Prolonged inhibition and incomplete recovery of mitochondrial function in oxazolidinone-treated megakaryoblastic cell lines.

Thrombocytopenia is commonly seen in patients receiving linezolid for >14 days. Linezolid is a reversible inhibitor of mitochondrial function in various cell types. This study investigated the inhibitory effects of linezolid and tedizolid, and their potential recovery on (i) CYTox I expression (subunit I of cytochrome c-oxidase; encoded by the mitochondrial genome), (ii) cytochrome c-oxidase activity and (iii) mitochondrial respiration (Seahorse bioanalysis) in two megakaryocytic cell lines [UT-7 WT (human acute megakaryoblastic leukaemia cells) and UT-7 MPL (transduced to express the thrombopoietin receptor)]. Cells were exposed to linezolid (0.5-25 mg/L) or tedizolid (0.1-5 mg/L) for up to 5 days and recovery followed after drug removal. Both oxazolidinones caused concentration- and time-dependent inhibition of CYTox I expression, cytochrome c-oxidase activity and mitochondrial spare capacity. On electron microscopy, mitochondria appeared dilated with a loss of cristae. Globally, tedizolid exerted stronger effects than linezolid. While CYTox I expression recovered completely after 6 days of drug washout, only partial (linezolid) or no (tedizolid) recovery of cytochrome c-oxidase activity, and no rescue of mitochondrial spare capacity (after 3 days) was observed. Thus, and in contrast to previous studies using a variety of cell lines unrelated to megakaryocytic lineages, the inhibitory effects exerted by oxazolidinones on the mitochondrial function of megakaryoblastic cells appear to be particularly protracted. Given the dynamics of platelet production and destruction, these results may explain why oxazolidinone-induced thrombocytopenia is one of the most common side effects in patients exposed to these antibiotics.

Loss of activity of ceftazidime-avibactam due to MexAB-OprM efflux and overproduction of AmpC cephalosporinase in Pseudomonas aeruginosa isolated from patients suffering from cystic fibrosis.

In Pseudomonas aeruginosa (P. aeruginosa) collected from cystic fibrosis (CF) patients, 24% resistance to ceftazidime-avibactam in isolates negative for carbapenemases and extended-spectrum ß-lactamases (ESBLs) has previously been observed. The current study aimed to unravel the underlying mechanism(s). Using the laboratory strain PAO1 and derivatives thereof, with ampC expression induced by a sub-minimum inhibitory concentration (MIC) of imipenem, a higher MIC of ceftazidime-avibactam was found for those overexpressing MexAB-OprM (quantitative polymerase chain reaction (PCR) of mexA) and, to a lesser extent, MexEF-OprN (PCR of mexE), or without OprD expression (SDS-Page and Coomassie blue staining). This was ascribed to (i) an efflux of avibactam (efflux mutants) and (ii) a lack of avibactam penetration (OprD mutants), respectively. We then used 10 CF clinical isolates resistant to ceftazidime (MIC ≥ 128 mg/L) and with (i) variable basal levels of ampC overexpression, (ii) mutations in mexA or mexB inactivating to variable extent the MexAB-OprM transport capacity (assessed by extrusion of N-phenyl-1-naphthylamine [NPN]), and (iii) expression or not of mexE and of OprD porin. The reduction of ceftazidime MIC in the presence of avibactam was partially lost for isolates with large efflux activity of MexAB-OprM and/or increased ampC expression, but not significantly with mexE expression or lack of OprD (non-parametric and parametric tests). This identified MexAB-OprM as a main avibactam efflux transporter in P. aeruginosa that, together with ampC overexpression, reduced avibactam potency. Since about 30% of CF isolates show mutations in MexAB-OprM compromising efflux (Chalhoub, et al. Sci Reports 2017;7:40208), routine susceptibility testing of CF P. aeruginosa with ceftazidime-avibactam is warranted.

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.

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.

Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic.

The ß-lactam antibiotic temocillin (6-α-methoxy-ticarcillin) shows stability to most extended spectrum ß-lactamases, but is considered inactive against Pseudomonas aeruginosa. Mutations in the MexAB-OprM efflux system, naturally occurring in cystic fibrosis (CF) isolates, have been previously shown to reverse this intrinsic resistance. In the present study, we measured temocillin activity in a large collection (n = 333) of P. aeruginosa CF isolates. 29% of the isolates had MICs ≤ 16 mg/L (proposed clinical breakpoint for temocillin). Mutations were observed in mexA or mexB in isolates for which temocillin MIC was ≤512 mg/L (nucleotide insertions or deletions, premature termination, tandem repeat, nonstop, and missense mutations). A correlation was observed between temocillin MICs and efflux rate of N-phenyl-1-naphthylamine (MexAB-OprM fluorescent substrate) and extracellular exopolysaccharide abundance (contributing to a mucoid phenotype). OpdK or OpdF anion-specific porins expression decreased temocillin MIC by ~1 two-fold dilution only. Contrarily to the common assumption that temocillin is inactive on P. aeruginosa, we show here clinically-exploitable MICs on a non-negligible proportion of CF isolates, explained by a wide diversity of mutations in mexA and/or mexB. In a broader context, this work contributes to increase our understanding of MexAB-OprM functionality and help delineating how antibiotics interact with MexA and MexB.

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.

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.

High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations.

High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine-glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg ß-naphthylamide (PAßN). ß-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64-128/16-32 mg/L and 128/128-256 mg/L in CF and HAP isolates, respectively; PAßN reduced meropenem MICs to 4-16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted.

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.

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.

Characterisation of a collection of Streptococcus pneumoniae isolates from patients suffering from acute exacerbations of chronic bronchitis: in vitro susceptibility to antibiotics and biofilm formation in relation to antibiotic efflux and serotypes/serogroups.

The correlation between Streptococcus pneumoniae serotypes, biofilm production, antibiotic susceptibility and drug efflux in isolates from patients suffering from acute exacerbations of chronic bronchitis (AECB) remains largely unexplored. Using 101 isolates collected from AECB patients for whom partial (n=51) or full (n=50) medical details were available, we determined serotypes (ST)/serogroups (SG) (Quellung reaction), antibiotic susceptibility patterns [MIC (microdilution) using EUCAST and CLSI criteria] and ability to produce biofilm in vitro (10-day model; crystal violet staining). The majority of patients were 55-75 years old and <5% were vaccinated against S. pneumoniae. Moreover, 54% showed high severity scores (GOLD 3-4), and comorbidities were frequent including hypertension (60%), cancer (24%) and diabetes (20%). Alcohol and/or tobacco dependence was >30%. Isolates of SG6-11-15-23, known for large biofilm production and causing chronic infections, were the most prevalent (>15% each), but other isolates also produced biofilm (SG9-18-22-27 and ST8-20 being most productive), except SG7, SG29 and ST5 (<2% of isolates each). Resistance (EUCAST breakpoints) was 8-13% for amoxicillin and cefuroxime, 35-39% for macrolides, 2-8% for fluoroquinolones and 2% for telithromycin. ST19A isolates showed resistance to all antibiotics, ST14 to all except moxifloxacin, and SG9 and SG19 to all except telithromycin, moxifloxacin and ceftriaxone (SG19 only). Solithromycin and telithromycin MICs were similar. No correlation was observed between biofilm production and MIC or efflux (macrolides, fluoroquinolones). S. pneumoniae serotyping may improve AECB treatment by avoiding antibiotics with predictable low activity, but it is not predictive of biofilm production.

Study of macrophage functions in murine J774 cells and human activated THP-1 cells exposed to oritavancin, a lipoglycopeptide with high cellular accumulation.

Oritavancin, a lipoglycopeptide antibiotic in development, accumulates to high levels in the lysosomes of eukaryotic cells. We examined specific functions of macrophages (phagocytic capacity, lysosomal integrity, metabolic activity, and production of reactive oxygen species [ROS]) in correlation with the cellular accumulation of the drug, using J774 mouse macrophages and THP-1 human monocytes differentiated into macrophages using phorbol 12-myristate 13-acetate. Oritavancin did not affect Pseudomonas aeruginosa phagocytosis, lysosomal integrity, or metabolic activity in cells incubated for 3 h with extracellular concentrations ranging from 5 to 50 µg/ml. At extracellular concentrations of ≥25 µg/ml, oritavancin reduced latex bead phagocytosis by approximately 50% and doubled ROS production in J774 macrophages only. This may result from the fact that the cellular accumulation of oritavancin was 15 times higher in J774 cells than in activated THP-1 cells at 3 h. Human pharmacokinetic studies estimate that the concentration of oritavancin in alveolar macrophages could reach approximately 560 µg/ml after administration of a cumulative dose of 4 g, which is below the cellular concentration needed in the present study to impair latex bead phagocytosis (1,180 µg/ml) or to stimulate ROS production (15,000 µg/ml) by J774 cells. The data, therefore, suggest that, in spite of its substantial cellular accumulation, oritavancin is unlikely to markedly affect macrophage functions under the conditions of use investigated in current phase III trials (a single dose of 1,200 mg).

Pharmacological characterization of 7-(4-(Piperazin-1-yl)) ciprofloxacin derivatives: antibacterial activity, cellular accumulation, susceptibility to efflux transporters, and intracellular activity.

PURPOSE: To evaluate pharmacological properties (antibacterial activity; accumulation in phagocytic cells; activity against intracellular bacteria; susceptibility to fluoroquinolone efflux transporters) of ciprofloxacin derivatives modified at C-7 of the piperazine ring. METHODS: N-acetyl- (1), N-benzoyl- (2), N-ethyl- (3), and N-benzyl- (4) ciprofloxacin were synthesized. MICs against Escherichia coli and Staphylococcus aureus were determined following CLSI guidelines. Cellular accumulation, subcellular distribution, and intracellular activity (towards S. aureus and Listeria monocytogenes) were determined in J774 mouse macrophages. Efflux in bacteria (NorA [S. aureus], Lde [L. monocytogenes]) and in macrophages (Mrp4) was assessed using the corresponding inhibitors reserpine and gemfibrozil, respectively. RESULTS: All derivatives were active, though less than ciprofloxacin. 2 and 3 accumulated 2-3 fold more than ciprofloxacin in mouse macrophages but remained substrates for efflux by Mrp4. 4 was insensitive to NorA and Lde, accumulated approx 50-fold more than ciprofloxacin in macrophages, was barely affected by Mrp4, localized in the soluble fraction of cells, and was equipotent to ciprofloxacin against intracellular bacteria. CONCLUSIONS: Benzyl substitution at C7 markedly affects the pharmacological profile of ciprofloxacin with respect to recognition by efflux transporters and cellular accumulation. N-benzyl-ciprofloxacin may serve as basis for designing molecules with higher intrinsic activity while remaining poorly susceptible to efflux.

Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC).

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.

Antibiotic activity against small-colony variants of Staphylococcus aureus: review of in vitro, animal and clinical data.

The pathogen Staphylococcus aureus uses various strategies for persisting in the host, among which switching to a small-colony variant (SCV) phenotype is of particular biological and therapeutic significance. Phenotypically, SCVs are characterized by a slow growth rate, atypical colony morphology and unusual biochemical features, constituting a real challenge for identification by the clinical microbiology laboratory. Their metabolic defects also alter their susceptibility to antibiotics, which, combined with the ability to survive intracellularly and, for some strains, to form biofilms, largely contributes to therapeutic failures. This paper reviews the available literature on antibiotic activity against SCVs of S. aureus in vitro, in animal models and in clinics. In vitro, aminoglycosides and antifolate agents show high MICs for electron-transport-defective and thymidine-dependent SCVs, respectively. The other antibiotic classes usually show MICs comparable to those measured for the parental strains, but they are less bactericidal. Intracellularly, auxotrophs for thymidine, haemin or menadione show contrasting behaviours with respect to their response to antibiotics, resulting from differences in their intracellular fate. In animal models, SCVs often persist in various locations, including metastatic ones, in spite of the administration of active antibiotics. In healthcare, several case reports mention the selection of SCVs after prolonged administration of not only aminoglycosides and antifolate agents, but also several other antibiotic classes. Apparent eradication requires several weeks or even months of aggressive polytherapy combined, whenever possible, with surgical intervention. Further research is thus warranted for optimizing the treatment of infections caused by SCVs.

Stability and compatibility of vancomycin for administration by continuous infusion.

BACKGROUND: Vancomycin is increasingly used by continuous infusion, but few specific data are available about stability under practical conditions of preparation and use, and compatibility with other intravenous drugs commonly used in the routine hospital setting. METHODS: Vancomycin stability [defined as recovery ≥ 93% of the original content (validated HPLC assay)] was examined throughout the whole process of centralized preparation, storage and use in the ward by infusion for up to 48 h, with allowances for deviations from recommended practice [exposure to high temperature; use of concentrated solutions (up to 83 g/L)]. Compatibility was assessed by mimicking co-administration in a single line via Y-shaped connectors with contact of 1 h at 25°C, followed by visual inspection (professional viewer), detection of particulate matter (particle analyser) and HPLC assay of vancomycin. RESULTS: Vancomycin was stable during the whole process and also during 72 h exposure of concentrated solutions at temperatures up to 37°C. Major incompatibilities were seen with ß-lactams (temocillin, piperacillin/tazobactam, ceftazidime, imipenem, cefepime and flucloxacillin) and moxifloxacin, but not with ciprofloxacin, aminoglycosides and macrolides. Propofol, valproic acid, phenytoin, theophylline, methylprednisolone and furosemide were also incompatible, whereas ketamine, sufentanil, midazolam, morphine, piritramide, nicardipine, urapidil, dopamine, dobutamine and adrenaline were compatible. No effect or incompatibility with N-acetyl-cysteine or amino acid solutions was detected. CONCLUSIONS: Centralized preparation of vancomycin and its use by continuous infusion in wards is safe concerning stability, but careful attention must be paid to incompatibilities. Several drugs (including all ß-lactams) require distinct intravenous lines or appropriate procedures to avoid undue contact.

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.

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 H(2)O(2). 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 H(2)O(2). 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.

Increased susceptibility of Pseudomonas aeruginosa to macrolides and ketolides in eukaryotic cell culture media and biological fluids due to decreased expression of oprM and increased outer-membrane permeability.

BACKGROUND: Macrolides show high minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa when tested in recommended media (cation-adjusted Muller-Hinton broth [CA-MHB]). Nevertheless, azithromycin is successfully used in cystic fibrosis patients, supposedly because of "nonantibiotic effects." METHODS: CA-MHB and Roswell Park Memorial Institute (RPMI) 1640 medium (used for growing eukaryotic cells) were compared for measuring azithromycin MICs (with or without Phe-Arg-ß-naphthylamide [PAßN], an efflux inhibitor), [(14)C]-clarithromycin accumulation, azithromycin-induced protein synthesis inhibition, oprM (encoding the outer-membrane protein coupled with MexAB and MexXY efflux systems) expression, outer-membrane permeability (tested with 1-N-phenylnaphthylamine and nitrocefin), and synergy (determined by checkerboard assay) between azithromycin and outer-membrane disrupting agents. Key experiments were repeated with CA-MHB supplemented with serum, mouse bronchoalveolar lavage fluid, other macrolides, and other gram-negative bacteria. RESULTS: Azithromycin MICs were ≥128 mg/L in CA-MHB, compared with 1-16 mg/L in RPMI 1640 medium, CA-MHB supplemented with serum, or bronchoalveolar lavage fluid (repeated for RPMI 1640 medium with clarithromycin, other macrolides, and other gram-negative bacteria). [(14)C]-clarithromycin accumulation was 2.2-fold higher in RPMI 1640 medium, compared with CA-MHB. Inhibition of >95% of protein synthesis was obtained with azithromycin at 16 mg/L in RPMI 1640 medium, compared with >512 mg/L in CA-MHB. Strains not expressing oprM showed an MIC of 4 mg/L in CA-MHB. PAßN decreased MICs in CA-MHB but not in RPMI 1640 medium. Real-time polymerase chain reaction showed downregulation of oprM by azithromycin in RPMI 1640 medium. Outer-membrane permeability was 3-4.5 times higher in RPMI 1640 medium or bronchoalveolar lavage fluid, compared with CA-MHB. Azithromycin combined with outer-membrane disrupting agents were synergistic in CA-MHB but indifferent in RPMI 1640 medium. CONCLUSIONS: Macrolides show antimicrobial activity against P. aeruginosa in eukaryotic media through increased uptake and reduced efflux. These data may help explain the clinical efficacy of macrolides against pseudomonal infections.