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ABSTRACTAnimal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureussurface adhesins and platelet aggregation in the infection process. In humans, however, S. aureusIE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureusvirulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 106CFU of Lactococcus lactispIL253 or a recombinant L. lactisstrain expressing an individual S. aureussurface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1a (IL-1a), IL-1ß, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis(ClfA strain) (52%; P= 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P< 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1ß and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureusIE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Staphylococcus aureusinfections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureusNewman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureusLPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureusNewman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureusphysiology should integrate all three approaches.

ABSTRACTStreptococcus uberisis an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis(MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberiscan readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberisin that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q554E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberismight follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.

ABSTRACTThe antifungal agent fluconazole (FLC) is widely used in clinical practice. Monitoring FLC levels is useful in complicated clinical settings and in experimental infection models. A bioassay usingCandida pseudotropicalis, a simple and cost-effective method, is validated only for FLC levels ranging from 5 to 40 mg/liter. An extension of the analytical range is needed to cover most yeast MICs. A new bioassay in RPMI agar containing methylene blue was developed using C. albicansDSY1024, a mutant rendered hypersusceptible to FLC constructed by the deletion of the multidrug efflux transporter genes CDR1, CDR2, CaMDR1, andFLU1. Reproducible standard curves were obtained with FLC concentrations in plasma ranging from 1 to 100 mg/liter (quadratic regression coefficient > 0.997). The absolute sensitivity was 0.026 μg of FLC. The method was internally validated according to current guidelines for analytical method validation. Both accuracy and precision lied in the required ±15% range. FLC levels measured by bioassay and by high-performance liquid chromatography (HPLC) performed with 62 plasma samples from humans and rats showed a strong correlation (coefficients, 0.979 and 0.995, respectively; percent deviations of bioassay from HPLC values, 0.44% ± 15.31% and 2.66% ± 7.54%, respectively). In summary, this newly developed bioassay is sensitive, simple, rapid, and inexpensive. It allows nonspecialized laboratories to determine FLC levels in plasma to within the clinically relevant concentration range and represents a useful tool for experimental treatment models.

ABSTRACTThe resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrataclinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism. The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates. For this purpose, C. glabratagenes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiaestrain in which the ATP binding cassette (ABC) transporter genePDR5was deleted. Three different genes were recovered, and among them, only C. glabrata CDR1(CgCDR1), a gene similar to the Candida albicansABC transporterCDRgenes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates. A correlation between upregulation of this gene and azole resistance was thus established. The deletion ofCgCDR1in an azole-resistant C. glabrataclinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate. When CgCDR1obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain. This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C. glabrataclinical isolates.