Staphylococcus aureus panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils.

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

Staphylococcal strains vary greatly in their ability to induce an inflammatory response in endothelial cells.

BACKGROUND: Staphylococcus aureus is an important human pathogen of endovascular diseases that can take an acute destructive course and/or develop into a chronic infection with a high rate of relapse. Despite good activity of the appropriate antimicrobial substances in vitro, these infections are often difficult to treat, perhaps because of the complex infection process of endovascular tissue. METHODS: We used the microarray technique to analyze the response of endothelial cells to different S. aureus strains, including highly virulent isolates (6850 and ST239); the Cowan I strain, which has a nonfunctioning accessory gene regulator (agr) quorum-sensing system; and various clinical isolates. RESULTS: All tested strains were equally invasive in endothelial cells and were found intracellularly, but they differed greatly in their ability to induce inflammation. Wild-type isolates (6850 and ST239) up-regulated a huge number of genes, including many genes involved in innate immunity. By contrast, Cowan I, which failed to express important virulence factors related to the agr system (eg, alpha-toxin and proteases), did not induce these dramatic changes in endothelial gene expression. Similar results were obtained with clinical isolates. CONCLUSION: Given that agr-defective strains are commonly recovered during colonization and infection, agr deficiency might represent a strategy of S. aureus to hide intracellularly without provoking the host immune system and causing relapsing infections.

Staphylococcal infections impair the mesothelial fibrinolytic system: the role of cell death and cytokine release.

Bacterial peritonitis is a serious complication of peritoneal dialysis patients and of patients after abdominal surgery. Especially episodes due to Staphylococcus aureus can harm the peritoneum severely, resulting in peritoneal fibrosis. Human peritoneal mesothelial cells play a critical role in maintaining the integrity of the peritoneum, as they release components of the fibrinolytic system and regulate the influx of immune cells by expressing chemokines and adhesion molecules. Using cultured human peritoneal mesothelial cells (HMCs) and blood mononuclear cells, we analyzed the effect of different staphylococcal strains on mesothelial fibrinolysis and on inflammatory reactions and show that only S. aureus strains with an invasive and hemolytic phenotype decrease the production of fibrinolytic system components, most likely via cell death induction. Furthermore, HMCs react to invading staphylococci by enhanced expression of chemokines and adhesion molecules. Mononuclear cells were activated by all staphylococcal strains tested, and their culture supernatants impaired mesothelial fibrinolysis. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, efficiently enhanced the mesothelial fibrinolytic capacity under these inflammatory conditions, but did not protect HMCs against S. aureus-induced cell death. We conclude that only selected S. aureus strains decrease the release of fibrinolytic system components and provoke a mesothelial inflammatory response. These factors most likely contribute to peritoneal fibrosis and might account for the severe clinical presentation of S. aureus peritonitis.

Thermonuclease gene as a target for specific identification of Staphylococcus intermedius isolates: use of a PCR-DNA enzyme immunoassay.

A PCR-DNA enzyme immunoassay (PCR-DEIA) was developed for identification of the coagulase-positive species Staphylococcus intermedius. Two PCR primers and a hybridization probe were designed to target specific sequences of the S. intermedius thermonuclease (nuc) gene. In addition to S. intermedius reference strains, the PCR-DEIA was tested using 295 veterinary and human S. intermedius isolates. A specific 933-bp DNA fragment was successfully amplified in 281 (94.9%) S. intermedius isolates. Five canine isolates showed an unexpected 2.8-kbp band. Except for 10 amplicons derived from equine, camel, and pigeon isolates, all positive PCR results (n = 288, 96.6%) were confirmed by the colorimetric microtiter plate DEIA hybridization. Isolates that failed both in amplification and DEIA hybridization were only observed in equine isolates (10/23, 43.5%). Except for the limitations with isolates of hoofed animals, the S. intermediusnuc PCR assay has potential for rapid identification of S. intermedius and differentiation from other coagulase-positive staphylococci including S. aureus.