BACTERIUM-HOST CELL-INTERACTIONS AT THE CELLULAR-LEVEL - FLUORESCENT LABELING OF BACTERIA AND ANALYSIS OF SHORT-TERM BACTERIUM-PHAGOCYTE INTERACTION BY FLOW-CYTOMETRY

Flow cytometry is a potentially powerful tool for analyzing the intera ctions of facultative intracellular bacteria and macrophages on a cell ular level, particularly when fluorochromes are used to label the bact eria. We labeled Listeria monocytogenes and Salmonella typhimurium wit h a lipophilic dye, PKH-2, and used flow cytometry to investigate phag ocytosis by J774A.1 cells and short-term bacterial survival. Labeled a nd unlabeled bacteria were identical in terms of viability, growth kin etics, and survival within macrophages, although recovery per macropha ge was much greater for L. monocytogenes than for S. typhimurium. Usin g L. monocytogenes as a prototypical facultative intracellular bacteri um, we estimated bacterial survival during phagocytosis on the basis o f linear fluorescence measurements of infected J774A.1 cells and recov ery of L. monocytogenes from sorted cells. The lower percentage of sur viving L. monocytogenes in macrophages containing higher bacterial loa ds indicated the accumulation of nonviable bacteria within phagocytes. Removal of the external source of viable bacteria by washes and genta micin treatment reduced the percentage of surviving intracellular L. m onocytogenes to a baseline level, and all baseline levels mere similar , regardless of bacterial load. Listeria enrichment recoveries, derive d from individually sorted J774A.1 cells, demonstrated the heterogenei ty of macrophages in intracellular bacterial survival, especially with in heavily infected cells. These results indicated that survival of L. monocytogenes was dependent on the adaptations of a small fraction of bacteria within a population of macrophages which permit intracellula r growth.