We recently described a novel high throughput screening technology (highlighted on the cover of the May issue of Assay Development and Technology) to measure contemporaneously and in real-time effects on both intracellular growth of Legionella and host cell viability. This methodology has allowed us to rapidly screen 200,000+ compounds for intracellular growth inhibition, while at the same time weeding out compounds that are toxic to eukaryotic cells. In this way, our screening hits are enriched for those with therapeutic antimicrobial potential.
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What is synergy? Synergy is the ability of two or more antimicrobials when you used together to have much greater effect than the sum of their separate effects. Or in other words: A + B (observed) >> A+B (expected).
Sometimes synergy is graphically represented by plotting the permutations of antibiotic concentrations that results in inhibition of bacterial growth. These plots are known as isobolograms. Using such analysis, we found that pairwise combinations of azithromycin, minocycline, and rifampin showed strong synergy. See example on the left showing pairwise synergy between minocycline and azithroymycin. In isobologram plots, a straight line connecting the minimal inhibitory concentration (MIC) of each drug indicates indifference, while a concave isobologram curve, such as the one shown, indicates synergy. In this plot, the right most contour line connects points of 99% inhibition relative to intracellular growth of untreated control cultures. Additional contour lines connecting antibiotic concentrations the lead to partial levels of intracellular growth inhibition are also shown. (Isobologram plots to the left and below were drawn with Mathematica. See our recent publication for additional details.) |
Intriguingly, when used in triple combination, azithromycin, minocyline, and rifampin showed even more potent three dimensional synergy. Specifically, when used in triple combination, only one tenth the concentration of each antimicrobial was required to inhibit intracellular growth compared to when each antimicrobial was tested separately. See graph to the right showing an isobologram surface, connecting points of 99% intracellular growth inhibition, demonstrating a high degree of surface concavity. It is intriguing to consider therapeutic implications and application of this data, especially in disease processes such as lung consolidation where penetrance of antimicrobials may be less than ideal. Just as importantly we found that combinations that might be used in severe community acquired pneumonia such as ceftriaxone plus azithromycin or ceftriaxone plus levofloxacin were not antagonistic. Furthermore, levofloxacin did not show synergy with azithromycin (or any other antimicrobial tested), indicating that the occasionally combined use of azithromycin plus levofloxacin for severe Legionella pneumonia is unlikely to provide benefit.
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