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ABSTRACT: 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.
Journal of Antimicrobial Chemotherapy 03/2013; · 5.07 Impact Factor
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ABSTRACT: BACKGROUND: Ceftaroline fosamil is approved for treatment of acute bacterial skin and skin structure infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We examined the activity of its active metabolite (ceftaroline) against intracellular forms of S. aureus in comparison with vancomycin, daptomycin and linezolid. METHODS: Two methicillin-susceptible S. aureus (MSSA) and 11 MRSA strains with ceftaroline MICs from 0.125 to 2 mg/L [two strains vancomycin- and one strain linezolid-resistant (EUCAST interpretative criteria); VISA and cfr+] were investigated. The activity was measured in broth and after phagocytosis by THP-1 monocytes in concentration-dependent experiments (24 h of incubation) to determine: (i) relative potencies (EC(50)) and static concentrations (C(s)) (mg/L and × MIC); and (ii) relative activities at human C(max) (E(C)(max)) and maximal relative efficacies (E(max)) (change in log(10) cfu compared with initial inoculum). Ceftaroline stability and cellular accumulation (at 24 h) were measured by mass spectrometry. RESULTS: Ceftaroline showed similar activities in broth and in monocytes compared with vancomycin, daptomycin and linezolid, with no impact of resistance mechanisms to vancomycin or linezolid. For all four antibiotics, intracellular E(C)(max) and E(max) were considerably lower than in broth (∼0.5 log(10) versus 4-5 log(10) cfu decrease), but the EC(50) and C(s) showed comparatively little change (all values between ∼0.3 and ∼6× MIC). The mean cellular to extracellular ceftaroline concentration ratios (20 mg/L; 24 h) were 0.66 ± 0.05 and 0.90 ± 0.36 in uninfected and infected cells, respectively. CONCLUSION: In vitro, ceftaroline controls the growth of intracellular MRSA to an extent similar to that of vancomycin, linezolid and daptomycin for strains with a ceftaroline MIC ≤2 mg/L.
Journal of Antimicrobial Chemotherapy 11/2012; · 5.07 Impact Factor
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ABSTRACT: In a previous paper (Garcia et al, 2012, 56 (7): 3700-3711), we studied the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the parental COL MRSA strain and the pharmacodynamic profile of 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. Post-phagocytosis 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 non-activated cells. This effect was inhibited when cells were incubated with N-acetylcysteine (scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been pre-exposed to H(2)O(2). In contrast, the intracellular potency of oritavancin was not different in activated and non-activated cells and was not decreased by the addition of N-acetylcysteine, regardless the phenotype of the strains. 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.
Antimicrobial Agents and Chemotherapy 09/2012; · 4.84 Impact Factor
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ABSTRACT: 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.
Clinical Infectious Diseases 04/2012; 54 Suppl 3:S229-32. · 9.15 Impact Factor
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ABSTRACT: Fluoroquinolones enter eukaryotic cells but the correlation between cellular accumulation and activity remains poorly established. Gemifloxacin is known to accumulate to a larger extent than most other fluoroquinolones in tissues. Using murine J774 macrophages and human THP-1 monocytes, we show that gemifloxacin accumulates more than ciprofloxacin and even moxifloxacin. Whilst showing indistinguishable kinetics of accumulation in J774 macrophages, gemifloxacin was released at an approximately two-fold slower rate than ciprofloxacin and its release was only partial. Gemifloxacin was also a weaker substrate than ciprofloxacin for the efflux transporter Mrp4 active in J774 macrophages. In cells infected with Listeria monocytogenes or Staphylococcus aureus (typical cytoplasmic and phagolysosomal organisms, respectively), gemifloxacin was equipotent to moxifloxacin and ciprofloxacin in concentration-dependent experiments if data are normalised based on the minimum inhibitory concentration (MIC) in broth. Thus, larger cellular concentrations of gemifloxacin than of moxifloxacin or ciprofloxacin were needed to obtain a similar target effect. Fractionation studies showed a similar subcellular distribution for all three fluoroquinolones, with approximately two-thirds of the cell-associated drug recovered in the soluble fraction (cytosol). These data suggest that cellular accumulation of fluoroquinolones is largely a self-defeating process as far as activity is concerned, with the intracellular drug made inactive in proportion to its accumulation level. Whilst these observations do not decrease the intrinsic value of fluoroquinolones for the treatment of intracellular infections, they indicate that ranking fluoroquinolones based on cell accumulation data without measuring the corresponding intracellular activity may lead to incorrect conclusions regarding their real potential.
International journal of antimicrobial agents 09/2011; 38(3):249-56. · 3.03 Impact Factor
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ABSTRACT: Finafloxacin, an 8-cyano-substituted fluoroquinolone, expresses enhanced activity at acidic pH and is less susceptible to several fluoroquinolone resistance determinants. In this study, we compared finafloxacin and ciprofloxacin for (i) activity against ciprofloxacin-susceptible and -resistant Staphylococcus aureus as well as wild-type and Lde efflux-positive (Lde+) Listeria monocytogenes, (ii) accumulation in THP-1 macrophages and (iii) intracellular activity towards phagocytised S. aureus, L. monocytogenes and Legionella pneumophila (developing in acidic, neutral and mildly acidic environments, respectively), using a pharmacological approach assessing drug potencies and maximal relative efficacies (E(max)). Finafloxacin minimum inhibitory concentrations (MICs) were two-fold lower than those of ciprofloxacin against meticillin-susceptible S. aureus ATCC 25923, were only modestly increased in an isogenic strain overexpressing NorA and were ≤0.25 mg/L for community-acquired meticillin-resistant S. aureus. No loss of activity was seen in Lde+ L. monocytogenes. An acidic pH decreased the MIC of finafloxacin and increased that of ciprofloxacin both for S. aureus and L. monocytogenes, in parallel with corresponding changes in drug accumulation (tested with S. aureus ATCC 25923 only). Finafloxacin accumulated less than ciprofloxacin in THP-1 cells, but the situation was reversed by exposure of cells to acid pH. In S. aureus-infected cells, acid pH increased the potency of finafloxacin without change of E(max), whilst decreasing the potency and the maximal relative efficacy of ciprofloxacin (less negative E(max)). Finafloxacin was more potent and showed larger E(max) than ciprofloxacin against phagocytised L. pneumophila, but was less potent against phagocytised L. monocytogenes. Finafloxacin appears to be an acid-pH-favoured antibiotic that may find useful applications in infections where the local pH is low.
International journal of antimicrobial agents 07/2011; 38(1):52-9. · 3.03 Impact Factor
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ABSTRACT: Emergence of multidrug-resistant Staphylococcus aureus has triggered a reassessment of fusidic acid (CEM-102, sodium fusidate).
Fusidic acid was examined for (1) activity against recent methicillin-resistant S. aureus (MRSA) isolates; (2) modulation of activity by acidic pH; and (3) accumulation by phagocytic cells and intracellular activity against methicillin-susceptible S. aureus (MSSA) and MRSA.
About 96% of strains (N = 94) were susceptible (European Committee on Antimicrobial Susceptibility Testing breakpoint [≤ 1 mg/L]). Activity was enhanced at pH 5.5 (6 dilutions decrease for minimum inhibitory concentration) in parallel with an increase of drug bacterial accumulation (opposite effects for clindamycin; linezolid remained unaffected). Fusidic acid accumulated in THP-1 cells (about 5.5 fold), with further accumulation at pH 5.5 vs pH 7.4. The intracellular activity of Fusidic acid was similar to that of clindamycin and linezolid (maximal relative activity, 0.4-0.6 log(10) colony-forming unit decrease). No cross-resistance to vancomycin or daptomycin was observed.
Fusidic acid is active against S. aureus in broth as well as intracellularly, with no cross-resistance to other antibiotics.
Clinical Infectious Diseases 06/2011; 52 Suppl 7:S493-503. · 9.15 Impact Factor
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ABSTRACT: 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 log(2) 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 log(2) 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.
Antimicrobial Agents and Chemotherapy 02/2011; 55(2):649-58. · 4.84 Impact Factor
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ABSTRACT: 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 log(10) 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.
Antimicrobial Agents and Chemotherapy 01/2011; 55(4):1443-52. · 4.84 Impact Factor
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ABSTRACT: Co-trimoxazole, clindamycin and linezolid are used to treat community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, but little is known about intracellular activity. Moxifloxacin is active against intracellular methicillin-susceptible S. aureus (MSSA), but CA-MRSA has not been studied.
We used 12 clinical CA-MRSA, 1 MSSA overexpressing norA and 2 hospital-acquired MRSA (moxifloxacin MICs: 0.03 to 4 mg/L). Activity was assessed in broth and after phagocytosis by THP-1 macrophages or keratinocytes {concentration-dependent experiments [24 h of incubation] to determine relative potencies [EC(50)], static concentrations [C(s)] and maximal relative efficacies [E(max) (change in log(10) cfu compared with initial inoculum)] and time-dependent experiments [0-72 h] at human C(max)}.
Concentration-dependent experiments: in broth, EC(50) and C(s) were correlated with the MIC for all antibiotics, but moxifloxacin achieved significantly (P < 0.01) greater killing (more negative E(max)) than the comparators; and in THP-1 cells and keratinocytes, moxifloxacin acted more slowly but still reached a near bactericidal effect (2 to 3 log(10) cfu decrease) at 24 h with unchanged EC(50) and C(s) as long as its MIC was ≤0.125 mg/L (recursive partitioning analysis). Clindamycin and linezolid were static, and co-trimoxazole was unable to suppress the intracellular growth of CA-MRSA. At human C(max) in broth, moxifloxacin killed more rapidly and more extensively (≥5 log(10) cfu decrease at 10 h) than clindamycin (4 log(10) cfu at 48 h) or co-trimoxazole and linezolid (1-2 log(10) cfu at 72 h).
Moxifloxacin is active against both extracellular and intracellular CA-MRSA if the MIC is low, and is more effective than clindamycin, co-trimoxazole and linezolid.
Journal of Antimicrobial Chemotherapy 12/2010; 66(3):596-607. · 5.07 Impact Factor
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ABSTRACT: Epicatechin gallate (ECg) sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin and other beta-lactam agents; it also reduces the secretion of virulence-associated proteins, prevents biofilm formation, and induces gross morphological changes in MRSA cells without compromising the growth rate. MRSA is resistant to oxacillin because of the presence of penicillin-binding protein 2a (PBP2a), which allows peptidoglycan synthesis to continue after oxacillin-mediated acylation of native PBPs. We show that ECg binds predominantly to the cytoplasmic membrane (CM), initially decreasing the fluidity of the bilayer, and induces changes in gene expression indicative of an attempt to preserve and repair a compromised cell wall. On further incubation, the CM is reorganized; the amount of lysylphosphatidylglycerol is markedly reduced, with a concomitant increase in phosphatidylglycerol, and the proportion of branched chain fatty acids increases, resulting in a more fluid structure. We found no evidence that ECg modulates the enzymatic activity of PBP2a through direct binding to the protein but determined that PBP2 is delocalized from the FtsZ-anchored cell wall biosynthetic machinery at the septal division site following intercalation into the CM. We argue that many features of the ECg-induced phenotype can be explained by changes in the fluid dynamics of the CM.
Journal of Biological Chemistry 07/2010; 285(31):24055-65. · 4.77 Impact Factor
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ABSTRACT: Treatment of chronic or recurrent Staphylococcus aureus infections may require using antibiotics with activity against intracellular multiresistant organisms. Quinupristin/dalfopristin (3:7) has been examined in this context.
Quinupristin and dalfopristin were used separately or mixed. Strains used were: (i) methicillin-susceptible and -resistant S. aureus (MSSA and MRSA); (ii) one vat(B) MSSA and msr(A/B) MRSA; (iii) erm(A)+ [MSSA, MRSA, vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA)]; and (iv) one erm(A/B)+ cfr+ MRSA resistant to quinupristin, dalfopristin and their combination. Assessment of activity was determined by: (i) MICs (CLSI method); and (ii) concentration-response curves in broth and after phagocytosis by THP-1 macrophages, with descriptors of the model (Emin) and the pharmacodynamic response [maximal relative efficacy (Emax), relative potency (EC50) and apparent static concentration (Cstatic)].
erm(A)-positive strains were all susceptible to quinupristin/dalfopristin (except strain CM05), with MICs not adversely influenced by acid pH or by the MRSA, VISA or VRSA character of the strain. In concentration-response experiments, quinupristin/dalfopristin showed similar patterns for all strains (except strain CM05), with a >3 log10 cfu decrease in broth and a 1.3 [erm(A) strain] to 2.6 [fully susceptible, vat(B) and msr(A/B) strains] log10 cfu decrease for intracellular bacteria at the maximal extracellular concentration tested (25 mg/L). Maximal extracellular and intracellular activity was obtained for a quinupristin/dalfopristin ratio of 3:7. For strain CM05, quinupristin/dalfopristin was static in all conditions.
Based on historical comparisons with rifampicin, fluoroquinolones, lipoglycopeptides and other antistaphylococcal drugs with a large accumulation in eukaryotic cells, quinupristin/dalfopristin appears to be one of the most active antibiotics against intracellular S. aureus studied in this model so far, largely irrespective of its resistance phenotype.
Journal of Antimicrobial Chemotherapy 04/2010; 65(6):1228-36. · 5.07 Impact Factor
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ABSTRACT: 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 approximately 60% of the drug localized in the cytosol and approximately 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 ( approximately 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.
Antimicrobial Agents and Chemotherapy 04/2010; 54(6):2549-59. · 4.84 Impact Factor
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ABSTRACT: 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 degrees C but was further increased in medium with low serum content. Subcellular fractionation studies demonstrated a dual subcellular distribution for radezolid, with approximately 60% of the drug colocalizing to the cytosol and approximately 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.
Antimicrobial Agents and Chemotherapy 04/2010; 54(6):2540-8. · 4.84 Impact Factor
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ABSTRACT: In Staphylococcus aureus, rsbU down-regulates agr and stimulates production of staphyloxanthin (STX), an antioxidant 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 human umbilical vein endothelial cells (more so for the latter) without change in subcellular localization and that inhibition of STX biosynthesis markedly reduces intracellular growth of the rsbU(+) strain (and of clinical isolates, including USA300; tested with macrophages only) without affecting internalization. Thus, rsbU is important for uptake and for STX biosynthesis and is critical for intracellular multiplication of S. aureus.
The Journal of Infectious Diseases 11/2009; 200(9):1367-70. · 6.41 Impact Factor
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ABSTRACT: Optimal treatment of infections caused by Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila requires antibiotics with intracellular activity. Linezolid accumulates poorly within cells. Torezolid (TR-700) is a novel methyltetrazolyl oxazolidinone with potentially different cellular pharmacokinetic properties. Our aim was to examine the accumulation and intracellular activities of torezolid in this context.
Measurement of torezolid cell content and antibacterial activity in comparison with linezolid using human macrophages (THP-1) and human endothelial cells [human umbilical vein endothelial cells (HUVECs)], applying models allowing for the quantitative evaluation of the pharmacodynamics of antibiotics towards intracellular bacteria.
Torezolid accumulated rapidly in THP-1 macrophages, reaching a stable intracellular to extracellular ratio of approximately 10 (compared with approximately 1-2 for linezolid) within 15 min. On a weight concentration basis (mg/L), torezolid was approximately 5- to 10-fold more potent intracellularly (lower concentration needed to achieve a bacteriostatic effect) than linezolid against phagocytosed S. aureus, L. monocytogenes and L. pneumophila, with no change in maximal efficacy (approximately 1 log(10) reduction of the original, post-phagocytosis inoculum). When drugs were compared at equipotent concentrations (multiples of the MIC), no difference was seen between linezolid and torezolid, but the higher potency of torezolid allowed control of intracellular infections caused by linezolid-resistant S. aureus.
Torezolid exerts intracellular activity at lower extracellular concentrations than linezolid because of its greater potency independent of its greater intracellular accumulation. This may confer an advantage to torezolid in vivo if the drug can be used at dosages creating serum concentrations similar to those achieved with linezolid.
Journal of Antimicrobial Chemotherapy 09/2009; 64(5):1035-43. · 5.07 Impact Factor
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ABSTRACT: 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 < or = 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.
Antimicrobial Agents and Chemotherapy 07/2009; 53(9):3734-43. · 4.84 Impact Factor
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ABSTRACT: Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, beta-lactams show activity against intracellular methicillin (methicillin)-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 beta-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 (E(max)), relative potency (the concentration causing a reduction of the inoculum halfway between E(0) and E(max) [EC(50)]), and static concentration (C(s))} were measured. All strains showed sigmoidal dose-responses, with E(max) being about a 1 log(10) CFU decrease from the postphagocytosis inoculum, and EC(50) and C(s) being 0.2 to 0.3x and 0.6 to 0.9x 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.
Antimicrobial Agents and Chemotherapy 04/2009; 53(6):2289-97. · 4.84 Impact Factor
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ABSTRACT: 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 beta-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 (0.01x to 100x 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], 6.09x 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 beta-lactams after phagocytosis is independent of the strain and the types of cells but varies between beta-lactams.
Antimicrobial Agents and Chemotherapy 08/2008; 52(8):2797-805. · 4.84 Impact Factor
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ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is a global scourge, and treatment options are becoming limited. The MRSA phenotype reverts to that of beta-lactam-sensitive S. aureus when bacteria are grown at pH 5.0 in broth and, more importantly from a medical perspective (protracted, relapsing infections), after phagocytosis by macrophages, where the bacteria thrive in the acidic environment of phagolysosomes. The central factor for the MRSA phenotype is the function of the penicillin-binding protein (PBP) 2a, which maintains transpeptidase activity while being poorly inhibited by beta-lactams because of a closed conformation of its active site. We document herein by binding, acylation/deacylation kinetics, and circular dichroism spectroscopy with purified PBP 2a that at acidic pH (i) beta-lactams interact with PBP 2a more avidly; (ii) the non-covalent pre-acylation complex exhibits a lower dissociation constant and an increased rate of acyl-enzyme formation (first-order rate constant) without change in hydrolytic deacylation rate; and (iii) PBP 2a undergoes a conformational change in the presence of the antibiotic consistent with the opening of the active site from the closed conformation. These observations argue that PBP 2a most likely evolved for its physiological function at pH 7 or higher by adopting a closed conformation, which is not maintained at acidic pH. Although at the organism level the effect of acidic pH on other biological processes in MRSA could not be discounted, our report should provide the impetus for closer examination of the properties of PBP 2a at low pH and thereby identifying novel points of intervention in combating this problematic organism.
Journal of Biological Chemistry 06/2008; 283(19):12769-76. · 4.77 Impact Factor
