Pharmacodynamics of ceftazidime/avibactam against extracellular and intracellular forms of Pseudomonas aeruginosa.

Objectives: When tested in broth, avibactam reverses ceftazidime resistance in many Pseudomonas aeruginosa that express ESBLs. We examined whether similar reversal is observed against intracellular forms of P. aeruginosa . Methods: Strains: reference strains; two engineered strains with basal non-inducible expression of AmpC and their isogenic mutants with stably derepressed AmpC; and clinical isolates with complete, partial or no resistance to reversion with avibactam. Pharmacodynamic model: 24 h concentration-response to ceftazidime [0.01-200 mg/L alone or with avibactam (4 mg/L)] of bacteria in broth or bacteria phagocytosed by THP-1 monocytes, with calculation of ceftazidime relative potency ( C s : concentration yielding a static effect) and maximal relative effect [ E max : cfu decrease at infinitely large antibiotic concentrations (efficacy in the model)] using the Hill equation. Cellular content of avibactam: quantification by LC-MS/MS. Results: For both extracellular and intracellular bacteria, ceftazidime C s was always close to its MIC. For ceftazidime-resistant strains, avibactam addition shifted ceftazidime C s to values close to the MIC of the combination in broth. E max was systematically below the detection limit (-5 log 10 ) for extracellular bacteria, but limited to -1.3 log 10 for intracellular bacteria (except for two isolates) with no effect of avibactam. The cellular concentration of avibactam reflected extracellular concentration and was not influenced by ceftazidime (0-160 mg/L). Conclusions: The potential for avibactam to inhibit ß-lactamases does not differ for extracellular and intracellular forms of P. aeruginosa , denoting an unhindered access to its target in both situations. The loss of maximal relative efficacy of ceftazidime against intracellular P. aeruginosa was unrelated to resistance via avibactam-inhibitable ß-lactamases.

[Information transmission to the community pharmacist after a patient's discharge from the hospital: setting up of a written medication discharge form, prospective evaluation of its impact, and survey of the information needs of the pharmacists]. / Transmission d'information au pharmacien d'officine après hospitalisation du patient: élaboration d'une feuille de transfert, évaluation prospective de son effet et enquête sur les besoins d'information des pharmaciens.

INTRODUCTION: Discharge from the hospital is a period at risk for the continuity of patient's medication (seamless pharmaceutical care). The community pharmacist is often the first health care professional seen by the patient after hospital discharge. The clinical pharmacist has potentially a key role in establishing an efficient information transfer from the hospital to the community pharmacy. OBJECTIVE: (1) To develop and, (2) to evaluate the impact of a structured discharge medication form prepared at hospital discharge by the clinical pharmacist and containing information items related to the medication regimen for the community pharmacist, and (3) to survey the information needs of the Belgian community pharmacists to ensure continuity of care after hospitalization. METHODS: (1) A structured discharge medication form has been developed based on a Literature review and on opinions expressed by community and clinical pharmacists, members of the Belgian Pharmaceutical Union (Association Pharmaceutique Belge) and an ethical committee. (2) A prospective study has been conducted with patients from geriatrics and orthopaedics wards of the University Hospital Dinant-Godinne returning home after hospital discharge with the discharge medication form to be given to their commuiity pharmacist; its use, the reasons for non-use, the perceived impact and the satisfaction of the community pharmacist have been assessed. (3) An on-line survey addressed to all Belgian community pharmacists evaluated their information needs. RESULTS: (1) The final version of the discharge medication form included key information items concerning the hospital, the patient, the discharge treatment (including the type of modifications made as compared to medications taken before admission), and on medication management at home. Some items were excluded because of Lack of perceived utility by pharmacists, confidentiality issues, and respect of patient's freedom of choice. (2) From the 71 medication forms given to patients, 48 were received by the community pharmacist. One quarter of respondents stated that they did not use the form, the main reason being that it was received after dispensing of the discharge treatment (n=6/11). The majority of the community pharmacists considered most of the information items as useful and the discharge medication form as being valuable for continuity of care. Requests for additional information were made (e.g., reason of admission and of treatment modifications, etc.). (3) The utility, benefits, and need for additional information items beyond what was included in the discharge medication form were highlighted by the respondents (n=309) of the national survey. Most of these respondents confirmed the value of the different information items included in the discharge medication form. CONCLUSION: The transmission of a structured medication form containing information about the medication regimen upon hospital discharge is of real interest and value for the community pharmacist because it goes beyond what is usually provided on a medical prescription. However, this discharge medication form should include more information items for effective pharmaceutical care.

Penicillin susceptibility breakpoints for Streptococcus pneumoniae and their effect on susceptibility categorisation in Germany (1997-2013).

Continuous nationwide surveillance of invasive pneumococcal disease (IPD) was conducted in Germany. From July 1, 1997, to June 30, 2013, data on penicillin susceptibility were available for 20,437 isolates. 2,790 of these isolates (13.7 %) originate from patients with meningitis and 17,647 isolates (86.3 %) are from non-meningitis cases. A slight decline in isolates susceptible at 0.06 and 0.12 µg/ml can be noticed over the years. Overall, 89.1 % of the isolates had minimum inhibitory concentrations (MICs) of ≤0.015 µg/ml. In 2012/2013, the first three isolates of Streptococcus pneumoniae with MICs of 8 µg/ml were found. The application of different guidelines with other MIC breakpoints for the interpretation of penicillin resistance leads to differences in susceptibility categorisation. According to the pre-2008 Clinical and Laboratory Standards Institute (CLSI) interpretive criteria, 5.3 % of isolates overall were intermediate and 1.4 % were resistant to penicillin. Application of the 2008-2014 CLSI interpretive criteria resulted in 7.6 % resistance among meningitis cases and 0.5 % intermediate resistance in non-meningitis cases. Referring to the 2009-2014 European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, 7.6 % of the isolates in the meningitis group were resistant to penicillin. In the non-meningitis group, 6.1 % of the isolates were intermediate and 0.5 % were resistant. These differences should be kept in mind when surveillance studies on pneumococcal penicillin resistance are compared.

A survey of beta-lactam antibiotics and vancomycin dosing strategies in intensive care units and general wards in Belgian hospitals.

Extended and continuous infusions with beta-lactam antibiotics have been suggested as a means of pharmacokinetic and pharmacodynamic optimisation of antimicrobial therapy. Vancomycin is also frequently administered in continuous infusion, although more for practical reasons. A survey was undertaken to investigate the recommendations by the local antibiotic management teams (AMTs) in Belgian acute hospitals concerning the administration (intermittent, extended or continuous infusion) and therapeutic drug monitoring of four beta-lactam antibiotics (ceftazidime, cefepime, piperacillin-tazobactam, meropenem) and vancomycin for adult patients with a normal kidney function. A structured questionnaire survey comprising three domains was developed and approved by the members of the Belgian Antibiotic Policy Coordination Committee (BAPCOC). The questionnaire was sent by e-mail to the official AMT correspondents of 105 Belgian hospitals, followed by two reminders. The response rate was 32 %, with 94 %, 59 %, 100 %, 100 % and 100 % of the participating Belgian hospitals using ceftazidime, cefepime, piperacillin-tazobactam, meropenem and vancomycin, respectively. Comparing intensive care unit (ICU) with non-ICU wards showed a higher implementation of extended or continuous infusions for ceftazidime (81 % vs. 41 %), cefepime (35 % vs. 10 %), piperacillin-tazobactam (38 % vs. 12 %), meropenem (68 % vs. 35 %) and vancomycin (79 % vs. 44 %) on the ICU wards. A majority of the hospitals recommended a loading dose prior to the first dose. For vancomycin, the loading dose and the trough target concentration were too low based on the current literature. This survey shows that extended and continuous infusions with beta-lactams and vancomycin are widely implemented in Belgian hospitals.

Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of Staphylococcus aureus in models of extracellular (broth) and intracellular (THP-1 monocytes) infections.

Staphylococcus aureus small-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistant S. aureus (MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for the menD SCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for the menD mutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

Long-term stability of temocillin in dextrose 5% and in sodium chloride 0.9% polyolefin bags at 5 ± 3°C after freeze-thaw treatment.

INTRODUCTION: The aim of this study was to investigate the stability of a mixture of temocillin 20mg/ml in 5% dextrose and in 0.9% sodium chloride polyolefin bags after freezing, microwave thawing and long-term storage at 5±3°C. METHODS: The stability of ten polyolefin bags containing 20mg/ml of temocillin, five bags in 5% dextrose and five bags in 0.9% sodium chloride, prepared under aseptic conditions was studied after freezing for 1 month at -20°C, thawing in a microwave oven with a validated cycle, and stored at 5±3°C. Over 30 days, temocillin concentrations were measured by high-pressure liquid chromatography. Visual inspections, microscope observation, spectrophotometric measurements and pH measurements were also performed. RESULTS AND DISCUSSION: No precipitation occurred in the preparations but minor colour change was observed. No microaggregate was observed with optical microscopy or revealed by a change of absorbance. Based on a shelf life of 95% residual potency, temocillin infusions were stable at least 11 days in 5% dextrose and 14 days in 0.9% sodium chloride after freezing and microwave thawing (corresponding at the period where 95% lower confidence limit of the concentration-time profile remained superior to 95% of the initial concentration). During this period, the pH values of drug solutions have been observed to decrease without affecting chromatographic parameters. CONCLUSION: Within these limits, temocillin in 5% dextrose and in 0.9% sodium chloride infusions may be prepared and frozen in advance by a centralized intravenous admixture service then thawed before use in clinical units.

Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes.

OBJECTIVES: Burkholderia pseudomallei, Yersinia pestis and Francisella tularensis are facultative intracellular bacteria causing life-threatening infections. We have (a) compared the activity of finafloxacin (a fluoroquinolone in development showing improved activity at acidic pH) with that of ciprofloxacin, levofloxacin and imipenem against the extracellular and intracellular (THP-1 monocytes) forms of infection by attenuated surrogates of these species (B. thailandensis, Y. pseudotuberculosis, F. philomiragia) and (b) assessed finafloxacin cellular pharmacokinetics (accumulation, distribution, efflux). METHODS: Bacteria in broth or in infected monocytes were exposed to antibiotics at pH 7.4 or 5.5 for 24 hr. Maximal relative efficacies (Emax) and static concentrations (Cs) were calculated using the Hill equation (concentration-response curves). Finafloxacin pharmacokinetics in cells at pH 7.4 or 5.5 was investigated using 14C-labelled drug. RESULTS: Extracellularly, all drugs sterilized the cultures, with finafloxacin being two to six times more potent at acidic pH. Intracellularly, Emax reached the limit of detection (4-5 log10 cfu decrease) for finafloxacin against all species, but only against B. thailandensis and F. philomiragia for ciprofloxacin and levofloxacin, while imipenem caused less than 2 log10 cfu decrease for all species. At acid pH, Cs shifted to two to five times lower values for finafloxacin and to one to four times higher values for the other drugs. Finafloxacin accumulated in THP-1 cells by approximately fivefold at pH 7.4 but up to 20-fold at pH 5.5, and distributed in the cytosol. CONCLUSIONS: Fluoroquinolones have proven to be effective in reducing the intracellular reservoirs of B. thailandensis, Y. pseudotuberculosis and F. philomiragia, with finafloxacin demonstrating an additional advantage in acidic environments.

Analytical fractionation of homogenates from cultured rat embryo fibroblasts.

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-beta-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized. In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.

Fate of plasma membrane during endocytosis. I. Uptake and processing of anti-plasma membrane and control immunoglobulins by cultured fibroblasts.

The uptake and processing by cultured rat embryo fibroblasts of control rabbit immunoglobulins (C IgG) or IgG directed against plasma membrane constituents (anti-PM IgG), and labeled with fluorescein (F) or with radioactive acetate (A), have been investigated by cell fractionation and immunological techniques. Both F and A anti-PM IgGs become bound to the cell surface, by a process that is slow, but largely temperature-independent. In the presence of an excess of high-affinity antibodies, binding reaches an absolute limit which corresponds to extensive coating of the plasma membrane. The anti-PM IgGs remain attached to the membrane for at least several days, even at 37 degrees C, with no significant transfer to lysosomes or degradation. In contrast, C IgGs are handled very differently by the fibroblasts, and their fate is strikingly affected by the type of labeling used. AC IgG is taken up slowly, at a rate proportional to its concentration, and is subsequently broken down in what appears to be lysosomes. Part of the AC IgG also binds to the plasma membrane. FC IgG is taken up many times faster than AC IgG, though with the same strict linearity as a function of concentration. Most of the FC IgG taken up is stored in cytoplasmic granules which behave like lysosomes. For reasons that are not understood, only about half of the stored FC IgG can be broken down. Cells exposed simulatnaously to AC IgG and FC IgG, or to A anti-PM IgG and FC IgG, handle each type of IgG in its characteristic fashion. Kinetic analysis of these results indicates that Ac IgG could be taken up by fluid endocytosis, but that FC IgG must be interiorized by a selective mechanism, presumably adsorptive in nature. That anti-PM antibodies remain stably bound to the plasma membrane and do not interfere with the uptake of FC IgG is interpreted to indicate either that two distinct membrane domains are involved in the two phenomena, or that membrane patches coated with anti-PM IgG participate in endocytosis, and are recycled back to the cell surface after delivering their contents intracellularly.

Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents.

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5'- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.

[Pharmacodynamics of antibiotics in CSF: principles and consequences (predictive factors of efficacy)]. / Pharmacodynamie des antibiotiques dans le LCR : principes et conséquences (facteurs prédictifs d'efficacité).

The rational selection of antibiotics for the treatment of meningitis must take into account several criteria, among which their intrinsic activity against the causative bacteria, and their pharmacokinetic and pharmacodynamic properties. The intrinsic activity is evaluated by the Minimal Inhibitory Concentration (MIC), which, however, does not give any information on the bactericidal potency of the drug (important property for infections localized in compartments with low immune defense such as the CSF). The capacity of the antibiotic to reach the infected compartment depends on its physicochemical properties (molecular weight, lipophilicity) and its protein binding capacity, but also on the properties of the blood-CSF barrier (permeability modulated by inflammation and activity of active transporters). Pharmacodynamics correlate intrinsic activity to pharmacokinetics by determining the optimal value of the ratio between MIC and time of exposure, area under the curve, or peak concentration. On these bases, beta-lactams appear as first-line antibiotics, if used with large and repeated doses (or even as a continuous infusion), because of their time-dependent activity. The choice of the molecule is based on the susceptibility of the bacterium. Potential alternatives include chloramphenicol (limited however by its toxicity), moxifloxacin (showing high bactericidal effect, a low MIC, and appropriate penetration) but little clinically documented, linezolid and vancomycin for Methicillin-Resistant Staphylococcus aureus (MRSA), and vancomycin for penicillin non-susceptible pneumococci. Other molecules in clinical development are being evaluated for this indication.

Development of clinical pharmacy in Belgian hospitals through pilot projects funded by the government.

Objectives The goal is to develop clinical pharmacy in the Belgian hospitals to improve drug efficacy and to reduce drug-related problems. Methods From 2007 to 2014, financial support was provided by the Belgian federal government for the development of clinical pharmacy in Belgian hospitals. This project was guided by a national Advisory Working Group. Each funded hospital was obliged to describe yearly its clinical pharmacy activities. Results In 2007, 20 pharmacists were funded in 28 pilot hospitals; this number was doubled in 2009 to 40 pharmacists over 54 institutions, representing more than half of all acute Belgian hospitals. Most projects (72%) considered patient-related activities, whereas some projects (28%) had a hospital-wide approach. The projects targeted patients at admission (30%), during hospital stay (52%) or at discharge (18%). During hospital stay, actions were mainly focused on geriatric patients (20%), surgical patients (15%), and oncology patients (9%). Experiences, methods, and tools were shared during meetings and workshops. Structure, process, and outcome indicators were reported and strengths, weaknesses, opportunities, and threats were described. The yearly reports revealed that the hospital board was engaged in the project in 87% of the cases, and developed a vision on clinical pharmacy in 75% of the hospitals. In 2014, the pilot phase was replaced by structural financing for clinical pharmacy in all acute Belgian hospitals. Conclusion The pilot projects in clinical pharmacy funded by the federal government provided a unique opportunity to launch clinical pharmacy activities on a broad scale in Belgium. The results of the pilot projects showed clear implementation through case reports, time registrations, and indicators. Tools for clinical pharmacy activities were developed to overcome identified barriers. The engagement of hospital boards and the results of clinical pharmacy activities persuaded the government to start structural financing of clinical pharmacy.

Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium.

Pseudomonas aeruginosa is a major cause of nosocomial infections. This organism shows a remarkable capacity to resist antibiotics, either intrinsically (because of constitutive expression of beta-lactamases and efflux pumps, combined with low permeability of the outer-membrane) or following acquisition of resistance genes (e.g., genes for beta-lactamases, or enzymes inactivating aminoglycosides or modifying their target), over-expression of efflux pumps, decreased expression of porins, or mutations in quinolone targets. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. Susceptibility testing is therefore crucial in clinical practice. Empirical treatment usually involves combination therapy, selected on the basis of known local epidemiology (usually a beta-lactam plus an aminoglycoside or a fluoroquinolone). However, therapy should be simplified as soon as possible, based on susceptibility data and the patient's clinical evolution. Alternative drugs (e.g., colistin) have proven useful against multiresistant strains, but innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been unsuccessful to date. Among broad-spectrum antibiotics in development, ceftobiprole, sitafloxacin and doripenem show interesting in-vitro activity, although the first two molecules have been evaluated in clinics only against Gram-positive organisms. Doripenem has received a fast track designation from the US Food and Drug Administration for the treatment of nosocomial pneumonia. Pump inhibitors are undergoing phase I trials in cystic fibrosis patients. Therefore, selecting appropriate antibiotics and optimising their use on the basis of pharmacodynamic concepts currently remains the best way of coping with pseudomonal infections.

Analytical fractionation of cultured hepatoma cells (HTC cells).

Homogenates of HTC cells have been fractionated by differential centrifugation (in four particulate fractions: N, M, L, P, and a supernatant S) or isopycnic banding in linear sucrose gradients. On this basis, the following subcellular organelles may be characterized: (i) Mitochondria, detected by cytochrome oxidase and succinodehydrogenase, are collected in the M and L fractions, and equilibrate, as a narrow band, at a median buoyant density of 1.18 g/cm3. (ii) Lysosomes, detected by the latent hydrolases beta-glycerophosphatase and N-acetyl-beta-glucosaminidase, are largely sedimented in the M and L fractions, and display a broad density distribution pattern with a median value of 1.17 g/cm3. This density is decreased or increased after cultivation of the cells in presence of Triton WR-1339 or Dextran 500, respectively. The behavior of cathepsin D is somewhat at variance with that of the two other hydrolases. (iii) Plasma membrane is tentatively detected by alkaline phosphodiesterase I. Largely recovered in the P fraction, this enzyme equilibrates at a median density close to that of the lysosomal hydrolases; the bulk of cholesterol and about half of the leucyl-2-naphthylamidase are closely associated with alkaline phosphodiesterase I; HTC cells do not contain typical 5'-nucleotidase. (iv) Catalase-bearing particles, of high buoyant density (1.22 g/cm3) are present, but 30-40% of the catalase is also found readily soluble. NADPH- and NADH: cytochrome c reductase, and RNA show more complex distributions. It is suggested that the former enzyme is associated with the endoplasmic reticulum; as in liver, NADH reductase activity is shared between the endoplasmic reticulum and the mitochondria; half of the RNA is associated with free ribosomes of polysomes. True glucose-6-phosphatase could not be detected.

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells).

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5'-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez-Saura, P., Trouet, A. and Tulkens, P. (1978) Biochim. Biophys. Acta 543, 430-449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4 degrees C and fractionated. Alkaline phosphodiesterase I and IgG showed superimposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome c reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm3, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome c reductase are increased only by 0.03 and 0.015 g/cm3, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome c reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.

Quinolones in 2005: an update.

Quinolones are one of the largest classes of antimicrobial agents used worldwide. This review considers the quinolones that are available currently and used widely in Europe (norfoxacin, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin) within their historical perspective, while trying to position them in the context of recent and possible future advances based on an understanding of: (1) their chemical structures and how these impact on activity and toxicity; (2) resistance mechanisms (mutations in target genes, efflux pumps); (3) their pharmacodynamic properties (AUC/MIC and Cmax/MIC ratios; mutant prevention concentration and mutant selection window); and (4) epidemiological considerations (risk of emergence of resistance, clonal spread). Their main indications are examined in relation to their advantages and drawbacks. Overall, it is concluded that these important agents should be used in an educated fashion, based on a careful balance between their ease of use and efficacy vs. the risk of emerging resistance and toxicity. However, there is now substantial evidence to support use of the most potent drug at the appropriate dose whenever this is required.

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.

Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts.

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.

Hyperactivity of cathepsin B and other lysosomal enzymes in fibroblasts exposed to azithromycin, a dicationic macrolide antibiotic with exceptional tissue accumulation.

Azithromycin accumulates in lysosomes where it causes phospholipidosis. In homogenates prepared by sonication of fibroblasts incubated for 3 days with azithromycin (66 microM), the activities of sulfatase A, phospholipase A1, N-acetyl-beta-hexosaminidase and cathepsin B increased from 180 to 330%, but not those of 3 non-lysosomal enzymes. The level of cathepsin B mRNA was unaffected. The hyperactivity induced by azithromycin is non-reversible upon drug withdrawal, prevented by coincubation with cycloheximide, affects the Vmax but not the Km, and is not reproduced with gentamicin, another drug also causing lysosomal phospholipidosis. The data therefore suggest that azithromycin increases the level of lysosomal enzymes by a mechanism distinct from the stimulation of gene expression but requiring protein synthesis, and is not in direct relation to the lysosomal phospholipidosis.