activity on NPPc but the ectopic expression of the CXCR2 receptor in U937 cells conferred cytotoxity to Luk-I. This is in agreement with the lack of Luk-I effect on NPPc and the previous report of Luk-I cytoxic activity on immune cells. Contrary to Luk-I, synthetic δ-toxin and PSMε had a strong cytotoxic activity on NPPc. The secretion of δ-toxin and PSMε at cytotoxic concentrations by S. pseudintermedius in culture supernatant was confirmed by HPLC-MS. In addition, the supplementation of such supernatants with human serum, known to inhibit PSM, induced a complete abolition of cytotoxicity which indicates that PSMs are the key players in the cytotoxic phenotype of NPPc. The results suggest that the severity of S. pseudintermedius infections is, at least in part, explained by a combined action of Luk-I that specifically targets immune cells expressing the CXCR2 receptor, and PSMs that disrupt cell membranes whatever the cell types. The present study strengthens the key role of PSMs in virulence of the different species belonging to Staphylococcus genus.</p

Data_Sheet_2_Understanding the Virulence of Staphylococcus pseudintermedius: A Major Role of Pore-Forming Toxins.DOCX

<p>Staphylococcus pseudintermedius is responsible for severe and necrotizing infections in humans and dogs. Contrary to S. aureus, the pathophysiological mechanisms involved in this virulence are incompletely understood. We previously showed the intracellular cytotoxicity induced after internalization of S. pseudintermedius. Herein, we aimed to identify the virulence factors responsible for this cytotoxic activity. After addition of filtered S. pseudintermedius supernatants in culture cell media, MG63 cells, used as representative of non-professional phagocytic cells (NPPc), released a high level of LDH, indicating that the cytotoxicity was mainly mediated by secreted factors. Accordingly, we focused our attention on S. pseudintermedius toxins. In silico analysis found the presence of two PSMs (δ-toxin and PSMε) as well as Luk-I leukotoxin, the presence of which was confirmed by PCR in all clinical strains tested (n = 17). Recombinant Luk-I leukotoxin had no cytotoxic activity on NPPc but the ectopic expression of the CXCR2 receptor in U937 cells conferred cytotoxity to Luk-I. This is in agreement with the lack of Luk-I effect on NPPc and the previous report of Luk-I cytoxic activity on immune cells. Contrary to Luk-I, synthetic δ-toxin and PSMε had a strong cytotoxic activity on NPPc. The secretion of δ-toxin and PSMε at cytotoxic concentrations by S. pseudintermedius in culture supernatant was confirmed by HPLC-MS. In addition, the supplementation of such supernatants with human serum, known to inhibit PSM, induced a complete abolition of cytotoxicity which indicates that PSMs are the key players in the cytotoxic phenotype of NPPc. The results suggest that the severity of S. pseudintermedius infections is, at least in part, explained by a combined action of Luk-I that specifically targets immune cells expressing the CXCR2 receptor, and PSMs that disrupt cell membranes whatever the cell types. The present study strengthens the key role of PSMs in virulence of the different species belonging to Staphylococcus genus.</p