Influence of Physiological State, Prolonged Dry Storage, and Passage through Simulated Digestion on the Survival and Gene Expression of Salmonella enterica sv. Tennessee

Abstract

Salmonella enterica serotypes have been linked to outbreaks associated with low water activity foods. The ability of biofilm forming pathogens, such as Salmonella, to survive thermal and chemical processes is improved; it is unclear if biofilms will also improve survival to desiccation and gastric stresses. The purpose of this study was to quantify the effect of physiological state (planktonic versus biofilm) and prior exposure to desiccation on Salmonella survival and gene expression after passage through an in-vitro digestion model.

Cells of Salmonella enterica serotype Tennessee were deposited onto membranes for planktonic cells or on glass beads to create biofilms. The cells were subsequently dried at room temperature and stored in dried milk powder (aw = 0.3) for up to 30 days. Salmonella survival was quantified by serial dilution onto brilliant green agar before desiccation, after desiccation, after 1-day storage and after 30-day storage. At each sampling both physiological states were tested for survival through a simulated gastrointestinal system. RNA was extracted at the identical time points and relative gene expression determined for genes associated with stress response (rpoS, otsB), virulence (hilA, hilD, invA, sipC) and a housekeeping gene 16S rRNA using quantitative real-time PCR.

The physiological state and length of storage effected the survival and gene expression of Salmonella within the desiccated milk powder environment and after passage through an in-vitro digestion system (p<0.05). Larger numbers of S. Tennessee were recovered by plate counts for biofilm cells, compared to planktonic cells. However, the numbers of 16S rRNA gene copies were not significantly different suggesting entry of S. Tennessee into a viable but non-culturable state. Prolonged storage in dry milk powder was not associated with increased cross-protection to gastric stress. Increased expression of stress response genes rpoS and otsB correlated with survival, indicating cross protection of low water activity and acid stress. Increased expression of virulence-associated genes was seen in cells exposed to short periods of dry storage, suggesting an increased virulence potential.