Binding of temocillin to plasma proteins in vitro and in vivo: the importance of plasma protein levels in different populations and of co-medications.

BACKGROUND: Temocillin plasma protein binding (PPB) in healthy individuals is reported to be ∼85% but had not been studied in patients. OBJECTIVES: To obtain normative data on temocillin PPB in patients in relation to infection and impact of co-medications widely used in ICU. METHODS: Plasma was obtained from healthy individuals (Group #1), non-ICU patients with UTI (Group #2), ICU patients with suspected/confirmed ventriculitis (Group #3) or with sepsis/septic shock (Group #4). Total and unbound temocillin concentrations were measured in spiked samples from temocillin-naive donors (in vitro) or in plasma from temocillin-treated subjects (in vivo). The impact of diluting plasma, using pharmaceutical albumin, or adding drugs potentially competing for PPB was tested in spiked samples. Data were analysed using a modified Hill-Langmuir equation taking ligand depletion into account. RESULTS: Temocillin PPB was saturable in all groups, both in vitro and in vivo. Maximal binding capacity (Bmax) was 1.2-2-fold lower in patients. At 20 and 200 mg/L (total concentrations), the unbound fraction reached 12%-29%, 23%-42% and 32%-52% in Groups #2, #3, #4. The unbound fraction was inversely correlated with albumin and C-reactive protein concentrations. Binding to albumin was 2-3-fold lower than in plasma and non-saturable. Drugs with high PPB but active at lower molar concentrations than temocillin caused minimal displacement, while fluconazole (low PPB but similar plasma concentrations to temocillin) increased up to 2-fold its unbound fraction. CONCLUSIONS: Temocillin PPB is saturable, 2-4-fold lowered in infected patients in relation to disease severity (ICU admission, hypoalbuminaemia, inflammation) and only partially reproducible with albumin. Competition with other drugs must be considered for therapeutic concentrations to be meaningful.

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Current and future options for treating complicated skin and soft tissue infections: focus on fluoroquinolones and long-acting lipoglycopeptide antibiotics.

Bacterial skin and soft tissue infections are among the most common bacterial infections and constitute a major burden for patients and healthcare systems. Care is complicated by the variety of potential pathogens, some with resistance to previously effective antimicrobial agents, the wide spectrum of clinical presentations and the risk of progression to life-threatening forms. More-efficient care pathways are needed that can reduce hospital admissions and length of stay, while maintaining a high quality of care and adhering to antimicrobial stewardship principles. Several agents approved recently for treating acute bacterial skin and skin structure infections have characteristics that meet these requirements. We address the clinical and pharmacological characteristics of the fourth-generation fluoroquinolone delafloxacin, and the long-acting lipoglycopeptide agents dalbavancin and oritavancin.

Profile of a Novel Anionic Fluoroquinolone-Delafloxacin.

Fluoroquinolones have been in clinical use for over 50 years with significant efficacy. However, increasing resistance and emergence of some marked adverse events have limited their usage. The most recently approved class member, delafloxacin, is the only available anionic (non-zwitterionic) fluoroquinolone. Its unique molecular structure provides improved in vitro activity against most Gram-positive pathogens, including quinolone-resistant strains, which is further enhanced at acid pH. Delafloxacin shows favorable pharmacological properties, with about 60% bioavailability after oral administration, only mild inhibition of cytochrome P450 3A, and no evidence of cardiac- or phototoxicity in healthy volunteers (tested against positive controls). Its twice daily dosing, suitability for intravenous, oral, or switch dosing, the lack of many clinically significant drug-drug interactions, and acceptable adverse event profile in registration clinical trials supports its use in the treatment of acute bacterial skin and skin structure infections, and potentially in other infections, where resistance to other agents, safety, and/or the need for early discharge is of concern.

Cellular Pharmacokinetics and Intracellular Activity of Gepotidacin against Staphylococcus aureus Isolates with Different Resistance Phenotypes in Models of Cultured Phagocytic Cells.

Gepotidacin (GSK2140944), a novel triazaacenaphthylene bacterial topoisomerase inhibitor, is currently in clinical development for the treatment of bacterial infections. This study examined in vitro its activity against intracellular Staphylococcus aureus (involved in the persistent character of skin and skin structure infections) by use of a pharmacodynamic model and in relation to cellular pharmacokinetics in phagocytic cells. Compared to oxacillin, vancomycin, linezolid, daptomycin, azithromycin, and moxifloxacin, gepotidacin was (i) more potent intracellularly (the apparent bacteriostatic concentration [Cs ] was reached at an extracellular concentration about 0.7× its MIC and was not affected by mechanisms of resistance to the comparators) and (ii) caused a maximal reduction of the intracellular burden (maximum effect) of about -1.6 log10 CFU (which was better than that caused by linezolid, macrolides, and daptomycin and similar to that caused by moxifloxacin). After 24 h of incubation of infected cells with antibiotics at 100× their MIC, the intracellular persisting fraction was <0.1% with moxifloxacin, 0.5% with gepotidacin, and >1% with the other drugs. The accumulation and efflux of gepotidacin in phagocytes were very fast (kin and kout, ∼0.3 min-1; the plateau was reached within 15 min) but modest (intracellular concentration-to-extracellular concentration ratio, ∼1.6). In cell fractionation studies, about 40 to 60% of the drug was recovered in the soluble fraction and ∼40% was associated with lysosomes in uninfected cells. In infected cells, about 20% of cell-associated gepotidacin was recovered in a sedimentable fraction that also contained bacteria. This study highlights the potential for further study of gepotidacin to fight infections where intracellular niches may play a determining role in bacterial persistence and relapses.

Activities of Combinations of Antistaphylococcal Antibiotics with Fusidic Acid against Staphylococcal Biofilms in In Vitro Static and Dynamic Models.

Staphylococcal biofilms are a major cause of therapeutic failure, especially when caused by multiresistant strains. Oral fusidic acid is currently being redeveloped in the United States for skin, skin structure, and orthopedic infections, in which biofilms play a major role. The aim of this study was to examine the activity of fusidic acid alone or combined with other antistaphylococcal drugs against biofilms made by a reference strain and five clinical isolates of Staphylococcus aureus or Staphylococcus epidermidis in in vitro static and dynamic models (microtiter plates and a CDC reactor) exposed to clinically relevant concentrations. In microtiter plates, antibiotics alone were poorly active, with marked differences among strains. At concentrations mimicking the free-drug human maximum concentration of drug in serum (Cmax), the combination of fusidic acid with linezolid, daptomycin, or vancomycin resulted in increased activity against 4 to 5 strains, while the combination with doxycycline, rifampin, or moxifloxacin increased activity against 1 to 3 strains only. In the CDC reactor, biofilms were grown under constant flow and antibiotic concentrations decreased over time according to human elimination rates. A bactericidal effect was obtained when fusidic acid was combined with daptomycin or linezolid, but not with vancomycin. The higher tolerance of biofilms to antibiotics in the CDC reactor is probably attributable to the more complex architecture they adopt when growing under constant flow. Because biofilms grown in the CDC reactor are considered more similar to those developing in vivo, the data support further testing of combinations of fusidic acid with daptomycin or linezolid in models pertinent to chronic skin, skin structure, or orthopedic infections.

Mitochondrial Alterations (Inhibition of Mitochondrial Protein Expression, Oxidative Metabolism, and Ultrastructure) Induced by Linezolid and Tedizolid at Clinically Relevant Concentrations in Cultured Human HL-60 Promyelocytes and THP-1 Monocytes.

Linezolid, the first clinically available oxazolidinone antibiotic, causes potentially severe toxicities (myelosuppression, lactic acidosis, and neuropathies) ascribed to impairment of mitochondrial protein synthesis and consecutive mitochondrial dysfunction. Tedizolid, a newly approved oxazolidinone, shows an enhanced activity compared to linezolid but is also a more potent inhibitor of mitochondrial protein synthesis. We compared linezolid and tedizolid for (i) inhibition of the expression of subunit I of cytochrome c-oxidase (CYTox I; Western blot analysis), (ii) cytochrome c-oxidase activity (biochemical assay), (iii) mitochondrial oxidative metabolism (Seahorse technology), and (iv) alteration of mitochondrial ultrastructure (electron microscopy) using HL-60 promyelocytes and THP-1 monocytes exposed to microbiologically (multiples of modal MIC against Staphylococcus aureus) and therapeutically (Cmin - Cmax) pertinent concentrations. Both drugs caused a rapid and complete (48 to 72 h) inhibition of CYTox I expression, cytochrome c-oxidase activity, and spare respiratory capacity, with conspicuous swelling of the mitochondrial matrix and loss of their cristae. Globally, tedizolid was a more potent inhibitor than linezolid. For both drugs, all effects were quickly (48 to 72 h) and fully reversible upon drug withdrawal. Using an alternation of exposure to and withdrawal from drug mimicking their approved schedule of administration (twice daily and once daily [qD] for linezolid and tedizolid, respectively), only partial inhibition of CYTox I expression was noted for up to 96 h. Thus, rapid reversal of toxic effects upon discontinuous administration may mitigate oxazolidinone toxicity. Since tedizolid is given qD, this may help to explain its reported lower preclinical and clinical toxicity.

Temocillin dosing in haemodialysis patients based on population pharmacokinetics of total and unbound concentrations and Monte Carlo simulations.

Objectives: To develop a population model describing temocillin pharmacokinetics (PK) in patients undergoing haemodialysis and investigate how pharmacokinetic/pharmacodynamic (PD) targets can be met with different dosage regimens. Patients and methods: Sixteen patients received the currently licenced dosing of 1, 2 or 3 g of temocillin (total of 61 doses) corresponding to an inter-dialytic period of 20, 44 or 68 h, respectively, and a dialysis period of 4 h. A non-linear mixed-effects model was developed jointly for total and unbound temocillin serum concentrations. The performance of clinically feasible dosing regimens was evaluated using a 5000-subject Monte Carlo (MC) simulation for determining the highest MIC for which the PK/PD target of 40%ƒT>MIC would be reached in 90% of patients [probability of target attainment (PTA)]. This PK study was registered at ClinicalTrials.gov (NCT02285075). Results: Temocillin unbound and total serum concentrations (429 samples) were used to fit an open two-compartment model with non-linear albumin binding and first-order elimination. In addition to total body clearance, dialysis clearance was modelled using the Michaels function. The currently licenced dosing achieved a 90% PTA for an MIC up to 8 mg/L. A new temocillin dosage regimen was designed that would achieve a 90% PTA for an MIC of 16 mg/L (MIC90 of target organisms) adjusted to patient weight and inter-dialytic period. Conclusions: Currently licensed dosage regimen is suboptimal for MICs >8 mg/L (frequently found in clinical isolates). Model-based simulations allowed suggestion of a new dosage regimen with improved probability of microbiological success, applicability in routine clinical practice and more appropriate for empirical therapy.

Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms.

Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosain vitro Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

Acquired resistance to macrolides in Pseudomonas aeruginosa from cystic fibrosis patients.

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that Pseudomonas aeruginosa, considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth.We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP).Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain.Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance.

Inhibition of the Injectisome and Flagellar Type III Secretion Systems by INP1855 Impairs Pseudomonas aeruginosa Pathogenicity and Inflammasome Activation.

With the rise of multidrug resistance, Pseudomonas aeruginosa infections require alternative therapeutics. The injectisome (iT3SS) and flagellar (fT3SS) type III secretion systems are 2 virulence factors associated with poor clinical outcomes. iT3SS translocates toxins, rod, needle, or regulator proteins, and flagellin into the host cell cytoplasm and causes cytotoxicity and NLRC4-dependent inflammasome activation, which induces interleukin 1ß (IL-1ß) release and reduces interleukin 17 (IL-17) production and bacterial clearance. fT3SS ensures bacterial motility, attachment to the host cells, and triggers inflammation. INP1855 is an iT3SS inhibitor identified by in vitro screening, using Yersinia pseudotuberculosis Using a mouse model of P. aeruginosa pulmonary infection, we show that INP1855 improves survival after infection with an iT3SS-positive strain, reduces bacterial pathogenicity and dissemination and IL-1ß secretion, and increases IL-17 secretion. INP1855 also modified the cytokine balance in mice infected with an iT3SS-negative, fT3SS-positive strain. In vitro, INP1855 impaired iT3SS and fT3SS functionality, as evidenced by a reduction in secretory activity and flagellar motility and an increase in adenosine triphosphate levels. As a result, INP1855 decreased cytotoxicity mediated by toxins and by inflammasome activation induced by both laboratory strains and clinical isolates. We conclude that INP1855 acts by dual inhibition of iT3SS and fT3SS and represents a promising therapeutic approach.

Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe.

Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. This study compared the antimicrobial susceptibilities of 153 P. aeruginosa isolates from the United Kingdom (UK) (n = 58), Belgium (n = 44), and Germany (n = 51) collected from 118 patients during routine visits over the period from 2006 to 2012. MICs were measured by broth microdilution. Genes encoding extended-spectrum ß-lactamases (ESBL), metallo-ß-lactamases, and carbapenemases were detected by PCR. Pulsed-field gel electrophoresis and multilocus sequence typing were performed on isolates resistant to ≥3 antibiotic classes among the penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility rates were ≤30%/≤40% (penicillins, ceftazidime, amikacin, and ciprofloxacin), 44 to 48%/48 to 63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independent of patient age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease Prevention and Control criteria). Genes encoding the most prevalent ESBL (BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo-ß-lactamases (VIM, IMP, and NDM), or carbapenemases (OXA-48 and KPC) were not detected. The Liverpool epidemic strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR sequence type 958 (ST958) isolates were found to be spread over the three countries. The other MDR clones were evidenced in ≤3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and nonclonal isolates with different susceptibility profiles were found in 20 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with meropenem, tobramycin, and colistin remaining the most active drugs.

Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum.

Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines.

Cellular pharmacokinetics and intracellular activity of the novel peptide deformylase inhibitor GSK1322322 against Staphylococcus aureus laboratory and clinical strains with various resistance phenotypes: studies with human THP-1 monocytes and J774 murine macrophages.

GSK1322322 is a peptide deformylase inhibitor active against Staphylococcus aureus strains resistant to currently marketed antibiotics. Our aim was to assess the activity of GSK1322322 against intracellular S. aureus using an in vitro pharmacodynamic model and, in parallel, to examine its cellular pharmacokinetics and intracellular disposition. For intracellular activity analysis, we used an established model of human THP-1 monocytes and tested one fully susceptible S. aureus strain (ATCC 25923) and 8 clinical strains with resistance to oxacillin, vancomycin, daptomycin, macrolides, clindamycin, linezolid, or moxifloxacin. Uptake, accumulation, release, and subcellular distribution (cell fractionation) of [(14)C]GSK1322322 were examined in uninfected murine J774 macrophages and uninfected and infected THP-1 monocytes. GSK1322322 demonstrated a uniform activity against the intracellular forms of all S. aureus strains tested, disregarding their resistance phenotypes, with a maximal relative efficacy (E max) of a 0.5 to 1 log10 CFU decrease compared to the original inoculum within 24 h and a static concentration (C s) close to its MIC in broth. Influx and efflux were very fast (<5 min to equilibrium), and accumulation was about 4-fold, with no or a minimal effect of the broad-spectrum eukaryotic efflux transporter inhibitors gemfibrozil and verapamil. GSK1322322 was recovered in the cell-soluble fraction and was dissociated from the main subcellular organelles and from bacteria (in infected cells). The results of this study show that GSK1322322, as a typical novel deformylase inhibitor, may act against intracellular forms of S. aureus. They also suggest that GSK1322322 has the ability to freely diffuse into and out of eukaryotic cells as well as within subcellular compartments.

Activities of antibiotic combinations against resistant strains of Pseudomonas aeruginosa in a model of infected THP-1 monocytes.

Antibiotic combinations are often used for treating Pseudomonas aeruginosa infections but their efficacy toward intracellular bacteria has not been investigated so far. We have studied combinations of representatives of the main antipseudomonal classes (ciprofloxacin, meropenem, tobramycin, and colistin) against intracellular P. aeruginosa in a model of THP-1 monocytes in comparison with bacteria growing in broth, using the reference strain PAO1 and two clinical isolates (resistant to ciprofloxacin and meropenem, respectively). Interaction between drugs was assessed by checkerboard titration (extracellular model only), by kill curves, and by using the fractional maximal effect (FME) method, which allows studying the effects of combinations when dose-effect relationships are not linear. For drugs used alone, simple sigmoidal functions could be fitted to all concentration-effect relationships (extracellular and intracellular bacteria), with static concentrations close to (ciprofloxacin, colistin, and meropenem) or slightly higher than (tobramycin) the MIC and with maximal efficacy reaching the limit of detection in broth but only a 1 to 1.5 (colistin, meropenem, and tobramycin) to 2 to 3 (ciprofloxacin) log10 CFU decrease intracellularly. Extracellularly, all combinations proved additive by checkerboard titration but synergistic using the FME method and more bactericidal in kill curve assays. Intracellularly, all combinations proved additive only based on both FME and kill curve assays. Thus, although combinations appeared to modestly improve antibiotic activity against intracellular P. aeruginosa, they do not allow eradication of these persistent forms of infections. Combinations including ciprofloxacin were the most active (even against the ciprofloxacin-resistant strain), which is probably related to the fact this drug was the most effective alone intracellularly.

RX-P873, a Novel Protein Synthesis Inhibitor, Accumulates in Human THP-1 Monocytes and Is Active against Intracellular Infections by Gram-Positive (Staphylococcus aureus) and Gram-Negative (Pseudomonas aeruginosa) Bacteria.

The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies (Emax values) and bacteriostatic concentrations (Cs values) on the basis of Hill equations of the concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/liter (S. aureus) and 2 to 8 mg/liter (P. aeruginosa), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units in infected THP-1 cells, respectively, after 24 h. Cs values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, and daptomycin for S. aureus; ciprofloxacin and ceftazidime for P. aeruginosa). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus. Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species.

Nonclinical and pharmacokinetic assessments to evaluate the potential of tedizolid and linezolid to affect mitochondrial function.

Prolonged treatment with the oxazolidinone linezolid is associated with myelosuppression, lactic acidosis, and neuropathies, toxicities likely caused by impairment of mitochondrial protein synthesis (MPS). To evaluate the potential of the novel oxazolidinone tedizolid to cause similar side effects, nonclinical and pharmacokinetic assessments were conducted. In isolated rat heart mitochondria, tedizolid inhibited MPS more potently than did linezolid (average [± standard error of the mean] 50% inhibitory concentration [IC50] for MPS of 0.31 ± 0.02 µM versus 6.4 ± 1.2 µM). However, a rigorous 9-month rat study comparing placebo and high-dose tedizolid (resulting in steady-state area under the plasma concentration-time curve values about 8-fold greater than those with the standard therapeutic dose in humans) showed no evidence of neuropathy. Additional studies explored why prolonged, high-dose tedizolid did not cause these mitochondriopathic side effects despite potent MPS inhibition by tedizolid. Murine macrophage (J774) cell fractionation studies found no evidence of a stable association of tedizolid with eukaryotic mitochondria. Monte Carlo simulations based on population pharmacokinetic models showed that over the course of a dosing interval using standard therapeutic doses, free plasma concentrations fell below the respective MPS IC50 in 84% of tedizolid-treated patients (for a median duration of 7.94 h) and 38% of linezolid-treated patients (for a median duration of 0 h). Therapeutic doses of tedizolid, but not linezolid, may therefore allow for mitochondrial recovery during antibacterial therapy. The overall results suggest that tedizolid has less potential to cause myelosuppression and neuropathy than that of linezolid during prolonged treatment courses. This, however, remains a hypothesis that must be confirmed in clinical studies.

Modulation of the activity of moxifloxacin and solithromycin in an in vitro pharmacodynamic model of Streptococcus pneumoniae naive and induced biofilms.

OBJECTIVES: Bacterial biofilms developing in the bronchial tree of patients experiencing acute exacerbations of chronic bronchitis (AECBs) are suggested to cause relapses and recurrences of the disease because the matrix barrier impairs antibiotic access to the offending organisms. We examined whether bronchodilators could modulate pneumococcal biofilm development and antibiotic action using an in vitro model. METHODS: Streptococcus pneumoniae strains from patients hospitalized for AECBs and two reference strains (ATCC 49619 and R6) were screened for biofilm formation (multi-well plates; 2-11 days of growth). Ipratropium and salbutamol (alone or in combination) were added at concentrations of 1.45 and 7.25 mg/L, respectively (mimicking those in the bronchial tree), and their effects were measured on biofilm formation and modulation of the activity of antibiotics [full antibiotic concentration-dependent effects (pharmacodynamic model)] with a focus on moxifloxacin and solithromycin. Bacterial viability and biomass were measured by the reduction of resazurin and crystal violet staining, respectively. Release of sialic acid (from biofilm) and neuraminidase activity were measured using enzymatic and HPLC-MS detection of sialic acid. RESULTS: All clinical isolates produced biofilms, but with fast disassembly if from patients who had received muscarinic antagonists. Ipratropium caused: (i) reduced biomass formation and faster biofilm disassembly with free sialic acid release; and (ii) a marked improvement of antibiotic activity (bacterial killing and biomass reduction). Salbutamol stimulated neuraminidase activity associated with improved antibiotic killing activity (reversed by zanamivir) but modest biomass reduction. CONCLUSIONS: Ipratropium and, to a lesser extent, salbutamol may cooperate with antibiotics for bacterial clearance and disassembly of pneumococcal biofilms.

Temocillin (6 g daily) in critically ill patients: continuous infusion versus three times daily administration.

OBJECTIVES: The growing incidence of infections caused by Enterobacteriaceae producing ESBLs has led to increased use of carbapenems. Temocillin, which resists most ß-lactamases, may be a useful alternative. The aim of this study was to assess the pharmacokinetics and target attainment rates of 6 g of temocillin daily divided into three administrations every 8 h (three times daily) or administered by continuous infusion in critically ill patients. PATIENTS AND METHODS: This was a prospective, two-centre, randomized, controlled study in patients with intra-abdominal or lower respiratory tract infections caused by Enterobacteriaceae. RESULTS: Thirty-two patients were included and analysed for clinical efficacy, and pharmacokinetics were measured in 29 of them. Four patients undergoing continuous veno-venous haemofiltration (CVVH) were analysed separately. Mean, median and range of percentages of the dosing interval during which the free drug concentration remained >16 mg/L were 76.4, 98 and 18.7-98.9 in patients treated three times daily and 98.9, 89.7 and 36.4-99.9 in patients with continuous infusion, respectively. Clinical cure rates were 79% and 93% in each of these groups, respectively (not significant). Patients with CVVH received a daily dose of 750 mg given by continuous infusion and had a mean free drug concentration of only 13.8 ±â€Š1.9 mg/L. No adverse event attributable to temocillin was observed. CONCLUSIONS: Temocillin (6 g daily) given by continuous infusion allows a larger proportion of critically ill patients to have free drug serum concentrations covering infections caused by Enterobacteriaceae with an MIC of 16 mg/L compared with administration three times daily. Clinical efficacy compared with carbapenems in documented severe infections needs to be further studied.