Induction of the viable but nonculturable state of Salmonella enterica serovar Enteritidis deficient in (p)ppGpp synthesis

Abstract

Salmonella enterica enters a viable but nonculturable (VBNC) state in adverse environmental conditions. Under nutritional stress, RelA and SpoT proteins synthesize (p)ppGpp, a molecule that controls the expression of genes important for the survival of a cell under those conditions. This study aimed to verify the involvement of (p)ppGpp in the entry of S. enterica serovar Enteritidis PT4 578 cells into the VBNC state by evaluating ΔrelA and ΔrelAΔspoT mutants. The wild-type and mutant strains deficient in (p)ppGpp synthesis were subjected to osmotic, nutritional and cold stress, after which the cells entered a VBNC state at different time periods, concurrent with reductions in cell diameter, volume and length, and conversion from a bacillary to a coccoid form. No difference in the culturability or cell viability of the wild-type and single and double mutants was observed. Expression of the rpoS gene was increased in the double mutant, while both mutant strains presented a reduction in mreB gene expression after 25 days under conditions of nutritional and cold stress. Surprisingly, (p)ppGpp was not necessary for induction of the VBNC state in Salmonella PT4 578 cells, but may be associated with regulation of genes that control septum formation during cell division, maintaining the bacillary cell morphology (mreB) and stress response (rpoS). Our findings contribute to the understanding of the mechanisms of resistance and survival of Salmonella under adverse conditions.