The bacterial cell envelope as delimiter of anti-infective bioavailability – An in vitro permeation model of the Gram-negative bacterial inner membrane

Graef, Florian; Vukosavljevic, Branko; Michel, Jean‐Philippe; Wirth, Marius; Ries, Oliver; Rossi, Chiara; Windbergs, Maike; Rosilio, Véronique; Ducho, Christian; Gordon, Sarah; Lehr, Claus Michael

doi:10.1016/j.jconrel.2016.10.018

Cited by 27 publications

(32 citation statements)

References 41 publications

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“…Gram-negative bacteria are intrinsically more resistant to antimicrobial compounds than Gram-positive organisms due to (i) the requirement for penetration of both the negatively charged outer and lipophilic inner membranes, (ii) stringent transcriptional and post-transcriptional control of porin expression in the outer membrane, and (iii) expression of multi-drug efflux pumps [4–6]. As a result of these barriers to the entry of small molecules into cells, many bioactive compounds discovered in cell-free, targeted screens do not retain their activity in whole cell assays [7].…”

Section: Introductionmentioning

confidence: 99%

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae

Liao

Mevers

et al. 2018

PLoS ONE

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Enteric Gram-negative rods (GNR), which are frequent causes of community-acquired and nosocomial infections, are increasingly resistant to the antibiotics in our current armamentarium. One solution to this medical dilemma is the development of novel classes of antimicrobial compounds. Here we report the development of a robust, whole cell-based, high-throughput metabolic assay that detects compounds with activity against carbapenem-resistant Klebsiella pneumoniae. We have used this assay to screen approximately 8,000 fungal extracts and 50,000 synthetic compounds with the goal of identifying extracts and compounds active against a highly resistant strain of Klebsiella pneumoniae. The primary screen identified 43 active fungal extracts and 144 active synthetic compounds. Patulin, a known fungal metabolite and inhibitor of bacterial quorum sensing and alanine racemase, was identified as the active component in the most potent fungal extracts. We did not study patulin further due to previously published evidence of toxicity. Three synthetic compounds termed O06, C17, and N08 were chosen for further study. Compound O06 did not have significant antibacterial activity but rather interfered with sugar metabolism, while compound C17 had only moderate activity against GNRs. Compound N08 was active against several resistant GNRs and showed minimal toxicity to mammalian cells. Preliminary studies suggested that it interferes with protein expression. However, its direct application may be limited by susceptibility to efflux and a tendency to form aggregates in aqueous media. Rapid screening of 58,000 test samples with identification of several compounds that act on CR-K. pneumoniae demonstrates the utility of this screen for the discovery of drugs active against this highly resistant GNR.

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Section: Introductionmentioning

confidence: 99%

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae

Liao

Mevers

et al. 2018

PLoS ONE

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“…Bacteriomimetic liposomes were prepared by thin film hydration, as previously reported, with minor modifications [36]. A 6% (w/v) phospholipid solution was prepared by dissolving 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (POPE), 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1 -rac-glycerol) (sodium salt) (POPG) and 1,1 ,2,2 -tetra-(9Z-octadecenoyl) cardiolipin (sodium salt) (CL) (Avanti Polar Lipids Inc., Alabaster, AL, USA) (weight ratio 70:20:10) in 5 mL of a chloroform-methanol blend (2:1) in a 250 mL round bottom flask.…”

Section: Bacteriomimetic Liposome Preparationmentioning

confidence: 99%

Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections

Goes

Lapuhs

Kuhn

et al. 2020

Cells

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In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.

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“…For staining of phospholipids within the IM, model preparation was carried out as previously described, with the addition of 0.1 mol% laurdan to the employed PL solution. 4 The PS model was prepared as described according to the standard protocol (see main manuscript), but on a separate Transwell ® insert. The formed gel was then incubated for 1.5 h with a 500 µg/mL solution of FITC-labeled concanavalin A, known to specifically interact with alginate.…”

Section: Structural Characterization Of the Overall Envelope Model (Cmentioning

confidence: 99%

In Vitro Model of the Gram-Negative Bacterial Cell Envelope for Investigation of Anti-Infective Permeation Kinetics

et al. 2018

Self Cite

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The cell envelope of Gram-negative bacteria is a formidable biological barrier, inhibiting the action of antibiotics by impeding their permeation into the intracellular environment. In-depth understanding of permeation through this barrier remains a challenge, despite its critical role in antibiotic activity. We therefore designed a divisible in vitro permeation model of the Gram-negative bacterial cell envelope, mimicking its three essential structural elements, the inner membrane and the periplasmic space as well as the outer membrane, on a Transwell setup. The model was characterized by contemporary imaging techniques and employed to generate reproducible quantitative and time-resolved permeation data for various fluorescent probes and anti-infective molecules of different structure and physicochemical properties. For a set of three fluorescent probes, the permeation through the overall membrane model was found to correlate with in bacterio permeation. Even more interestingly, for a set of six Pseudomonas quorum sensing inhibitors, such permeability data were found to be predictive for their corresponding in bacterio activities. Further exploration of the capabilities of the overall model yielded a correlation between the permeability of porin-independent antibiotics and published in bacterio accumulation data; a promising ability to provide structure-permeability information was also demonstrated. Such a model may therefore constitute a valuable tool for the development of novel anti-infective drugs.

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The bacterial cell envelope as delimiter of anti-infective bioavailability – An in vitro permeation model of the Gram-negative bacterial inner membrane

Cited by 27 publications

References 41 publications

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae

Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections

In Vitro Model of the Gram-Negative Bacterial Cell Envelope for Investigation of Anti-Infective Permeation Kinetics

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