High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

被引:3
作者
Chiaraviglio, Lucius [1 ]
Kang, Yoon-Suk [1 ]
Kirby, James E. [1 ]
机构
[1] Beth Israel Deaconess Med Ctr, Dept Pathol, 330 Brookline Ave, Boston, MA 02215 USA
来源
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2016年 / 117期
基金
美国国家卫生研究院;
关键词
Infection; Issue; 117; synergy; high-throughput screening; real-time; Legionella pneumophila; intracellular growth; antimicrobial; antibiotic; minimal inhibitory concentration; dose-response; cytotoxicity; isobologram; SINGLE GENETIC-LOCUS; LEGIONELLA-PNEUMOPHILA; FLUORESCENT PROTEINS; ESCHERICHIA-COLI; RED; PERSISTENCE; GROWTH;
D O I
10.3791/54841
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.
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页数:13
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