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Background Active efflux is a common mechanism of resistance to fluoroquinolones in Streptococcus pneumoniae. Two efflux systems have been described so far in this species: PmrA, a member of the major facilitator superfamily; and the two ABC transporters PatA and PatB. We studied the inducibility of expression of pmrA, patA and patB by using subinhibitory concentrations of fluoroquinolones. Methods A wild-type susceptible strain, two clinical isolates resistant to fluoroquinolones and two efflux mutants selected in vitro after exposure to ciprofloxacin were studied. MICs were determined for these strains and their mutants in which pmrA, patA or patB had been disrupted. Gene expression was determined after exposure to half the MIC of norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin or gemifloxacin and quantified by real-time PCR. Results Increased MICs of norfloxacin, ciprofloxacin and levofloxacin (to a lesser extent) and increased expression of patA and patB were seen for all resistant strains; these were reduced in patA or patB disruptants or in the presence of reserpine. Exposure to any of the five fluoroquinolones caused a reversible increase in expression of patA and patB, but not of pmrA. Mitomycin C, an inducer of the competence system in S. pneumoniae, also induced patA and patB expression in the two strains tested. Conclusion The ABC efflux system PatA/PatB is induced upon exposure to subinhibitory concentrations of fluoroquinolones, whether substrates of the transporter or not. This effect, possibly resulting from the activation of the competence pathway, may contribute to resistance.

Background and aims Optimal treatment of infections caused by Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila requires antibiotics with intracellular activity. Linezolid accumulates poorly within cells. Torezolid (TR-700) is a novel methyltetrazolyl oxazolidinone with potentially different cellular pharmacokinetic properties. Our aim was to examine the accumulation and intracellular activities of torezolid in this context. Methods Measurement of torezolid cell content and antibacterial activity in comparison with linezolid using human macrophages (THP-1) and human endothelial cells [human umbilical vein endothelial cells (HUVECs)], applying models allowing for the quantitative evaluation of the pharmacodynamics of antibiotics towards intracellular bacteria. Results Torezolid accumulated rapidly in THP-1 macrophages, reaching a stable intracellular to extracellular ratio of approximately 10 (compared with approximately 1-2 for linezolid) within 15 min. On a weight concentration basis (mg/L), torezolid was approximately 5- to 10-fold more potent intracellularly (lower concentration needed to achieve a bacteriostatic effect) than linezolid against phagocytosed S. aureus, L. monocytogenes and L. pneumophila, with no change in maximal efficacy ( approximately 1 log(10) reduction of the original, post-phagocytosis inoculum). When drugs were compared at equipotent concentrations (multiples of the MIC), no difference was seen between linezolid and torezolid, but the higher potency of torezolid allowed control of intracellular infections caused by linezolid-resistant S. aureus. Conclusions Torezolid exerts intracellular activity at lower extracellular concentrations than linezolid because of its greater potency independent of its greater intracellular accumulation. This may confer an advantage to torezolid in vivo if the drug can be used at dosages creating serum concentrations similar to those achieved with linezolid.

Resistance in Gram-negative pathogens is an increasing concern, with carbapenems often appearing as the only acceptable treatment option in serious infections. Reviving older compounds that have fallen into disuse may help to alleviate this burden. Temocillin (6-alpha-methoxy-ticarcillin) is resistant to most if not all classical and extended-spectrum beta-lactamases and to AmpC enzymes. It is also chemically stable, allowing administration by continuous infusion. Pharmacokinetic/pharmacodynamic analysis, aided by Monte-Carlo simulations, suggests a breakpoint of 8 mg/L for the registered maximum dosage of 4 g daily. Temocillin's weaknesses, explaining its limited previous use, are a lack of activity against Gram-positive organisms, anaerobes and Pseudomonas. In settings where these are unlikely or are covered by other agents, temocillin may be useful, potentially 'sparing' carbapenems and having little apparent potential to select for Clostridium difficile.

AIMS: In endothelial cells, caveolin-1 (cav-1) is known to negatively modulate the activation of endothelial nitric oxide synthase, a key regulator of blood pressure (BP). However, the impact of genetic alteration of cav-1 on vascular nitric oxide (NO) production and BP homeostasis in vivo is unknown. METHODS AND RESULTS: We used spectral analysis of systolic blood pressure (SBP) variability in mice chronically equipped with telemetry implants to identify frequency ranges (0.05-0.4 Hz; very low frequency, VLF) specifically responding to NO, independently of changes in absolute BP or systemic neurohormone levels. VLF variability was inversely correlated to aortic vasodilator-stimulated Ser(239) phosphoprotein (VASP) phosphorylation, reflecting NO bioactivity. We show that mice deficient in cav-1 have decreased VLF variability paralleled with enhanced systemic and vascular production of NO at unchanged mean SBP levels. Conversely, VLF variability was increased upon acute injection of mice, with a peptide containing the caveolin-scaffolding domain (CSD; residues 82-101) fused to an internalization sequence of antennapedia that decreased vascular and circulating NO in vivo. CONCLUSION: These data highlight the functional importance of cav-1 for the production of bioactive NO in conduit arteries and its control of central BP variability. Given the impact of the latter on target organ damage, this raises the interest for genetic, pharmacological, or molecular interventions that modulate cav-1 expression in diseases with NO-dependent endothelial dysfunction.

Background Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin). Methods J774 macrophages and rat embryo fibroblasts were exposed for up to 24 and 72 h to telavancin (5-90 mg/L). The following studies were performed: measurement of (14)C-labelled telavancin cellular uptake and subcellular distribution (cell fractionation), determination of pericellular membrane integrity (lactate dehydrogenase release), electron microscopy with morphometric analysis of changes in lysosome size and determination of total phospholipid and cholesterol content. Results The uptake of telavancin proceeded linearly as a function of time and concentration in both cell types (clearance rate of approximately 10 mL/g of protein/h). Efflux (macrophages) was approximately 5.7-fold slower. Telavancin subcellular distribution was superimposable on that of a lysosomal marker (N-acetyl-beta-hexosaminidase). It did not cause an increase in the release of lactate dehydrogenase and did not induce significant increases in total phospholipid or cholesterol content. It caused only mild morphological lysosomal alterations (similar to vancomycin and much less than oritavancin by morphometric analysis). Conclusions Telavancin is taken up by eukaryotic cells and localizes in lysosomes, causing mild morphological alterations without evidence of lipid metabolism alterations. These data support our observations that telavancin is active against intracellular S. aureus.

Background and aims: Temocillin, a 6 alpha-methoxy-penicillin stable towards most beta-lactamases (including extended-spectrum beta-lactamase), is presented as an alternative to carbapenems for susceptible Enterobacteriaceae in microbiological surveys. We aimed at documenting its potential clinical usefulness in intensive care (IC) patients using pharmacokinetic/pharmacodynamic approaches applied to conventional (twice daily) and continuous infusion (CI) modes of administration. Methods: (i) In vitro evaluation of temocillin stability and compatibility with other drugs under conditions pertinent of CI in IC patients; (ii) pharmacokinetic study in patients treated by CI (4 g/day; n = 6) versus [twice daily (2 g every 12 h); n = 6]; (iii) population pharmacokinetic analysis of twice daily with Monte Carlo simulations to determine 95% probability of target attainment (PTA(95)) versus MIC (based on time above MIC >= 40% for measured free drug). Results: Temocillin was stable at 37 degrees C in 8.34% solutions for 24 h and compatible with flucloxacillin and aminoglycosides, but not with several other antibiotic and non-antibiotic drugs. With CI, stable total serum concentrations were 73.5 +/- 3.0 mg/L (SEM) and free concentration 29.3 +/- 2.8 mg/L. With twice daily, C-max (total drug) was 147 +/- 12.3 mg/L (SEM; free drug: 50.3 +/- 15.8 mg/L), lowest trough (total drug) 12.3 mg/L, and PTA(95) (free drug) obtained for MIC <= 8 mg/L. Conclusions: Temocillin (4 g/day) by CI yields stable free serum concentrations above the current breakpoint (16 mg/L), although individual variations may suggest lowering the breakpoint to 8 mg/L (as for twice daily) unless the daily dose or the frequency of administration is increased.

Over the last decades, numerous papers have appeared-and still are appearing-that describe concentrations in tissues in an effort to predict the efficacy of an antimicrobial agent based on these concentrations and MICs for microorganisms. A common method is to use measurements of concentrations in tissue homogenates, comparing these with values derived from the corresponding blood samples and on that basis draw conclusions with respect to the potential clinical use of the drug. This approach is not justifiable for a number of reasons that includes both pharmacokinetic as well as pharmacodynamic causes. This way of presenting data with the derived conclusions is often misleading and may ultimately be harmful in patient care.

OBJECTIVES: Does exposure to subinhibitory concentrations of quinolones favour overexpression of efflux pumps or selection of target site mutations? METHODS: ATCC 49,619 (fully susceptible) and SP32 (clinical isolate with PmrA-mediated efflux and mutation in ParE) were exposed for 24 h in broth to ciprofloxacin, levofloxacin, moxifloxacin or garenoxacin at concentrations of 0.5x the MIC, with daily re-adjustments for up to 13 days. Efflux was detected phenotypically (decrease in MIC in the presence of reserpine), and expression of pmrA and patA/patB was measured by real-time PCR and comparative RT-PCR, respectively. Target site mutations were detected by sequencing of the quinolone resistance determining regions in parC, parE and gyrA. The clonal identity of isolates was checked by PFGE of genomic DNA. RESULTS: Ciprofloxacin selected for stable mutants with 2.5-5-fold MIC increases for ciprofloxacin, 2-3-fold for levofloxacin and 1.3-2-fold for garenoxacin and moxifloxacin [partial reversion with reserpine for ciprofloxacin, gemifloxacin and levofloxacin (SP32 strain only), but not for garenoxacin and moxifloxacin]. Increased MICs were associated with overexpression of patA/B but not pmrA. In contrast, exposure to levofloxacin, moxifloxacin or garenoxacin selected target site mutations (gyrA, parC, parE) in both strains. Increases in MIC caused by efflux were similar to those caused by target site mutations. CONCLUSIONS: Exposure of Streptococcus pneumoniae to subinhibitory MICs of ciprofloxacin, a substrate for efflux pumps, results in patA/B-mediated efflux whatever the initial level of expression of pmrA of the strain. Quinolones that are poorly (levofloxacin) or not affected (moxifloxacin, garenoxacin) in their activity by efflux transporters preferentially select for target site mutants.

BACKGROUND: Staphylococcus aureus survives in acid media, including phagolysosomes. Conflicting in vitro/in vivo data exist on its susceptibility to antibiotics in such environments. METHODS: Oxacillin and gentamicin activities against methicillin-susceptible S. aureus ATCC 25923 were compared extracellularly (broth; different pH) and assessed intracellularly (THP-1 macrophages), using a pharmacological approach (antibiotic concentrations: 0.01-1000 x MIC). Antibiotic cellular contents were determined by microbiological assay. RESULTS: MICs and MBCs increased 72-fold for gentamicin, and decreased 8-fold for oxacillin between pH 7.4 and 5.0. Plots of log(10) colony-forming unit changes at 24 h versus log(10) of antibiotic concentration followed sigmoidal shapes, allowing calculation of EC(50) (relative potency) and apparent E(max) (relative efficacy) in all conditions. In broth, the EC(50) of gentamicin rose 316-fold and that of oxacillin decreased 15-fold with unchanged apparent E(max) [-5 log (limit of detection)] between pH 7.4 and 5. Intracellularly, EC(50)s were similar to those observed extracellularly at pH 7.4, but E(max) values were much lower (-1 log) for both antibiotics. Calculations based on the assumed pH in phagolysosomes (5.4) and on local accumulation of antibiotics (gentamicin, 23-fold; oxacillin, 0.05-fold) suggest that the contrasting effects of acid pH on relative potencies of gentamicin and oxacillin could be almost exactly compensated for by differences in accumulation. CONCLUSIONS: The weak activity of gentamicin and oxacillin towards intraphagocytic S. aureus compared with extracellular forms is not related to an overall decrease of their relative potencies but to impaired efficacy, suggesting the need for new approaches to improve the eradication of intracellular S. aureus.

OBJECTIVES: Poor solubility and toxicity severely hinder the clinical use of amphotericin B (AmB), in spite of its attractive chemotherapeutic properties. Water-soluble complexes of AmB and polyvinylpyrrolidone (AmB-PVP) could display lower cytotoxicity while maintaining antifungal activity. METHODS: AmB-PVP [with PVP of 10, 24 and 40 kDa (AC1, AC2 and AC4)] were compared with free AmB for (i) activity against Candida spp. (five albicans; nine non-albicans) and Aspergillus spp. (four strains), (ii) haemolysis of sheep red blood cells, and (iii) release of lactate dehydrogenase from J774 macrophages [with further comparison with free PVP and a liposomal formulation of amphotericin (AmBisome)]. RESULTS: MICs and MFCs of AC1, AC2 and AC4 against Candida spp. and of AC2 and AC4 against Aspergillus spp. were similar to those of AmB (and even lower for some Candida strains). Killing kinetics (24 h) were also similar. Haemolytic activity of AC2 and AC4 was 2-fold lower than that of free AmB. Cytotoxicity of AC2 towards J774 macrophages was 8-fold lower, and that of AC4 5-fold lower than that of AmB and not significantly different from that of AmBisome. The lower cytotoxicity of AC2, AC4 was correlated with a lower cellular accumulation of amphotericin. Spectroscopic analysis shows that the lower toxicity of AmB-PVP was not owing to significant change in the monomeric/polymeric forms ratio of the drug. CONCLUSIONS: AmB-PVP complexes compared favourably with AmB for antifungal activity, were less haemolytic and cytotoxic than AmB, and show a similar cytotoxicity profile to AmBisome.

OBJECTIVES: Assessment of the activity of three beta-lactams [ertapenem (a carbapenem with a prolonged half-life), meropenem and ampicillin] against intraphagocytic Listeria monocytogenes and Staphylococcus aureus. METHODS: Quantitative measurements of cfu changes in broth and in THP-1 macrophages (post-phagocytosis) over time (5 and 24 h) at concentrations spanning from sub-MICs to C(max) (maximal concentration typically observed in patients' serum upon administration of conventional doses); morphological studies using an electron microscope; evaluation of drug stability (HPLC), protein binding (equilibrium dialysis) and measurement of drug cellular accumulation (microbiological assay). RESULTS: Ertapenem was unable to control L. monocytogenes growth in THP-1 macrophages at all concentrations and times tested, even under conditions where ampicillin and meropenem were bactericidal. This behaviour could not be ascribed to drug instability, protein binding or lack of cell accumulation in comparison with ampicillin or meropenem. Ertapenem, ampicillin and meropenem were equally effective at reducing the post-phagocytosis inoculum of S. aureus ( approximately 1 log cfu), and caused conspicuous changes in the morphology of intracellular bacteria consistent with their lysis. These effects were obtained, however, only at large multiples (100-fold or more) of the MIC maintained over 24 h. Because of the high intrinsic antimicrobial potency of the beta-lactams studied, these concentrations were below the C(max). CONCLUSIONS: Ertapenem will probably be ineffective against intraphagocytic forms of L. monocytogenes for reasons that remain to be discovered. Conversely, ertapenem could be an alternative to ampicillin and meropenem against intraphagocytic S. aureus since its longer half-life may allow high concentrations to be maintained for more prolonged times.

Several homeodomains and homeodomain-containing proteins enter live cells through a receptor- and energy-independent mechanism. Translocation through biological membranes is conferred by the third alpha-helix of the homeodomain, also known as Penetratin. Biophysical studies demonstrate that entry of Penetratin into cells requires its binding to surface lipids but that binding and translocation are differentially affected by modifications of some physico-chemical properties of the peptide, like helical amphipathicity or net charge. This suggests that the plasma membrane lipid composition affects the internalization of Penetratin and that internalization requires both lipid binding and other specific properties. Using a phase transfer assay, it is shown that negatively charged lipids promote the transfer of Penetratin from a hydrophilic into a hydrophobic environment, probably through charge neutralization. Accordingly, transfer into a hydrophobic milieu can also be obtained in the absence of negatively charged lipids, by the addition of DNA oligonucleotides. Strikingly, phase transfer by charge neutralization was also observed with a variant peptide of same charge and hydrophobicity in which the tryptophan at position 6 was replaced by a phenylalanine. However, Penetratin, but not its mutant version, is internalized by live cells. This underscores that charge neutralization and phase transfer represent only a first step in the internalization process and that further crossing of a biological membrane necessitates the critical tryptophan residue at position 6.

Regarding in vivo L-band dosimetry with human teeth, a number of preliminary experiments were carried out that were linked to the resonators response and the relative contribution of enamel to the EPR signal intensity of irradiated whole teeth. The sensitivity of the extended loop resonator varies in the antenna plane, but this variation tends to vanish when the sample is moved away from this plane. When the loop antenna is placed just above the highly irradiated molar, around 88% of the dosimetric signal is due to the crown enamel. The sensitivity inside a birdcage cavity is approximately equal over the volume of a molar; only 30% of the molar's total dosimetric signal results from enamel. Some decrease in the intensity of the dosimetric signal from enamel is observed after irradiation. At room temperature, the signal is reduced by about 20% within 90 days and approaches a plateau with a time constant of about 35 days.

Thirty-two green- and/or nutty-smelling compounds, most of them pyrazine and thiazole derivatives, were tested in ligand binding assays with purified 19 kDa bovine and 22 kDa porcine olfactive binding proteins (OBPs). Unlike the nutty ones, all the green odorants were found to be good ligands for both proteins. Lipophilicity appears as an interesting discriminating physicochemical parameter, predictive of the affinity for OBPs and of the related odour profile.

To better understand the role of free radicals in liver carcinogenesis, endogenous antioxidant defense systems and the susceptibility of membranes to lipid peroxidation were evaluated in early lesions and in malignant tumors induced by the Solt-Farber resistant hepatocyte protocol. These parameters were also measured in the liver surrounding these tumors. In comparison with the normal liver, both nodules and carcinomas show a different biochemical pattern consisting of decreased glutathione peroxidase (GSH peroxidase) and catalase activities plus increased glutathione reductase (GSSG reductase) activity. In contrast, 1 week after the application of the initiation-selection protocol, the liver displays a high level of glutathione (GSH), high GSSG reductase activity, a reduced production of malondialdehyde and no changes in superoxide dismutase and GSH peroxidase activities. These data suggest that the liver is well protected against reactive oxygen species. During the carcinogenic process, the liver parenchyma surrounding the altered foci recovers from most of the modifications induced by the initiation-selection treatment. These results add additional support for the hypothesis that the appearance of early alterations in the liver, after a carcinogenic treatment, might be an adaptive response to a hazardous environment in which selected cell populations are transformed into nodules and/or carcinomas.