Molecular models of human P-glycoprotein in two different catalytic states

P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Increased azithromycin susceptibility of multidrug-resistant gram-negative bacteria on RPMI-1640 agar assessed by disk diffusion testing

Increasing antibiotic resistances and a lack of new antibiotics render the treatment of Gram-negative bacterial infections increasingly difficult. Therefore, additional approaches are being investigated. Macrolides are not routinely used against Gram-negative bacteria due to lack of evidence of in vitro effectiveness. However, it has been shown that Pseudomonas spp. are susceptible to macrolides in liquid RPMI-1640 and clinical data suggest improvement in patients' outcomes. So far, these findings have been hardly applicable to the clinical setting due to lack of routine low-complexity antimicrobial susceptibility testing (AST) for macrolides. We therefore optimized and compared broth microdilution and disk diffusion AST. Multidrug-resistant Gram-negative bacteria (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa) were tested for azithromycin susceptibility by disk diffusion and broth microdilution in Mueller-Hinton and RPMI-1640 media. Azithromycin susceptibility of Enterobacteriaceae and a subgroup of P. aeruginosa increased significantly on RPMI-1640 agar compared to Mueller-Hinton agar. Further, a significant correlation (Kendall, τ, p) of zone diameters and minimal inhibitory concentrations (MICs) was found on RPMI-1640 agar for E. coli (-0.4279, 0.0051), E. cloacae (-0.3783, 0.0237) and P. aeruginosa (-0.6477, <0.0001). Performing routine disk diffusion AST on RPMI-1640 agar may lead to the identification of additional therapeutic possibilities for multidrug-resistant bacterial infections in the routine clinical diagnostic setting

Penicillin-binding Proteins (PBP) and Lmo0441 (a PBP-like protein) play a role in Beta-lactam sensitivity of Listeria monocytogenes

While seven penicillin-binding proteins (PBPs) or PBP-like proteins have been identified either by radiolabelled penicillin binding studies or genomic analysis, only PBP3 has been considered of interest for Beta-lactams activity against Listeria monocytogenes. Herein we reveal that both PBP4 and Lmo0441 (a PBP-like protein) play a direct role in cephalosporin activity in L. monocytogenes while PBP4 additionally has a protective affect against both penicillin and carbapenem

Cellular pharmacokinetics of telavancin, a novel lipoglycopeptide antibiotic, and analysis of lysosomal changes in cultured eukaryotic cells (J774 mouse macrophages and rat embryonic fibroblasts)

Background Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin). Methods J774 macrophages and rat embryo fibroblasts were exposed for up to 24 and 72 h to telavancin (5-90 mg/L). The following studies were performed: measurement of (14)C-labelled telavancin cellular uptake and subcellular distribution (cell fractionation), determination of pericellular membrane integrity (lactate dehydrogenase release), electron microscopy with morphometric analysis of changes in lysosome size and determination of total phospholipid and cholesterol content. Results The uptake of telavancin proceeded linearly as a function of time and concentration in both cell types (clearance rate of approximately 10 mL/g of protein/h). Efflux (macrophages) was approximately 5.7-fold slower. Telavancin subcellular distribution was superimposable on that of a lysosomal marker (N-acetyl-beta-hexosaminidase). It did not cause an increase in the release of lactate dehydrogenase and did not induce significant increases in total phospholipid or cholesterol content. It caused only mild morphological lysosomal alterations (similar to vancomycin and much less than oritavancin by morphometric analysis). Conclusions Telavancin is taken up by eukaryotic cells and localizes in lysosomes, causing mild morphological alterations without evidence of lipid metabolism alterations. These data support our observations that telavancin is active against intracellular S. aureus

Antibacterial activity of 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol against antibiotic-resistant strains of diverse bacterial pathogens, biofilms and in pre-clinical infection models

We recently described the novel anti-persister compound 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol (SPI009), capable of directly killing persister cells of the Gram-negative pathogen Pseudomonas aeruginosa. This compound also shows antibacterial effects against non-persister cells, suggesting that SPI009 could be used as an adjuvant for antibacterial combination therapy. Here, we demonstrate the broad-spectrum activity of SPI009, combined with different classes of antibiotics, against the clinically relevant ESKAPE pathogens Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterococcus faecium and Burkholderia cenocepacia and Escherichia coli. Importantly, SPI009 re-enabled killing of antibiotic-resistant strains and effectively lowered the required antibiotic concentrations. The clinical potential was further confirmed in biofilm models of P. aeruginosa and S. aureus where SPI009 exhibited effective biofilm inhibition and eradication. Caenorhabditis elegans infected with P. aeruginosa also showed a significant improvement in survival when SPI009 was added to conventional antibiotic treatment. Overall, we demonstrate that SPI009, initially discovered as an anti-persister molecule in P. aeruginosa, possesses broad-spectrum activity and is highly suitable for the development of antibacterial combination therapies in the fight against chronic infections

Cellular pharmacokinetics and intracellular activity against Listeria monocytogenes and Staphylococcus aureus of chemically modified and nanoencapsulated gentamicin

OBJECTIVES: The aim of this study was to investigate different hydrophobic gentamicin formulations [gentamicin-bis(2-ethylhexyl) sulfosuccinate (GEN-AOT), microstructured GEN-AOT (PCA GEN-AOT) and GEN-AOT-loaded poly(lactide-co-glycolide) acid (PLGA) nanoparticles (NPs)] in view of improving its therapeutic index against intracellular bacteria. The intracellular accumulation, subcellular distribution and intracellular activity of GEN-AOT and NPs in different monocytic-macrophagic cell lines were studied. METHODS: Human THP-1 and murine J774 phagocytic cells were incubated with GEN-AOT formulations at relevant extracellular concentrations [from 1× MIC to 18 mg/L (human C(max))], and their intracellular accumulation, subcellular distribution and toxicity were evaluated and compared with those of conventional unmodified gentamicin. Intracellular activity of the formulations was determined against bacteria showing different subcellular localizations, namely Staphylococcus aureus (phagolysosomes) and Listeria monocytogenes (cytosol). RESULTS: GEN-AOT formulations accumulated 2-fold (GEN-AOT) to 8-fold (GEN-AOT NPs) more than gentamicin in phagocytic cells, with a predominant subcellular localization in the soluble fraction (cytosol) and with no significant cellular toxicity. NP formulations allowed gentamicin to exert its intracellular activity after shorter incubation times and/or at lower concentrations. With an extracellular concentration of 10× MIC, a 1 log(10) decrease in S. aureus intracellular inoculum was obtained after 12 h instead of 24 h for NPs versus free gentamicin, and a static effect was observed against L. monocytogenes at 24 h with NPs, while free gentamicin was ineffective. CONCLUSIONS: GEN-AOT formulations yielded a high cellular accumulation, especially in the cytosol, which resulted in improved efficacy against both intracellular S. aureus and L. monocytogenes

The Putative De-N-acetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1–3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5–7 fold) and mexX (3.6–5.4 fold) in the parental strain but to a lower extent (3–4-fold for gyrA and 1.8–2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target

Correlation between cytotoxicity induced by Pseudomonas aeruginosa clinical isolates from acute infections and IL-1β secretion in a model of human THP-1 monocytes

Type III secretion system (T3SS) in Pseudomonas aeruginosa is associated with poor clinical outcome in acute infections. T3SS allows for injection of bacterial exotoxins (e.g. ExoU or ExoS) into the host cell, causing cytotoxicity. It also activates the cytosolic NLRC4 inflammasome, activating caspase-1, inducing cytotoxicity and release of mature IL-1β, which impairs bacterial clearance. In addition, flagellum-mediated motility has been suggested to also modulate inflammasome response and IL-1β release. Yet the capacity of clinical isolates to induce IL-1β release and its relation with cytotoxicity have never been investigated. Using 20 clinical isolates from acute infections with variable T3SS expression levels and human monocytes, our aim was to correlate IL-1β release with toxin expression, flagellar motility and cytotoxicity. ExoU-producing isolates caused massive cell death but minimal release of IL-1β, while those expressing T3SS but not ExoU (i.e. expressing ExoS or no toxins) induced caspase-1 activation and IL-1β release, the level of which was correlated with cytotoxicity. Both effects were prevented by a specific caspase-1 inhibitor. Flagellar motility was not correlated with cytotoxicity or IL-1β release. No apoptosis was detected. Thus, T3SS cytotoxicity is accompanied by a modification in cytokine balance for P. aeruginosa clinical isolates that do not express Exo