title: High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila.
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.
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Lucius
authors.initials: L
authors.lastname: Chiaraviglio
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA jekirby@bidmc.harvard.edu.
authors.forename: James E
authors.initials: JE
authors.lastname: Kirby
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005456:Fluorescent Dyes / Q000737:chemistry; D024841:Fluoroquinolones / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006801:Humans; D016952:Legionella pneumophila / Q000187:drug effects* / Q000254:growth & development; D018942:Macrolides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D009930:Organic Chemicals / Q000737:chemistry; D013754:Tetracyclines / Q000494:pharmacology; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D024841:Fluoroquinolones; D018942:Macrolides; D009930:Organic Chemicals; C402795:SYTOX Green; D013754:Tetracyclines; D047090:beta-Lactams