Virulence and antimicrobial resistance genes in human MRSA ST398 isolates in Austria.

This study determined the genetic background of virulence and resistance genes of MRSA ST398 in Austria. From 2004 up to 2008 a total of 41 human isolates of MRSA ST398 were investigated for virulence and resistance gene patterns using DNA microarray chip analysis. Highly similar virulence gene profiles were found in 29 (70·7%) of the isolates but genes encoding Panton-Valentine leukocidin, enterotoxins, or toxic shock syndrome toxin were not detected. Genes conferring resistance to tetracycline and erythromycin-lincosamide were common as all but one of the isolates exhibited tetM and/or tetK, which are involved in tetracycline resistance, and 12 (29·9%) were positive for ermC, conferring resistance to erythromycin/lincosamide. SplitsTree analysis showed that 40 isolates were closely related. Changes in virulence and resistance gene patterns were minimal over the observed time period.

Antimicrobial resistance of Streptococcus pneumoniae in Southeast Austria, 1997-2008.

Antibiotic resistance in Streptococcus pneumoniae has increased worldwide but varies within geographical regions. We conducted a retrospective analysis of resistance in S. pneumoniae over a 12-year period to assess local and temporal trends in antibacterial resistance. From 1997 to 2008, a total of 1814 non-duplicate S. pneumoniae isolates were identified at the Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Austria. Antibiotic resistance was determined by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion test. For penicillin, the minimum inhibitory concentration was determined by Etest. Susceptibility was defined according to CLSI interpretive criteria. For penicillin, resistance rates were consistently low at 0.2% over the 12-year study period. An increase in resistance was remarkable for erythromycin (3.5% in 1997; 14.7% in 2008), clindamycin (1.8% in 1997; 10.6% in 2008) and tetracycline (1.8% in 2000; 11.0% in 2008). For trimethoprim/sulfamethoxazole, resistance increased slightly to 9.2% in 2008. Quinolones showed a low resistance rate of 0.2% that persisted over the whole study period. In contrast to previously published national data, resistance to penicillin was observed to remain at a remarkably low and constant level. Although international surveillance programmes have set up sustainable and interlinked data networks, our results suggest that regional surveillance may still be needed as decision support for appropriate empirical antibiotic therapy in the local health setting.

Use of automated repetitive-sequence-based PCR for rapid laboratory confirmation of nosocomial outbreaks.

OBJECTIVE: Rapid and reliable diagnosis of genetic relatedness of clinical isolates in microbiologic laboratory is essential in case of nosocomial outbreak investigation. Most molecular techniques used to type microorganisms are technically demanding and time consuming. Currently repetitive-sequence-based PCR (rep-PCR) technique has been adapted to an automated format on the DiversiLab system (bioMérieux, Marcy l'Etoile, France). Aim of this study was to compare the performance of the DiversiLab system to that of pulsed-field gel electrophoresis (PFGE) in nosocomial outbreaks. METHODS: 122 clinical isolates (28 Methicillin-resistant Staphylococcus aureus (MRSA), 26 Acinetobacter baumannii, 45 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae and 13 ESBL-producing Klebsiella oxytoca) were investigated. 70 isolates originated from six well-documented outbreaks, 52 were non-outbreak isolates. RESULTS: Concordant results for identification of outbreak and non-outbreak MRSA, A. baumannii and ESBL-producing K. pneumoniae strains were achieved with both methods. In the outbreak of ESBL-producing K. oxytoca automated rep-PCR was slightly more discriminatory than PFGE. Rep-PCR identified investigated ESBL-producing K. oxytoca outbreak-strains as indistinguishable or closely related, showing similarity of >90%, while PFGE identified these strains as indistinguishable. CONCLUSION: Automated rep-PCR assays on the DiversiLab system were used for MRSA, A. baumannii and for the first time ESBL-producing Klebsiella spp. and proved as a rapid and reliable method for molecular analysis of nosocomial outbreaks.

Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways.

The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.

Effect of malathion on apoptosis of murine L929 fibroblasts: a possible mechanism for toxicity in low dose exposure.

While acute organophosphorous compound poisoning due to inhibition of acetylcholinesterase is a well-established clinical entity, the existence of chronic poisoning due to exposure to low levels of organophosphorous compounds (below the threshold required for cholinergic clinical symptoms) is a hotly debated issue. In this study, we have evaluated the effects of noncholinergic doses of malathion (0.01-20 microM) on apoptosis of murine L929 fibroblasts. Employing flow cytometric and caspase activation analyses we demonstrate that malathion induces apoptosis in L929 cells in a dose- and time-dependent manner. The initiator caspases (caspase-8 and caspase-9) as well as the effector caspase (caspase-3) were activated by the treatment of L929 cells with malathion. Exposure of L929 cells to malathion in the presence of a general inhibitor of caspase, z-VAD-FMK abolished the apoptotic effect of the compound. In addition, malathion induced an increase in the expression of the pro-apoptotic protein p53. However, the induction of p53 expression was subsequent to activation of the caspase cascades. The present findings suggest, that the cytotoxicity of malathion at noncholinergic doses is mediated through caspase-dependent apoptosis.