Jacques Schrenzel

A RecA-LexA-dependent Pathway Mediates Ciprofloxacin-induced Fibronectin Binding in Staphylococcus aureus

Subinhibitory concentrations of ciprofloxacin (CPX) raise the fibronectin-mediated attachment of fluoroquinolone-resistant Staphylococcus aureus by selectively inducing fnbB coding for one of two fibronectin-binding proteins: FnBPB. To identify candidate regulatory pathway(s) linking drug exposure to up-regulation of fnbB, we disrupted the global response regulators agr, sarA, and recA in the highly quinolone-resistant strain RA1. Whereas agr and sarA mutants of RA1 exposed to CPX still displayed increased adhesion to fibronectin, the CPX-triggered response was abolished in the uvs-568 recA mutant, but was restored following complementation with wild type recA. Steady-state levels of recA and fnbB, but not fnbA, mRNA were co-coordinately increased >3-fold in CPX-exposed strain RA1. Electrophoretic mobility shift assays revealed specific binding of purified S. aureus SOS-repressor LexA to recA and fnbB, but not to fnbA or rpoB promoters. DNase I footprint analysis showed LexA binding overlapping the core promoter elements in fnbB. We conclude that activation of recA and derepression of lexA-regulated genes by CPX may represent a response to drug-induced damage that results in a novel induction of a virulence factor leading to increased bacterial tissue adherence.

Highly cooperative Ca2+ elevations in response to Ins(1,4,5)P3 microperfusion through a patch-clamp pipette

To study the initial kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations with a high time resolution and to avoid the problem of cell-to-cell heterogeneity, we have used the combined patch-clamp/microfluorimetry technique. The mathematical description of the microperfusion of Ins(1,4,5)P3 and the subsequent Ca2+ release consists of a monoexponential decay (cytosolic Ins(1,4,5)P3 concentration) and a Hill equation (Ins(1,4,5)P3 dose-response curve). Two additional Hill equations and an integration were necessary to include a putative dependence of Ins(1,4,5)P3-induced Ca2+ release on [Ca2+]i. Best-fitting analysis assuming [Ca2+]i-independent Ca2+ release yielded Hill coefficients between 4 and 12. The high cooperativity was also observed with the poorly metabolizable analog Ins(2,4,5)P3 and was independent of extracellular [Ca2+]. Best-fitting analysis including a positive [Ca2+]i feedback suggested a cooperativity on the level of Ins(1,4,5)P3-induced channel opening (n = 2) and an enhancement of Ins(1,4,5)P3-induced Ca2+ release by [Ca2+]i. In summary, the onset kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations in single HL-60 granulocytes showed a very high cooperativity, presumably because of a cooperativity on the level of channel opening and a positive Ca2+ feedback, but not because of Ca2+ influx or Ins(1,4,5)P3 metabolism. This high cooperativity, acting in concert with negative feedback mechanisms, might play an important role in the fine-tuning of the cellular Ca2+ signal.

From Genotype to Phenotype: A Clinical Perspective for Hospital-Acquired Pneumonia

Clinical metagenomics (CMg), referred to as the application of next-generation sequencing (NGS) to clinical samples, is a promising tool for the diagnosis of hospital-acquired pneumonia (HAP). Indeed, CMg allows identifying pathogens and antibiotic resistance genes (ARGs), thereby providing the information required for the optimization of the antibiotic regimen. Hence, provided that CMg would be faster than conventional culture, the probabilistic regimen used in HAP could be tailored faster, which should lead to an expected decrease of mortality and morbidity. While the inference of the antibiotic susceptibility testing from metagenomic or even genomic data is challenging, a limited number of antibiotics are used in the probabilistic regimen of HAP (namely beta-lactams, aminoglycosides, fluoroquinolones, glycopeptides and oxazolidinones). Accordingly in the perspective of applying CMg to the early diagnostic of HAP, we aimed at reviewing the performances of whole genomic sequencing (WGS) of the main HAP-causing bacteria (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia and Staphylococcus aureus) for the prediction of susceptibility to the antibiotic families advocated in the probabilistic regimen of HAP.

Rapid Screening and Identification of Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. While the first S. aureus isolates displaying resistance to methicillin (MRSA) were reported in the early 1960s [1], endemic strains of MRSA carrying multiple resistance determinants have become a worldwide nosocomial problem only in the early 1980s [2]. The presence of MRSA in an institution is paralleled by an increased rate of bacteremia, or other severe MRSA infections [3]. MRSA-related bacteremia carries a threefold attributable cost and a threefold excess length of hospital stay when compared with methicillin-susceptible S. aureus (MSSA) bacteremia [4].