Behavior of Salmonella Enteritidis and Shigella flexneri during induction and recovery of the viable but nonculturable state

Abstract

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

Keywords: VBNC; bacterial culturability; bacterial stress; bacterial viability; reactive oxygen species; starvation.