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. 2015 Dec;59(12):7517-29.
doi: 10.1128/AAC.01248-15. Epub 2015 Sep 21.

High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella Pneumophila

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Free PMC article

High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella Pneumophila

Lucius Chiaraviglio et al. Antimicrob Agents Chemother. .
Free PMC article

Abstract

Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche.

Figures

FIG 1
FIG 1
Correlation between replicate antimicrobial screening wells. Each antimicrobial test compound from screening libraries was tested in duplicate in two separate screening plates. Plotted are the relative light units (RLU) on day 2 after macrophage infection for the first replicate versus the second replicate for each antimicrobial test compound listed in Table S1 in the supplemental material.
FIG 2
FIG 2
Dose-response antimicrobial activity against intracellular and axenic L. pneumophila. Serial 2-fold dilutions of select antimicrobials were tested for effect on intracellular and axenic growth, respectively. (A) Effects of antimicrobials mostly commonly used for clinical therapy. (B) Comparison of selected macrolides. (C) Comparison of selected tetracyclines, β-lactams, and additional antimicrobials with activity detected in the primary screen. Data points shown are the averages and standard deviations of three separate test wells per condition.
FIG 3
FIG 3
Isobolograms of minocycline versus azithromycin (A), rifampin versus minocycline (B), rifampin versus azithromycin (C), rifampin versus chloramphenicol (D), levofloxacin versus azithromycin (E), and rifampin versus levofloxacin (F). Isocontours were drawn using Mathematica based on regions of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% growth relative to growth in the untreated control. The 99% inhibition contour (1% growth, combinatorial MIC) is the contour line farthest to the right in each plot. Color shading between isocontours was added for clarity. Subinhibitory stimulation of luminescence led to isocontours greater than 100% in some plots. Data points used to generate isocontours were based on the means of three separate test wells per antimicrobial combination.
FIG 4
FIG 4
Three-dimensional synergy. The combinatorial effects of azithromycin, minocycline, and rifampin were tested against intracellular growth of L. pneumophila. The plotted surface contour overlies data points for antimicrobial combinations with <99% intracellular growth inhibition relative to that of the uninfected control wells. A high degree of surface concavity suggests three-dimensional synergy. Concentrations along axes are in nanograms/milliliter. A representative experiment is shown; the data set used for graph generation was based on 512 antimicrobial combinations tested in triplicate.

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