Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

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BackgroundCarbapenemase-producing Enterobacteriaceae (CPE), particularly carbapenemase-producing Klebsiella pneumoniae isolates, are important causative agents of nosocomial infections associated with significant mortality rates mostly in critical wards. The rapid detection and typing of these strains is critical either for surveillance purposes and to prevent outbreaks and optimize antibiotic therapy. In this study, the MALDI-TOF MS method was used to detect rapidly these isolates from blood cultures (BCs) and to obtain proteomic profiles enable to discriminate between carbapenemase-producing and non-carbapenemase-producing strains.ResultsFifty-five K. pneumoniae strains were tested. Identification and carbapenemase-production detection assay using Ertapenem were performed both from bacterial pellets extracted directly from BCs flasks and from subcultures of these strains. For all isolates, a complete antimicrobial susceptibility testing and a genotypic characterization were performed.We found 100% agreement between the carbapenemase-producing profile generated by MALDI TOF MS and that obtained using conventional methods. The assay detected and discriminated different carbapenemase-producing K. pneumoniae isolates within 30 min to 3 h after incubation with Ertapenem.ConclusionsMALDI-TOF MS is a promising, rapid and economical method for the detection of carbapenemase-producing K. pneumoniae strains that could be successfully introduced into the routine diagnostic workflow of clinical microbiology laboratories.

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Rapid detection of microorganisms in respiratory specimens is of paramount importance to drive the proper antibiotic regimen to prevent complications and transmission of infections. In the present study, the respiFISH® HAP Gram (-) Panel (miacom diagnostics GmbH, Duesseldorf, Germany) for the etiological diagnosis of hospital-acquired pneumonia was compared with the traditional culture method for the detection of major Gram-negative pathogens in respiratory specimens. respiFISH® combined the classical fluorescence in situ hybridization (FISH) technology with fluorescence-labeled DNA molecular beacons as probes. From September 2011 to January 2012, 165 samples were analyzed: the sensitivity and specificity were 94.39 and 87.93%, respectively. Only six pathogens (3.6%) were not identified with respiFISH®, while seven specimens (3%) provided false-positive results. This beacon-based identification shortens the time to result by at least one work day, providing species-level identification within half an hour. Considering the high sensitivity and specificity and the significant time saving, the introduction of bbFISH® assays could effectively complement traditional systems in microbiology laboratories.