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

authors:

avatar Ali Nouri Baghi ORCID 1 , * , avatar Mohammad Roayaei Ardakani ORCID 1 , avatar Elham Rezatofighi ORCID 1

Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Jundishapur Journal of Microbiology: Vol.17, issue 7; e147096
published online: August 28, 2024
article type: Research Article
revised: July 31, 2024
DOI: https://doi.org/10.5812/jjm-147096

how to cite: Nouri Baghi A, Roayaei Ardakani M, Rezatofighi E. Induction of Viable but Non-Culturable (VBNC) State in Shigella flexneri under Osmotic and Nutritional Stresses at Low Temperature. Jundishapur J Microbiol. 2024;17(7):e147096. https://doi.org/10.5812/jjm-147096.

Abstract

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 are metabolically active. 
Objectives: Considering the importance of 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: S. flexneri was inoculated in distilled water and NaCl solutions with concentrations of 0, 5, 10, 15, 20, 25 and 30% at 4 °C. Cultivability of bacteria was checked daily. After observing the uncultivability of bacteria, entering the VBNC state was checked by RT-PCR, flow cytometry and fluorescence microscope. 
Results: After cultivation of S. flexneri in different concentrations of salt solution, the time of non-cultivability was observed in concentrations of 0, 5, 10, 15, 20, 25 and 30%, respectively, on day 19, 20, 21, 22, 22, 24 and 24. Expression results of ipaH and ipaD genes with RT-PCR and observing the presence of living cells with flow cytometry and fluorescent staining methods indicated the entry of cells into the VBNC state. 
Conclusion: S. flexneri could survive and enter in the VBNC state under nutritional and osmotic stresses at low temperature (4 °C). In this case, although the bacterium is alive, it cannot be detected by conventional culture-dependent methods. Due to the possible risk of bacterial recovery, the evaluation of water and foods by conventional techniques may not be sufficient to detect the presence of pathogens in the VBNC state.