Simultaneous growth, survival and death: The trimodal behavior of Salmonella cells under osmotic stress giving rise to “Phoenix phenomenon”

Time-lapse microscopy methods were used to monitor growth, survival and death of Salmonella enterica serotype Agona individual cells on solid laboratory medium (tryptone soy agar) in the presence of various salt concentrations (0.5%, 3.5%, 4.5% and 5.7% NaCl). The results showed a highly heterogeneous behavior. As NaCl concentration increased, the distribution of the first division time was shifted to higher values and became wider. The mean first division time increased from 1.8 h at 0.5% NaCl to 5.48 h, 16.2 h, and 35.9 h at 3.5%, 4.5% and 5.7% NaCl, respectively. The concentration of NaCl in the growth medium also affected the ability of the cells to divide. The percentage of cells able to grow decreased from 88.9% at 0.5% NaCl to 66.5%, 32.8%, and 6.9% at 3.5%, 4.5% and 5.7% NaCl, respectively. In the latter case (5.7% NaCl), 74 cells out of 406 cells tested (18%) died with mean time to death 5.03 h and standard deviation 6.70 h. To investigate the effect of the behavior of individual cells on the dynamics of the whole population, simulation analysis was used. The simulation results showed that the simultaneous growth, survival and death of cells observed under osmotic stress can lead to a total population behavior known as the “Phoenix” phenomenon. The simulation findings were confirmed by validation experiments using both viable counts and time lapse microscopy. The results of the present study show the high heterogeneity of individual cell responses and the complexity in the behavior of microbial populations at conditions approaching the boundaries of growth.