Induction of Viable but Non-culturable (VBNC) State in Shigella flexneri Under Osmotic and Nutritional Stresses at Low Temperature

Background: Viable but non-culturable (VBNC) is a specific physiological state in which living bacteria lose the ability to grow and form colonies on conventional bacterial growth media; however, these cells remain metabolically active. Objectives: Considering the importance of the pathogenicity of Shigella flexneri and the possibility of its transmission through contaminated water and food, we aimed to study the possibility of this bacterium entering the VBNC state under osmotic and nutritional stresses at low temperature (4°C). Methods: Shigella flexneri was inoculated in distilled water and NaCl solutions with concentrations of 0, 5, 10, 15, 20, 25, and 30% at 4°C. The cultivability of the bacteria was checked daily. After observing the loss of cultivability, entry into the VBNC state was assessed using RT-PCR, flow cytometry, and fluorescence microscopy. Results: After cultivation of S. flexneri in different concentrations of salt solution, the time of non-cultivability was observed at concentrations of 0, 5, 10, 15, 20, 25, and 30%, respectively, on days 19, 20, 21, 22, 22, 24, and 24. Expression results of ipaH and ipaD genes using RT-PCR, along with the presence of live cells detected by flow cytometry and fluorescent staining, indicated that the cells had entered the VBNC state. Conclusions: Shigella flexneri can survive and enter the VBNC state under nutritional and osmotic stresses at low temperatures (4°C). In this state, although the bacterium is alive, it cannot be detected by conventional culture-dependent methods. Due to the potential risk of bacterial recovery, conventional techniques may be insufficient to detect the presence of pathogens in the VBNC state when evaluating water and food.