F. Van Bambeke

Macrolides: pharmacokinetics and pharmacodynamics

Three pharmacokinetic/pharmacodynamic parameters--(i) the peak concentration to the minimum inhibitory concentration ratio (C(max)/MIC); (ii) the area under the concentration-time curve to MIC ratio (AUC(24h)/MIC); and (iii) the time the concentration exceeds the MIC (T>MIC)--are important predictors of the clinical efficacy of antibiotics. For antibiotics with pronounced concentration-dependent killing, such as the fluoroquinolones or the aminoglycosides, C(max)/MIC and AUC(24)/MIC are the main factors that establish efficacy. Antibiotics with a weak, or no, concentration dependency generally have their efficacy linked to T>MIC, and these include the beta-lactams and the conventional macrolides. Antibiotics with weak concentration-dependent effects, but with prolonged persistent effects, such as tetracyclines and azithromycin, have their activity mostly related to the AUC(24)/MIC. By applying these concepts to current antibiotics, and also to the development of novel agents, it is possible to optimise their dosages and administration schedules. This will maximise therapeutic efficacy, may prevent or delay the emergence of bacterial resistance to antibiotics, and can certainly minimise side-effects.

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.

ABSTRACT 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.

Activities of antistaphylococcal antibiotics towards the extracellular and intraphagocytic forms of Staphylococcus aureus isolates from a patient with persistent bacteraemia and endocarditis

Decreased susceptibility of Staphylococcus aureus to antistaphylococcal agents may be associated with inability to eradicate intracellular forms, which could explain therapeutic failures. This hypothesis was tested using clinical isolates obtained from a patient with persistent staphylococcal bacteraemia under therapy. Four isogenic isolates (three from tissue, one from blood) with increased MICs for vancomycin (1-4 mg/L) and for daptomycin (1-4 mg/L) were collected after an initial 16-day treatment with vancomycin-rifampicin-gentamicin, followed by 13-20 days of treatment with daptomycin-rifampicin-gentamicin. Isolates were tested for MICs and for: (i) vancomycin (BODIPY-FL-vancomycin) and daptomycin binding; (ii) cell wall turnover (loss of N-acetyl-d-[1-(14)C]glucosamine in 30 min after 1 h of labelling); and (iii) Triton X-100-induced autolysis. Extracellular (broth) and intracellular (THP-1 macrophages) activities of rifampicin, linezolid and fusidic acid at C(max), and of vancomycin, daptomycin, quinupristin-dalfopristin and oritavancin over a wide range of extracellular concentrations (with pharmacological modelling to determine E(max)), were measured at 24 h. Increases in vancomycin MICs correlated with increased drug binding, and decreased cell wall turnover and detergent-induced autolysis. Increases in daptomycin MICs correlated with decreased daptomycin binding. Intracellular activity was weak (E(max) <1 log(10) CFU decrease) for vancomycin against all isolates, and for daptomycin against isolates with MICs >1 mg/L. Among all antibiotics tested, only quinupristin-dalfopristin and oritavancin provided close to bactericidal intracellular activities (1.6-2.5 log(10) CFU decreases at C(max)). Determination of the intracellular susceptibility of S. aureus, combined with improved methods of diagnosis, could be useful when dealing with persistent staphylococcal infections and could improve therapy.

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.

Pseudomonas aeruginosa : résistance et options thérapeutiques à l’aube du deuxième millénaire☆

Pseudomonas aeruginosa is a major cause of nosocomial infections. Due to its genetic plasticity, it can adapt to a wide variety of environments, causing infections in almost all body sites (with however a predilection for the respiratory tract, especially in cystic fibrosis patients). It also shows a remarkable capacity to resist to antibiotics, either intrinsically (through constitutive expression of β-lactamases and efflux pumps, or low permeability of the outer membrane), or upon exposure to antibiotics through acquisition of resistance genes (coding fo antibiotic-degrading enzymes inactivating or target modifications), overexpression of efflux pumps, decreased expression of porins, or target mutations. Worryingly also, these mechanisms are often present simultaneously, conferring multiresistant phenotypes. Susceptibility testing is therefore crucial in clinical practice. Empiric treatment is usually initiated using a bitherapy, selected based on local epidemiology (β-lactam plus aminoglycoside or fluoroquinolone). However, it should be streamlined as soon as possible, based on susceptibility data and patient’s evolution. Alternative drugs (colistin, e.g.) have proven useful for multiresistant strains. Innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been so far unsuccessful. Among broad-spectrum antibiotics in development, ceftobiprole, sitafloxacin and doripenem show interesting in vitro activity. The two first molecules, however, are evaluated in the clinics towards Gram-positive only. Pump inhibitors are under study but proved much more difficult to develop than originally anticipated. Therefore, selecting appropriate antibiotics and optimizing their use based on pharmacodynamic concepts remains the only way to cope with pseudomonal infections.

O31 Radezolid (RX-1741), a novel oxazolidinone, accumulates extensively within human macrophages and PMNs and shows activity towards intracellular linezolid-sensitive and linezolid-resistant Staphylococcus aureus

Background: Intracellular S. aureus cause chronic, difficult to treat, and relapsing infections that require antibiotics capable of accumulating and expressing activity in the infected compartment. Linezolid, which accumulates poorly within cells, shows only modest activity against intracellular S. aureus (Barcia-Macay et al, AAC, 2006). The aim of the present study was to compare the cellular accumulation and intracellular activity of radezolid, a new oxazolidinone in development, with those of linezolid. Methods: Cellular accumulation was determined in human THP-1 macrophages and PMNs, with radezolid assayed by scintillation counting (using 14C RX-1741) and linezolid by microbiological assay (using S. aureus as a test organism). Intracellular activity was determined against bacteria internalized by human THP-1 macrophages as previously described (Barcia-Macay et al, AAC, 2006, 50:841 851) and the results expressed as the change in the intracellular inoculum at 24 h compared to time zero (postphagocytosis). Results: Radezolid accumulates quickly (half-life of 5 10min) and extensively within THP-1 macrophages and PMNs, reaching an apparent cellular to extracellular concentration ratio of ~10 12 in both cell types vs 1 2 for linezolid (determined in THP-1 cells only). MICs (in broth) and intracellular activities are shown in the table.

P703 Comparative activity of moxifioxacin vs. trimethoprim-sulfamethoxazole, cloxacillin, linezolid, clindamycin, and ciprofioxacin against intracellular methicillin-sensitive and community-acquired methicillin-resistant S. aureus

emergence of resistance. We show that the activity of antibiotics may markedly differ when tested against extracellular and intracellular bacteria (AAC 2006, 50:841-851). In this context, we have examined the intraphagocytic activity of moxifloxacin (MXF, recently approved for skin and soft-tissues infections) in comparison to (i) antibiotics commonly used for the treatment of CAMRSA infections (trimethoprim-sulfamethoxazole [TMP-SMX], cloxacillin [CLX], linezolid [LNZ], clindamycin [CLI], and (ii) ciprofloxacin [CIP] against fully-sensitive S. aureus (MSSA) and the newly-emerging commynity-acquired methicillin-resistant S. aureus (CA-MRSA).

P313 Analysis of guidelines for treatment of community-acquired pneumonia (CAP) in outpatients

Background: CAP, usually treated by General Practitioners (GPs) without precise knowledge of the offending organism and of its susceptibility (if bacterial), remains an important cause of morbidity and mortality. Guidelines are supposed to help in selecting safe and effective drugs, taking local epidemiology and resistance data into account while avoiding unnecessary costs. Our aim was to analyze current guidelines for non-hospitalized CAP patients in this context. Methods: Guidelines (published or updated after 2004; written in English or another major European language) were identified from www.guideline.gov, by systematic search of pertinent literature, and by direct contact. Resistance data for S. pneumoniae (most critical organism in this context) were obtained from the literature and main congresses abstracts (2007-2008), and from the main surveillance networks (EARSS, PROTEKT and ALEXANDER). Safety of the recommended drugs was assessed from the warnings mentioned in their respective official labeling (package insert). Costs were estimated by using the recommended dosages and treatment durations, and using lowest [generic] and highest [branded product] prices in Belgium. Results: 13 guidelines for adults and 6 for children were reviewed. Major differences were noted between North American (favoring beta-lactam/macrolides combination and/or fluoroquinolones [adults]) and European guidelines (mainly recommending amoxicillin alone as 1st line therapy). Current resistance patterns of S. pneumoniae were often only poorly taken into account in guidelines (typical examples: macrolides and tetracyclines). Safety issues with several of the recommended antibiotics could also be identified (typical example: cotrimoxazole in children). Acquisition costs of the 1st line drugs often reached that of 2d line antibiotics (considered as most costly) because of the necessity to use larger doses (beta-lactams, e.g.) and longer durations of treatment. Conclusion: In spite of the efforts made to construct effective guidelines, several issues were identified that may explain why GPs often do not follow them. The main problem seems to be a difficulty for guidelines to keep in pace with the ever moving bacterial resistance patterns and too much focus on the use of low cost drugs. Analysis of Guidelines for Treatment of Community Acquired Pneumonia (CAP) in Outpatients Sylviane Carbonnelle MD, PhD, Ann Lismond MSc, Françoise Van Bambeke PharmD, PhD, Paul M. Tulkens MD, PhD

P2067 Ciprofloxacin and doxorubicin are substrates of different multidrug resistance-related proteins efflux transporters in J774 macrophages

Objectives: Ciprofloxacin (CIP) and doxorubicin (DOX) are both substrates of the Multidrug Resistance-related Proteins efflux transporters (MRP), a subfamily of the ATP-Binding Cassette transporters (ABC) in eukaryotic cells. This contributes to a reduction of their cellular accumulation, and, therefore, of their intracellular activity (Int J Cancer. 2001;93:107-13; J Antimicrob Chemother. 2003;51:1167-73). Our objective was to determine whether CIP and DOX could share the same MRP transporter in macrophages since this could be the basis of potential, unanticipated drug interactions in cancer patients receiving both drugs either simultaneously (by direct competition for transport) or in succession (trough drug-induced overexpression of the transporter). Methods: We used J774 macrophages, a cell line that spontaneously expel CIP (Antimicrob Agents Chemother. 2004;48:2673-82; wild-type cells) and a cell line derived thereof by continuous exposure to CIP (CIP-resistant cells) and which overexpresses the corresponding MRP transporter (Antimicrob Agents Chemother. 2006;50:1689-95). Cells were incubated with CIP or DOX alone, or in competition with each other, or in the presence of probenecid (PB), a non-specific inhibitor of MRP transporters. Cell-associated CIP and DOX were measured by fluorimetry and their accumulation recorded as cellular to extracellular concentration ratios (Cc/Ce). Results: In wild-type cells, (i) PB increased both CIP and DOX Cc/Ce but not to the same extent; (ii) excess of CIP but not of DOX caused an increase in CIP Cc/Ce; (iii) excess of DOX had no effect on CIP and only a marginal effect on DOX Cc/Ce. In CIP-resistant cells, accumulation of CIP was markedly reduced compared to wild-type cells (and only partially restored by PB), but not that of DOX (with PB exerting a similar effect to that observed in wild-type cells). Conclusions: CIP and DOX are substrates of distinct MRP efflux transporters in J774 cells. Drug interactions related to competition for transport or overexpression of CIP transporter are, therefore, unlikely. AIMS OF THE STUDY To compare the cellular accumulation of ciprofloxacin and doxorubicin in wild-type macrophages and in ciprofloxacin-resistant macrophages, which overexpress the ciprofloxacin MRP transporter. To examine whether these two drugs compete for a same MRP transporter in these two cell lines.