Dynamics of growth and death within aSalmonella typhimuriumpopulation during infection of macrophages

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Abstract
Survival of Salmonella typhimurium within macrophages is associated with virulence. Most data on the fate of Salmonella during infection of macrophages are derived from viable counts of intracellular bacteria. These counts are a result of a combination of bacterial death and growth within the intracellular population but may not reflect the true levels of either macrophage killing of Salmonella or bacterial growth inside cells. In this study, two independent methods have been used to obtain a more accurate measurement of absolute levels of both death and growth of Salmonella inside macrophages. A purine auxotroph (purD) was used to measure Salmonella death in the absence of bacterial growth and then bacterial growth was measured by supplementing the purD cultures with adenosine. Numbers of dead and live Salmonella were also quantitated using the BacLight staining system, which distinguishes dead from live bacteria. Both methods demonstrate that killing of Salmonella by macrophages is considerably greater than detected using traditional cell counts and that bacterial inactivation occurs throughout the infection period. Salmonella was inactivated at a similar rate in both J774 macrophages (most permissive macrophages) and peritoneal exuadate macrophages (least permissive macrophages), suggesting that the major difference between these cells is the ability to limit bacterial growth. These studies also demonstrate that growth of Salmonella within murine macrophages occurs simultaneously with significant amounts of bacterial death. Identifying the factors responsible for shifting the interaction between macrophages and bacteria toward conditions that favor bacterial growth will be critical to understanding Salmonella virulence.Key words: Salmonella, macrophage, purD, purine auxotroph.