Antibiotic transport kinetics in Gram-negative bacteria revealed via single-cell uptake analysis and mathematical modelling (original) (raw)
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Communications Biology, 2020
With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.
Biophysical Journal, 2008
We present novel microfluidic experiments to quantify population-scale transport parameters (chemotactic sensitivity x 0 and random motility m) of a population of bacteria. Previously, transport parameters have been derived theoretically from single-cell swimming behavior using probabilistic models, yet the mechanistic foundations of this upscaling process have not been verified experimentally. We designed a microfluidic capillary assay to generate and accurately measure gradients of chemoattractant (a-methylaspartate) while simultaneously capturing the swimming trajectories of individual Escherichia coli bacteria using videomicroscopy and cell tracking. By measuring swimming speed and bias in the swimming direction of single cells for a range of chemoattractant concentrations and concentration gradients, we directly computed the chemotactic velocity V C and the associated chemotactic sensitivity x 0 . We then show how m can also be readily determined using microfluidics but that a population-scale microfluidic approach is experimentally more convenient than a single-cell analysis in this case. Measured values of both x 0 [(12.4 6 2.0) 3 10 À4 cm 2 s À1 ] and m [(3.3 6 0.8) 3 10 À6 cm 2 s À1 ] are comparable to literature results. This microscale approach to bacterial chemotaxis lends experimental support to theoretical derivations of population-scale transport parameters from single-cell behavior. Furthermore, this study shows that microfluidic platforms can go beyond traditional chemotaxis assays and enable the quantification of bacterial transport parameters.
Fluorescence enlightens RND pump activity and the intrabacterial concentration of antibiotics
Research in Microbiology, 2018
To understand the antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation which is defined by influx and efflux. Several methods exist to evaluate the membrane permeability and efflux pump activity but they present some disadvantages and limitations. An optimized spectrofluorimetric method using the intrinsic tryptophan fluorescence as internal standard as well as a complementary microfluorimetric assay following the time-course accumulation in intact individual cells have been developed. Comparing the latter population and single cell approaches can lead to the understanding of the phenotypic heterogeneity within a population. The two methodologies lead to the determination of parameters, concentration, accumulation rates, localization that contribute to emerging concepts (RTC2T, SICAR) with the aim to identify and detail the antibiotic chemotypes that are involved in influx/efflux.
Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli
Journal of the American Chemical Society, 2015
Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF ...
Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level
Scientific reports, 2015
Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and ...
Antimicrobial Agents and Chemotherapy
A mathematical model of the passive permeation of a novel solute into bacteria that explicitly accounts for intracellular dilution through growth was developed. A bacterial cell envelope permeability coefficient of approximately >10 −8 cm 2 · s −1 is predicted to ensure passive permeation into rapidly replicating bacterial cells. The relative importance of the permeability coefficients of the cytoplasmic and outer membranes of Gram-negative bacteria in determining the overall envelope permeability coefficient was analyzed quantitatively. A mathematical description of the balance between passive influx and active efflux was also developed and shows that bacterial expansion through growth can usually be neglected for compounds likely to be prepared in antibacterial drug discovery programs and the balance between passive inward permeation and active outwardly directed efflux predominates. A new parameter, efflux efficiency (η, where η is equal to k / P , in which k is the rate coeff...
Anomalous diffusion ofE. coliunder microfluidic confinement and chemical gradient
bioRxiv (Cold Spring Harbor Laboratory), 2022
We report a two-layer microfluidic device to study the combined effect of confinement and chemical gradient on the motility of wild type E. coli. We track individual E. coli in 50µm and 10µm wide channels, with a channel height of 2.5µm, to generate quasi-2D conditions. We find that contrary to expectations, bacterial trajectories are super-diffusive even in absence of a chemical (glucose) gradient. The superdiffusive behaviour becomes more pronounced on introduction of a chemical gradient or on strengthening the lateral confinement. Runlength distributions for weak confinement in absence of chemical gradients follow an exponential distribution. Both confinement and chemoattraction induce deviations from this behaviour, with the runlength distributions approaching a power-law form under these conditions. Both confinement and chemoattraction suppress large angle tumbles as well. Our results suggest that wild-type E. coli modulates both its runs and tumbles in a similar manner under physical confinement and chemical gradient.
Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes
Journal of Molecular Biology, 2019
The double membrane cell envelope of Gram negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negatives are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target based screening programmes have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarise the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.
Nature Protocols, 2018
the efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. this is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMsF). these different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (oM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. the three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (Wcls) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.
ACS infectious diseases, 2018
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, fo...