The virulence of Staphylococcus aureus is essentially determined by cell wall associated proteins and secreted toxins that are regulated and expressed according to growth phases and/or growth conditions. Gene expression is regulated by specific and sensitive mechanisms, most of which act at the transcriptional level. Regulatory factors constitute numerous complex networks, driving specific interactions with target gene promoters. These factors are largely regulated by two-component regulatory systems, such as the agr, saeRS, srrAB, arlSR and lytRS systems. These systems are sensitive to environmental signals and consist of a sensor histidine kinase and a response regulator protein. DNA-binding proteins, such as SarA and the recently identified SarA homologues (SarR, Rot, SarS, SarT, SarU), also regulate virulence factor expression. These homologues might be intermediates in the regulatory networks. The multiple pathways generated by these factors allow the bacterium to adapt to environmental conditions rapidly and specifically, and to develop infection. Precise knowledge of these regulatory mechanisms and how they control virulence factor expression would open up new perspectives for antimicrobial chemotherapy using key inhibitors of these systems.

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/ DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.

Summary. DNA hybridisation of 309 consecutive Staphylococcus aureus clinical isolates with oligonucleotide probes specific for genes encoding Panton-Valentine leucocidin (luk-PV) and y-haemolysin (hlg) revealed that 99% of randomly selected strains carried the hlg locus whereas only 2 YO harboured the luk-PV as well as the hlg loci. Only 1 YO of the strains did not possess either gene. In a clinical prospective study of independent S . aureus strains, 58 Panton-Valentine leucocidin (PVL)-producing isolates were shown to be responsible for primary skin infections, mainly furuncles (86 YO). Phage susceptibility patterns and pulsed field gel electrophoresis (PFGE) profiles of DNA were shown to be polymorphic epidemiological markers of PVL-producing strains. In eight patients with recurrent furuncles, the PVL-producing strains isolated either from furuncles or from the anterior nares were considered to be identical in each based upon phage sensitivity profiles or PFGE patterns.

A new member of the staphylococcal bi-component leucotoxins family, LukE (32 kDa) and LukD (34.3 kDa) has been characterized from Staphylococcus aureus strain Newman. LukE was 58^68% identical with the class S proteins, whereas LukD was 71^77% identical with the class F proteins of the family. A partial immunoreactivity with the various affinitypurified antibodies specific for the other proteins was observed. Immunoprecipitation assay and gene probing confirmed a 30% frequency among human clinical isolates, differing from the distribution of the other known leucotoxins (P 6 0.005). LukE+LukD was as effective as the Panton-Valentine leucocidin for inducing dermonecrosis when injected in the rabbit skin, but not hemolytic and poorly leucotoxic compared to other leucotoxins expressed by Staphylococcus aureus.z 1998 Federation of European Biochemical Societies.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now widely used for marker/multi-biomarker detection in medical diagnosis. We tested a new protocol for bacterial identification from blood culture broths in hospital routine by using collection tubes with separator gels on 503 included samples examined over 3 months, where 1.5 mL was injected by a syringe into BD Vacutainer tubes from BACTEC-positive bottles, before processing for bacterial protein extraction. Samples were loaded in duplicate onto the MALDI MS target, allowing a series of 12 samples to be processed in duplicate within 80 min by using Biflex III and BioTyper 2.0 software (Bruker). Including polymicrobial samples, 193 of 213 of Gram-negative bacteria (91.08%) and 284 of 319 of Gram-positive bacteria (89.02%) were correctly identified at the species level. Enterobacteriaceae constituted 35.15% of all species found, Staphylococaceae 37.96%, Streptococaceae and Enterococaceae 20.85%, Pseudomonadaceae 1.69%, and anaerobes 2.44%. In most of the polymicrobial samples, one of the species present was identified (80.9%). Seven isolates remained misidentified as Streptococcus pneumoniae, all belonging to Streptococcus mitis. Staphylococcus aureus was identified better when grown on anaero-aerobic medium, and MALDI BioTyper identification scores as low as 1.4 were pertinent, provided that four successive proposals of the same species were given. This new protocol correlates with conventional microbiology procedures by up to 90%, and by >95% for only monomicrobial samples, and provides a decreased turn-around time for identification of bacteria isolated from blood cultures, making this technology suitable also for blood cultures, with less delay and cost decreases in bacterial diagnostics, and favouring better care of patients.