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1.  A Combined Pharmacodynamic Quantitative and Qualitative Model Reveals the Potent Activity of Daptomycin and Delafloxacin against Staphylococcus aureus Biofilms 
Biofilms are associated with persistence of Staphylococcus aureus infections and therapeutic failures. Our aim was to set up a pharmacodynamic model comparing antibiotic activities against biofilms and examining in parallel their effects on viability and biofilm mass. Biofilms of S. aureus ATCC 25923 (methicillin-sensitive S. aureus [MSSA]) or ATCC 33591 (methicillin-resistant S. aureus [MRSA]) were obtained by culture in 96-well plates for 6 h/24 h. Antibiotic activities were assessed after 24/48 h of exposure to concentrations ranging from 0.5 to 512 times the MIC. Biofilm mass and bacterial viability were quantified using crystal violet and the redox indicator resazurin. Biofilms stained with Live/Dead probes were observed by using confocal microscopy. Concentration-effect curves fitted sigmoidal regressions, with a 50% reduction toward both matrix and viability obtained at sub-MIC or low multiples of MICs against young biofilms for all antibiotics tested. Against mature biofilms, maximal efficacies and potencies were reduced, with none of the antibiotics being able to completely destroy the matrix. Delafloxacin and daptomycin were the most potent, reducing viability by more than 50% at clinically achievable concentrations against both strains, as well as reducing biofilm depth, as observed in confocal microscopy. Rifampin, tigecycline, and moxifloxacin were effective against mature MRSA biofilms, while oxacillin demonstrated activity against MSSA. Fusidic acid, vancomycin, and linezolid were less potent overall. Antibiotic activity depends on biofilm maturity and bacterial strain. The pharmacodynamic model developed allows ranking of antibiotics with respect to efficacy and potency at clinically achievable concentrations and highlights the potential utility of daptomycin and delafloxacin for the treatment of biofilm-related infections.
PMCID: PMC3716136  PMID: 23571532
2.  Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PAO1 in a Model of THP-1 Human Monocytes 
Pseudomonas aeruginosa invades epithelial and phagocytic cells, which may play an important role in the persistence of infection. We have developed a 24-h model of THP-1 monocyte infection with P. aeruginosa PAO1 in which bacteria are seen multiplying in vacuoles by electron microscopy. The model has been used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by using a pharmacodynamic approach (concentration-dependent experiments over a wide range of extracellular concentrations to calculate bacteriostatic concentrations [Cs] and maximal relative efficacies [Emax]; Hill-Langmuir equation). Using 16 antipseudomonal antibiotics (three aminoglycosides, nine β-lactams, three fluoroquinolones, and colistin), dose-response curves were found to be undistinguishable for antibiotics of the same pharmacological class if data were expressed as a function of the corresponding MICs. Extracellularly, all of the antibiotics reached a bacteriostatic effect at their MIC, and their Emax exceeded the limit of detection (−4.5 log10 CFU compared to the initial inoculum). Intracellularly, Cs values remained unchanged for β-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas Emax values were markedly reduced (less negative), reaching −3 log10 CFU for fluoroquinolones and only −1 to −1.5 log10 CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher Cs) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the Emax may reflect a reversible alteration of bacterial responsiveness to antibiotics in the intracellular milieu. The model may prove useful for comparison of antipseudomonal antibiotics to reduce the risk of persistence or relapse of pseudomonal infections.
PMCID: PMC3632903  PMID: 23478951
3.  Influence of the Protein Kinase C Activator Phorbol Myristate Acetate on the Intracellular Activity of Antibiotics against Hemin- and Menadione-Auxotrophic Small-Colony Variant Mutants of Staphylococcus aureus and Their Wild-Type Parental Strain in Human THP-1 Cells 
Antimicrobial Agents and Chemotherapy  2012;56(12):6166-6174.
In a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700–3711, 2012), we evaluated the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistant Staphylococcus aureus strain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, its hemB mutant, and the genetically complemented strain in PMA-activated cells and against the menD strain in both activated and nonactivated cells. This effect was inhibited when cells were incubated with N-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H2O2. In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition of N-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H2O2. Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.
PMCID: PMC3497167  PMID: 22985883
4.  Macrophage Killing of Bacterial and Fungal Pathogens Is Not Inhibited by Intense Intracellular Accumulation of the Lipoglycopeptide Antibiotic Oritavancin 
Intact phagocytic effector function is fundamental to host defense against microbial pathogens. Concern has been raised regarding the potential that accumulation of certain agents, including cationic amphiphilic antibiotics, within macrophages could cause a mixed-lipid storage disorder, resulting in macrophage dysfunction in recipients. The ability of 2 macrophage cell lines (HL-60; RAW 264.7) to kill archetypal Gram-positive (Staphylococcus aureus), Gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens was tested following exposure of the macrophages to the lipoglycopeptide antibiotic oritavancin. Oritavancin did not affect killing of C. albicans but markedly enhanced killing of S. aureus by both macrophages. Oritavancin modestly reduced killing of A. baumannii by HL-60 cells but not by RAW 264.7 cells. Thus, macrophage killing of microbes remains intact despite substantial intracellular accumulation with a lipoglycopeptide antibiotic.
PMCID: PMC3307583  PMID: 22431853
5.  Analysis of the Membrane Proteome of Ciprofloxacin-Resistant Macrophages by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) 
PLoS ONE  2013;8(3):e58285.
Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter.
PMCID: PMC3591400  PMID: 23505477
6.  Moxifloxacin Safety 
Drugs in R&d  2012;12(2):71-100.
Moxifloxacin, a fluoroquinolone antibiotic, is used for the treatment of respiratory tract, pelvic inflammatory disease, skin, and intra-abdominal infections. Its safety profile is considered favorable in most reviews but has been challenged with respect to rare but potentially fatal toxicities (e.g. hepatic, cardiac, or skin reactions).
To analyze and compare the safety profile of moxifloxacin versus comparators in the entire clinical database of the manufacturer.
Data on the valid-for-safety population from phase II–IV actively controlled studies (performed between 1996 and 2010) were analyzed. Studies were either double blind (n = 22 369) or open label (n = 7635) and included patients with indications that have been approved in at least one country [acute bacterial sinusitis, acute exacerbation of chronic bronchitis, community-acquired pneumonia, uncomplicated pelvic inflammatory disease, complicated and uncomplicated skin and skin structure infections, and complicated intra-abdominal infections] (n = 27 824) and patients with other indications (n = 2180), using the recommended daily dose (400 mg) and route of administration (oral, intravenous/oral, intravenous only). The analysis included patients at risk (age ≥65 years, diabetes mellitus, renal impairment, hepatic impairment, cardiac disorders, or body mass index <18 kg/m2). Patients with known contraindications were excluded from enrollment by study protocol design, but any patient having entered a study, even if inappropriately, was included in the analysis.
Main Outcome Measure
Crude incidences and relative risk estimates (Mantel-Haenszel analysis) of patients with any adverse event (AE), adverse drug reaction (ADR), serious AE (SAE), serious ADR (SADR), treatment discontinuation due to an AE or ADR, and fatal outcomes related to an AE or ADR.
Overall incidence rates of AEs were globally similar in the moxifloxacin and comparator groups. By filtering the data for differences in disfavor of moxifloxacin (i) at ≥2.5% for events with an incidence ≥2.5% or at ≥2-fold for events with an incidence <2.5% in one or both groups and (ii) affecting ≥10 patients in either group, we observed slightly more (i) AEs in double-blind intravenous-only and open-label oral studies, (ii) SAEs in double-blind intravenous-only studies, (iii) ADRs and SADRs in open-label oral studies, (iv) SADRs in open-label intravenous/oral studies, and (v) premature discontinuation due to AEs in open-label intravenous-only studies. The actual numbers of SADRs (in all studies) were small, with clinically relevant differences noted only in intravenous/oral studies and mainly driven by ‘gastrointestinal disorders’ (15 versus 7 patients) and ‘changes observed during investigations’ (23 versus 7 patients [asymptomatic QT prolongation: 11 versus 4 patients in double-blind studies]). Analysis by comparator (including another fluoroquinolone) did not reveal medically relevant differences, even in patients at risk. Incidence rates of hepatic disorders, tendon disorders, clinical surrogates of QT prolongation, serious cutaneous reactions, and Clostridium difficile-associated diarrhea were similar with moxifloxacin and comparators.
The safety of moxifloxacin is essentially comparable to that of standard therapies for patients receiving the currently registered dosage and for whom contraindications and precautions of use (as in the product label) are taken into account.
PMCID: PMC3585838  PMID: 22715866
7.  Continuous infusion of antibiotics in the critically ill: The new holy grail for beta-lactams and vancomycin? 
The alarming global rise of antimicrobial resistance combined with the lack of new antimicrobial agents has led to a renewed interest in optimization of our current antibiotics. Continuous infusion (CI) of time-dependent antibiotics has certain theoretical advantages toward efficacy based on pharmacokinetic/pharmacodynamic principles. We reviewed the available clinical studies concerning continuous infusion of beta-lactam antibiotics and vancomycin in critically ill patients. We conclude that CI of beta-lactam antibiotics is not necessarily more advantageous for all patients. Continuous infusion is only likely to have clinical benefits in subpopulations of patients where intermittent infusion is unable to achieve an adequate time above the minimal inhibitory concentration (T > MIC). For example, in patients with infections caused by organisms with elevated MICs, patients with altered pharmacokinetics (such as the critically ill) and possibly also immunocompromised patients. For vancomycin CI can be chosen, not always for better clinical efficacy, but because it is practical, cheaper, associated with less AUC24h (area under the curve >24 h)-variability, and easier to monitor.
PMCID: PMC3532155  PMID: 22747633
Continuous infusion; Intermittent infusion; Vancomycin; Beta-lactam; Antibiotic(s); Critically ill; Pharmacokinetic/pharmacodynamic
8.  Characterization of Abcc4 Gene Amplification in Stepwise-Selected Mouse J774 Macrophages Resistant to the Topoisomerase II Inhibitor Ciprofloxacin 
PLoS ONE  2011;6(12):e28368.
Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410–2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 µM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 µM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 µM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 µM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.
PMCID: PMC3230599  PMID: 22162766
9.  Intra- and Extracellular Activities of Dicloxacillin and Linezolid against a Clinical Staphylococcus aureus Strain with a Small-Colony-Variant Phenotype in an In Vitro Model of THP-1 Macrophages and an In Vivo Mouse Peritonitis Model ▿  
The small-colony-variant (SCV) phenotype of Staphylococcus aureus has been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT) S. aureus strain and its counterpart with an SCV phenotype both in vitro and in vivo, using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in S. aureus pathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log10 in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present.
PMCID: PMC3067142  PMID: 21282430
10.  Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin against Staphylococcus aureus ▿ †  
In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log2 dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log2 dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections.
PMCID: PMC3028753  PMID: 21135179
11.  Hepatic safety of antibiotics used in primary care 
Antibiotics used by general practitioners frequently appear in adverse-event reports of drug-induced hepatotoxicity. Most cases are idiosyncratic (the adverse reaction cannot be predicted from the drug's pharmacological profile or from pre-clinical toxicology tests) and occur via an immunological reaction or in response to the presence of hepatotoxic metabolites. With the exception of trovafloxacin and telithromycin (now severely restricted), hepatotoxicity crude incidence remains globally low but variable. Thus, amoxicillin/clavulanate and co-trimoxazole, as well as flucloxacillin, cause hepatotoxic reactions at rates that make them visible in general practice (cases are often isolated, may have a delayed onset, sometimes appear only after cessation of therapy and can produce an array of hepatic lesions that mirror hepatobiliary disease, making causality often difficult to establish). Conversely, hepatotoxic reactions related to macrolides, tetracyclines and fluoroquinolones (in that order, from high to low) are much rarer, and are identifiable only through large-scale studies or worldwide pharmacovigilance reporting. For antibiotics specifically used for tuberculosis, adverse effects range from asymptomatic increases in liver enzymes to acute hepatitis and fulminant hepatic failure. Yet, it is difficult to single out individual drugs, as treatment always entails associations. Patients at risk are mainly those with previous experience of hepatotoxic reaction to antibiotics, the aged or those with impaired hepatic function in the absence of close monitoring, making it important to carefully balance potential risks with expected benefits in primary care. Pharmacogenetic testing using the new genome-wide association studies approach holds promise for better understanding the mechanism(s) underlying hepatotoxicity.
PMCID: PMC3112029  PMID: 21586591
idiosyncratic; adverse event; clavulanic acid; co-trimoxazole; flucloxacillin
12.  Intra- and Extracellular Activities of Dicloxacillin against Staphylococcus aureus In Vivo and In Vitro▿  
Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fTMIC]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fTMIC is the index that is the most predictive of the outcome of infection both intra- and extracellularly.
PMCID: PMC2876366  PMID: 20308386
13.  Cellular Pharmacodynamics of the Novel Biaryloxazolidinone Radezolid: Studies with Infected Phagocytic and Nonphagocytic cells, Using Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Legionella pneumophila▿  
Radezolid is a novel biaryloxazolidinone in clinical development which shows improved activity, including against linezolid-resistant strains. In a companion paper (29), we showed that radezolid accumulates about 11-fold in phagocytic cells, with ∼60% of the drug localized in the cytosol and ∼40% in the lysosomes of the cells. The present study examines its activity against (i) bacteria infecting human THP-1 macrophages and located in different subcellular compartments (Listeria monocytogenes, cytosol; Legionella pneumophila, vacuoles; Staphylococcus aureus and Staphylococcus epidermidis, mainly phagolysosomal), (ii) strains of S. aureus with clinically relevant mechanisms of resistance, and (iii) isogenic linezolid-susceptible and -resistant S. aureus strains infecting a series of phagocytic and nonphagocytic cells. Radezolid accumulated to similar levels (∼10-fold) in all cell types (human keratinocytes, endothelial cells, bronchial epithelial cells, osteoblasts, macrophages, and rat embryo fibroblasts). At equivalent weight concentrations, radezolid proved consistently 10-fold more potent than linezolid in all these models, irrespective of the bacterial species and resistance phenotype or of the cell type infected. This results from its higher intrinsic activity and higher cellular accumulation. Time kill curves showed that radezolid's activity was more rapid than that of linezolid both in broth and in infected macrophages. These data suggest the potential interest of radezolid for recurrent or persistent infections where intracellular foci play a determinant role.
PMCID: PMC2876393  PMID: 20385852
14.  Cellular Pharmacokinetics of the Novel Biaryloxazolidinone Radezolid in Phagocytic Cells: Studies with Macrophages and Polymorphonuclear Neutrophils▿  
Radezolid (RX-1741) is the first biaryloxazolidinone in clinical development. It shows improved activity, including against linezolid-resistant strains. Radezolid differs from linezolid by the presence of a biaryl spacer and of a heteroaryl side chain, which increases the ionization and hydrophilicity of the molecule at physiological pH and confers to it a dibasic character. The aim of this study was to determine the accumulation and subcellular distribution of radezolid in phagocytic cells and to decipher the underlying mechanisms. In THP-1 human macrophages, J774 mouse macrophages, and human polymorphonuclear neutrophils, radezolid accumulated rapidly and reversibly (half-lives of approximately 6 min and 9 min for uptake and efflux, respectively) to reach, at equilibrium, a cellular concentration 11-fold higher than the extracellular one. This process was concentration and energy independent but pH dependent (accumulation was reduced to 20 to 30% of control values for cells in medium at a pH of <6 or in the presence of monensin, which collapses pH gradients between the extracellular and intracellular compartments). The accumulation at equilibrium was not affected by efflux pump inhibitors (verapamil and gemfibrozil) and was markedly reduced at 4°C but was further increased in medium with low serum content. Subcellular fractionation studies demonstrated a dual subcellular distribution for radezolid, with ∼60% of the drug colocalizing to the cytosol and ∼40% to the lysosomes, with no specific association with mitochondria. These observations are compatible with a mechanism of transmembrane diffusion of the free fraction and partial segregation of radezolid in lysosomes by proton trapping, as previously described for macrolides.
PMCID: PMC2876419  PMID: 20385873
15.  Plectasin Shows Intracellular Activity against Staphylococcus aureus in Human THP-1 Monocytes and in a Mouse Peritonitis Model▿  
Antimicrobial Agents and Chemotherapy  2009;53(11):4801-4808.
Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [Emax], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (Emax, >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an Emax of a 1.1-log CFU reduction. The parameter most important for activity was fCpeak/MIC, where fCpeak is the free peak concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.
PMCID: PMC2772343  PMID: 19738011
16.  Cellular Accumulation and Pharmacodynamic Evaluation of the Intracellular Activity of CEM-101, a Novel Fluoroketolide, against Staphylococcus aureus, Listeria monocytogenes, and Legionella pneumophila in Human THP-1 Macrophages ▿ †  
CEM-101 is a novel fluoroketolide with lower MICs than those of telithromycin and macrolides. Our aim was to assess the cellular accumulation and intracellular activity of CEM-101 using models developed for analyzing the pharmacokinetics and pharmacological properties of antibiotics against phagocytized bacteria. We used THP-1 macrophages and Staphylococcus aureus (ATCC 25923 [methicillin (meticillin) sensitive]), Listeria monocytogenes (strain EGD), and Legionella pneumophila (ATCC 33153). CEM-101 reached cellular-to-extracellular-concentration ratios of about 350 within 24 h (versus approximately 20, 30, and 160 for telithromycin, clarithromycin, and azithromycin, respectively). This intracellular accumulation was suppressed by incubation at a pH of ≤6 and by monensin (proton ionophore) and was unaffected by verapamil (P-glycoprotein inhibitor; twofold accumulation increase for azithromycin) or gemfibrozil. While keeping with the general properties of the macrolide antibiotics in terms of maximal efficacy (Emax; approximately 1-log10-CFU decrease compared to the postphagocytosis inoculum after a 24-h incubation), CEM-101 showed significantly greater potency against phagocytized S. aureus than telithromycin, clarithromycin, and azithromycin (for which the 50% effective concentration [EC50] and static concentrations were about 3-, 6-, and 15-fold lower, respectively). CEM-101 was also about 50-fold and 100-fold more potent than azithromycin against phagocytized L. monocytogenes and L. pneumophila, respectively. These differences in EC50s and static concentrations between drugs were minimized when data were expressed as multiples of the MIC, demonstrating the critical role of intrinsic drug activity (MIC) in eliciting the antibacterial intracellular effects, whereas accumulation per se was unimportant. CEM-101 should show enhanced in vivo potency if used at doses similar to those of the comparators tested here.
PMCID: PMC2737860  PMID: 19564365
17.  Penicillin-binding Proteins (PBP) and Lmo0441 (a PBP-like protein) play a role in Beta-lactam sensitivity of Listeria monocytogenes 
Gut Pathogens  2009;1:23.
While seven penicillin-binding proteins (PBPs) or PBP-like proteins have been identified either by radiolabelled penicillin binding studies or genomic analysis, only PBP3 has been considered of interest for Beta-lactams activity against Listeria monocytogenes. Herein we reveal that both PBP4 and Lmo0441 (a PBP-like protein) play a direct role in cephalosporin activity in L. monocytogenes while PBP4 additionally has a protective affect against both penicillin and carbapenem.
PMCID: PMC2801671  PMID: 20003484
18.  Identification of the Efflux Transporter of the Fluoroquinolone Antibiotic Ciprofloxacin in Murine Macrophages: Studies with Ciprofloxacin-Resistant Cells▿  
Ciprofloxacin, the most widely used totally synthetic antibiotic, is subject to active efflux mediated by a MRP-like transporter in wild-type murine J774 macrophages. To identify the transporter among the seven potential Mrps, we used cells made resistant to ciprofloxacin obtained by long-term exposure to increasing drug concentrations (these cells show less ciprofloxacin accumulation and provide a protected niche for ciprofloxacin-sensitive intracellular Listeria monocytogenes). In the present paper, we first show that ciprofloxacin-resistant cells display a faster efflux of ciprofloxacin which is inhibited by gemfibrozil (an unspecific MRP inhibitor). Elacridar, at a concentration known to inhibit P-glycoprotein and breast cancer resistance protein (BCRP), only slightly increased ciprofloxacin accumulation, with no difference between resistant and wild-type cells. Analysis at the mRNA (real-time PCR) and protein (Western blotting) levels revealed an overexpression of Mrp2 and Mrp4. Mrp4 transcripts, however, were overwhelmingly predominant (45% [wild-type cells] to 95% [ciprofloxacin-resistant cells] of all Mrp transcripts tested [Mrp1 to Mrp7]). Silencing of Mrp2 and Mrp4 with specific small interfering RNAs showed that only Mrp4 is involved in ciprofloxacin transport in both ciprofloxacin-resistant and wild-type cells. The study therefore identifies Mrp4 as the most likely transporter of ciprofloxacin in murine macrophages but leaves open a possible common upregulation mechanism for both Mrp4 and Mrp2 upon chronic exposure of eukaryotic cells to this widely used antibiotic.
PMCID: PMC2687251  PMID: 19307362
19.  Activities of Ceftobiprole and Other Cephalosporins against Extracellular and Intracellular (THP-1 Macrophages and Keratinocytes) Forms of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus▿ †  
Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, β-lactams show activity against intracellular methicillin (meticillin)-susceptible S. aureus (MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistant S. aureus (MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these β-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (Emax), relative potency (the concentration causing a reduction of the inoculum halfway between E0 and Emax [EC50]), and static concentration (Cs)} were measured. All strains showed sigmoidal dose-responses, with Emax being about a 1 log10 CFU decrease from the postphagocytosis inoculum, and EC50 and Cs being 0.2 to 0.3× and 0.6 to 0.9× the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.
PMCID: PMC2687181  PMID: 19289525
20.  Role of rsbU and staphyloxanthin in phagocytosis and intracellular growth of Staphylococcus aureus in human macrophages and endothelial cells 
The Journal of infectious diseases  2009;200(9):1367-1370.
In Staphylococcus aureus, rsbU dowregulates agr and stimulates production of staphyloxanthin (STX), an anti-oxidant that may contribute to intracellular survival after phagocytosis. Using isogenic rsbU- and rsbU+ strains, we show that rsbU causes increased internalization and intracellular growth in both THP-1 macrophages and HUVEC cells (more so for the latter) without change in sub-cellular localization. Inhibition of STX biosynthesis markedly reduces intracellular growth of the rsbU+ strain (and of clinical isolates, including USA300; A5 tested with macrophages only), without affecting internalization. rsbU is important for uptake, and for STX biosynthesis, critical for intracellular multiplication of S. aureus.
PMCID: PMC2762113  PMID: 19817587
S. aureus; rsbU; staphyloxanthin; intracellular; macrophages; HUVEC cells
21.  Intracellular Activity of Antibiotics in a Model of Human THP-1 Macrophages Infected by a Staphylococcus aureus Small-Colony Variant Strain Isolated from a Cystic Fibrosis Patient: Study of Antibiotic Combinations▿ † 
In a companion paper (H. A. Nguyen et al., Antimicrob. Agents Chemother. 53:1434-1442, 2009), we showed that vancomycin, oxacillin, fusidic acid, clindamycin, linezolid, and daptomycin are poorly active against the intracellular form of a thymidine-dependent small-colony variant (SCV) strain isolated from a cystic fibrosis patient and that the activity of quinupristin-dalfopristin, moxifloxacin, rifampin, and oritavancin remains limited (2- to 3-log CFU reduction) compared to their extracellular activity. Antibiotic combination is a well-known strategy to improve antibacterial activity, which was examined here against an intracellular SCV strain using combinations with either rifampin or oritavancin. Time-kill curve analysis using either concentrations that caused a static effect for each antibiotic individually or concentrations corresponding to the maximum concentration in human serum showed largely divergent effects that were favorable when antibiotics were combined with rifampin at low concentrations only and with oritavancin at both low and high concentrations. The nature of the interaction between rifampin, oritavancin, and moxifloxacin was further examined using the fractional maximal effect method, which allows categorization of the effects of combinations when dose-effect relationships are not linear. Rifampin and oritavancin were synergistic at all concentration ratios investigated. Oritavancin and moxifloxacin were also synergistic but at high oritavancin concentrations only. Rifampin and moxifloxacin were additive. This approach may help in better assessing and improving the activity of antibiotics against intracellular SCV strains.
PMCID: PMC2663110  PMID: 19188397
22.  Intracellular Activity of Antibiotics in a Model of Human THP-1 Macrophages Infected by a Staphylococcus aureus Small-Colony Variant Strain Isolated from a Cystic Fibrosis Patient: Pharmacodynamic Evaluation and Comparison with Isogenic Normal-Phenotype and Revertant Strains▿ †  
Small-colony variant (SCV) strains of Staphylococcus aureus show reduced antibiotic susceptibility and intracellular persistence, potentially explaining therapeutic failures. The activities of oxacillin, fusidic acid, clindamycin, gentamicin, rifampin, vancomycin, linezolid, quinupristin-dalfopristin, daptomycin, tigecycline, moxifloxacin, telavancin, and oritavancin have been examined in THP-1 macrophages infected by a stable thymidine-dependent SCV strain in comparison with normal-phenotype and revertant isogenic strains isolated from the same cystic fibrosis patient. The SCV strain grew slowly extracellularly and intracellularly (1- and 0.2-log CFU increase in 24 h, respectively). In confocal and electron microscopy, SCV and the normal-phenotype bacteria remain confined in acid vacuoles. All antibiotics tested, except tigecycline, caused a net reduction in bacterial counts that was both time and concentration dependent. At an extracellular concentration corresponding to the maximum concentration in human serum (total drug), oritavancin caused a 2-log CFU reduction at 24 h; rifampin, moxifloxacin, and quinupristin-dalfopristin caused a similar reduction at 72 h; and all other antibiotics had only a static effect at 24 h and a 1-log CFU reduction at 72 h. In concentration dependence experiments, response to oritavancin was bimodal (two successive plateaus of −0.4 and −3.1 log CFU); tigecycline, moxifloxacin, and rifampin showed maximal effects of −1.1 to −1.7 log CFU; and the other antibiotics produced results of −0.6 log CFU or less. Addition of thymidine restored intracellular growth of the SCV strain but did not modify the activity of antibiotics (except quinupristin-dalfopristin). All drugs (except tigecycline and oritavancin) showed higher intracellular activity against normal or revertant phenotypes than against SCV strains. The data may help rationalizing the design of further studies with intracellular SCV strains.
PMCID: PMC2663071  PMID: 19188393
23.  Cooperation between Prokaryotic (Lde) and Eukaryotic (MRP) Efflux Transporters in J774 Macrophages Infected with Listeria monocytogenes: Studies with Ciprofloxacin and Moxifloxacin▿ †  
Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01× to 100× MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target.
PMCID: PMC2533462  PMID: 18573933
24.  Restoration of Susceptibility of Intracellular Methicillin-Resistant Staphylococcus aureus to β-Lactams: Comparison of Strains, Cells, and Antibiotics▿ †  
Staphylococcus aureus invades eukaryotic cells. When methicillin-resistant S. aureus (MRSA) ATCC 33591 is phagocytized by human THP-1 macrophages, complete restoration of susceptibility to cloxacillin and meropenem is shown and the strain becomes indistinguishable from MSSA ATCC 25923 due to the acid pH prevailing in phagolysosomes (S. Lemaire et al., Antimicrob. Agents Chemother. 51:1627-1632, 2007). We examined whether this observation can be extended to (i) strains of current clinical and epidemiological interest (three hospital-acquired MRSA [HA-MRSA] strains, two community-acquired MRSA [CA-MRSA] strains, two HA-MRSA strains with the vancomycin-intermediate phenotype, one HA-MRSA strain with the vancomycin-resistant phenotype, and one animal [porcine] MRSA strain), (ii) activated THP-1 cells and nonprofessional phagocytes (keratinocytes, Calu-3 bronchial epithelial cells), and (iii) other β-lactams (imipenem, oxacillin, cefuroxime, cefepime). All strains showed (i) a marked reduction in MICs in broth at pH 5.5 compared with the MIC at pH 7.4 and (ii) sigmoidal dose-response curves with cloxacillin (0.01× to 100× MIC, 24 h of incubation) after phagocytosis by THP-1 macrophages that were indistinguishable from each other and from the dose-response curve for methicillin-susceptible S. aureus (MSSA) ATCC 25923 (relative potency [50% effect], 6.09× MIC [95% confidence interval {CI}, 4.50 to 8.25]; relative efficacy [change in bacterial counts over the original inoculum for an infinitely large cloxacillin concentration, or maximal effect], −0.69 log CFU [95% CI, −0.79 to −0.58]). Similar dose-response curves for cloxacillin were also observed with MSSA ATCC 25923 and MRSA ATCC 33591 after phagocytosis by activated THP-1 macrophages, keratinocytes, and Calu-3 cells. By contrast, there was a lower level of restoration of susceptibility of MRSA ATCC 33591 to cefuroxime and cefepime after phagocytosis by THP-1 macrophages, even when the data were normalized for differences in MICs. We conclude that the restoration of MRSA susceptibility to β-lactams after phagocytosis is independent of the strain and the types of cells but varies between β-lactams.
PMCID: PMC2493141  PMID: 18519727
25.  Molecular models of human P-glycoprotein in two different catalytic states 
P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies.
We present here three-dimensional models of two different catalytic states of P-glycoprotein that were developed based on the crystal structures of two bacterial multidrug transporters. Our models are supported by a large body of biochemical data. Measured inter-residue distances correlate well with distances derived from cross-linking data. The nucleotide-free model features a large cavity detected in the protein core into which ligands of different size were successfully docked. The locations of docked ligands compare favorably with those suggested by drug binding site mutants.
Our models can interpret the effects of several mutants in the nucleotide-binding domains (NBDs), within the transmembrane domains (TMDs) or at the NBD:TMD interface. The docking results suggest that the protein has multiple binding sites in agreement with experimental evidence. The nucleotide-bound models are exploited to propose different pathways of signal transmission upon ATP binding/hydrolysis which could lead to the elaboration of conformational changes needed for substrate translocation. We identified a cluster of aromatic residues located at the interface between the NBD and the TMD in opposite halves of the molecule which may contribute to this signal transmission.
Our models may characterize different steps in the catalytic cycle and may be important tools to understand the structure-function relationship of P-glycoprotein.
PMCID: PMC2661087  PMID: 19159494

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