iTRAQ-Based Global Proteomic Analysis of Salmonella enterica Serovar Typhimurium in Response to Desiccation, Low Water Activity, and Thermal Treatment.

In this study, the changes in the global proteome of Salmonella in response to desiccation and thermal treatment were investigated by using an iTRAQ multiplex technique. A Salmonella enterica serovar Typhimurium strain was dried, equilibrated at high (1.0) and low (0.11) water activity (aw), and thermally treated at 75°C. The proteomes were characterized after every treatment. The proteomes of the different treatments differed in the expression of 175 proteins. On the basis of their proteomic expression profiles, the samples were clustered into two major groups, namely, "dry" samples and "moist" samples. The groups had different levels of proteins involved in DNA synthesis and transcription and in metabolic reactions, indicating that cells under either of the aw conditions need to strictly control energy metabolism, the rate of replication, and protein synthesis. The proteins with higher expression levels in moist samples were flagellar proteins (FlgEFGH), membrane proteins, and export systems (SecF, SecD, the Bam complex), as well as stress response proteins, suggesting that rehydration can trigger stress responses in moist cells. Dry samples had higher levels of ribosomal proteins, indicating that ribosomal proteins might be important for additional regulation of the cellular response, even when the synthesis of proteins is slowed down. At both aws, no differences in protein expression were observed between the thermally treated samples and the nonheated cells. In conclusion, our study indicates that the preadaptation to a dry condition was linked to increased thermal tolerance, while reversion from a dry state to a moist state induced a significant change in protein expression, possibly linked to the observed loss of thermal tolerance.IMPORTANCESalmonella enterica is able to survive in dry environments for very long periods. While it is well known that the initial exposure to desiccation is fundamental to trigger thermal tolerance in this organism, the specific physiological and molecular processes involved in this cross-protection phenomenon have not been fully characterized. Several studies have focused on the low-aw transcriptome of this pathogen when inoculated in different food matrices or on abiotic surfaces, but proteomic analyses have not been reported in the literature. Our study investigated the changes in proteomic expression in Salmonella enterica serovar Typhimurium during desiccation, exposure to low aw, and thermal treatment. A better knowledge of the systems involved in the response to desiccation and thermal tolerance, as well as a better understanding of their interplay, is fundamental to identify the most effective combination of interventions to prevent Salmonella's contamination of foods.

Thermal Death Kinetics of Three Representative Salmonella enterica Strains in Toasted Oats Cereal.

Several reports have indicated that the thermal tolerance of Salmonella at low-water activity increases significantly, but information on the impact of diverse food matrices is still scarce. The goal of this research was to determine the kinetic parameters (decimal reduction time, D; time required for the first decimal reduction, δ) of thermal resistance of Salmonella in a previously cooked low water activity food. Commercial toasted oats cereal (TOC) was used as the food model, with or without sucrose (25%) addition. TOC samples were inoculated with 108 CFU/mL of a single strain of one of three Salmonella serovars (Agona, Tennessee, Typhimurium). TOC samples were ground and equilibrated to aw values of 0.11, 0.33 and 0.53, respectively. Ground TOC was heated at temperatures between 65 °C and 105 °C and viable counts were determined over time (depending on the temperature for up to 6 h). Death kinetic parameters were determined using linear and Weibull regression models. More than 70% of Weibull's adjusted regression coefficients (Radj2) and only 38% of the linear model's Radj2 had values greater than 0.8. For all serovars, both D and δ values increased consistently at a 0.11 aw compared to 0.33 and 0.53. At 0.33 aw, the δ values for Typhimurium, Tennessee and Agona were 0.55, 1.01 and 2.87, respectively, at 85 °C, but these values increased to 65, 105 and 64 min, respectively, at 0.11 aw. At 100 °C, δ values were 0.9, 5.5 and 2.3 min, respectively, at 0.11 aw. The addition of sucrose resulted in a consistent reduction of eight out of nine δ values determined at 0.11 aw at 85, 95 and 100 °C, but this trend was not consistent at 0.33 and 0.53 aw. The Z values (increase of temperature required to decrease δ-value one log) were determined with modified δ values for a fixed ß (a fitting parameter that describes the shape of the curve), and ranged between 8.9 °C and 13.4 °C; they were not influenced by aw, strain or sugar content. These findings indicated that in TOC, high thermal tolerance was consistent among serovars and thermal tolerance was inversely dependent on aw.

General response of Salmonella enterica serovar Typhimurium to desiccation: A new role for the virulence factors sopD and sseD in survival.

Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella's cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella's survival during desiccation.