Genotypic and phenotypic analysis of clinical isolates of Staphylococcus aureus revealed production patterns and hemolytic potentials unlinked to gene profiles and source.

BACKGROUND: Nosocomial infections caused by the bacterial pathogen Staphylococcus aureus can lead to serious complications due to the varying presence of secreted toxins. Comparative studies of genomic information and production rates are needed to assess the pathogenic potential of isolated strains. Genotypic and phenotypic profiling of clinical and colonising isolates of S. aureus was used to characterise the release of exotoxins. Blood isolates were compared with colonisation strains to determine similarities and differences of single strains and clusters. RESULTS: Fifty-one fresh isolates obtained from colonised individuals (n = 29) and S. aureus bacteremia (SAB) patients (n = 22) were investigated. The prevalence of genes encoding for three cytolysins (alpha/beta/gamma toxin) and twenty-four superantigens (SEA-SElX) was determined. Isolates exhibited eighteen distinct combinations of superantigens. Sequence analysis identified mutated open reading frames in hla in 13.7% of all strains, in selw (92.2%) and in selx (15.7%). All corrupted genes were associated with specific clonal complexes. Functional assessment of alpha toxin activity by a rabbit erythrocyte lysis assay revealed that supernatants lacking alpha toxin still displayed hemolysis. This was due to the presence of gamma toxin, as proven by inhibition experiments using antisera raised against the respective recombinant proteins. Alpha toxin, SEC, and TSST1 production was quantified by enzyme-linked immunosorbent assays on supernatants of all hla, sec, and tst positive isolates. Blood isolates and colonising strains showed comparable amounts of secreted proteins within a wide range. Agr types I to IV were identified, but did not allow a prediction of high or low production rates. In contrast, alpha toxin production rates between distinct clonal complexes clearly differed. Spa typing was performed and revealed thirty-two unique spa gene patterns and eight small clusters comprising nineteen isolates. Recognised spa-typing clusters displayed highly similar production rates. CONCLUSION: Production rates of the three most prevalent exotoxins varied within both groups of blood isolates and colonising strains. By comparing genotypes and secretion, we found that identical complex gene patterns did not allow predictions of toxin production and function. However, identification of spa typing clusters was suitable to predict similar quantities of released exotoxins.

Functional and Immunological Studies Revealed a Second Superantigen Toxin in Staphylococcal Enterotoxin C Producing Staphylococcus aureus Strains.

Staphylococcus aureus is a human and animal pathogen as well as a commensal bacterium. It can be a causative agent of severe, life-threatening infections with high mortality, e.g., toxic shock syndrome, septic shock, and multi-organ failure. S. aureus strains secrete a number of toxins. Exotoxins/enterotoxins are considered important in the pathogenesis of the above-mentioned conditions. Exotoxins, e.g., superantigen toxins, cause uncontrolled and polyclonal T cell activation and unregulated activation of inflammatory cytokines. Here we show the importance of genomic analysis of infectious strains in order to identify disease-causing exotoxins. Further, we show through functional analysis of superantigenic properties of staphylococcal exotoxins that even very small amounts of a putative superantigenic contaminant can have a significant mitogenic effect. The results show expression and production of two distinct staphylococcal exotoxins, SEC and SEL, in several strains from clinical isolates. Antibodies against both toxins are required to neutralise the superantigenic activity of staphylococcal supernatants and purified staphylococcal toxins.

Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits.

Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SElN, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SElN led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSElN led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens.