Heat resistance of Salmonella Enteritidis under prolonged exposure to acid-salt combined stress and subsequent refrigeration.

Salmonella Enteritidis is a major foodborne pathogen exposed to various environmental and preservation stresses in the food chain. Because adaptive responses of viable bacterial cells in the presence of sublethal stress can induce cross-protection against different stresses, we investigated the heat resistance of Salmonella Enteritidis at 60 °C under prolonged exposure to acid-salt combined stress and subsequent refrigeration. Salmonella Enteritidis was grown in tryptic soy broth at four pH values (4.5, 5.4, 6.4, and 7.3) and four NaCl concentrations (0%, 1%, 2%, and 3%) at 37 °C for 24 h and then incubated at 4 °C for 0, 1, 4, or 7 days. For 0 and 1 day-refrigerated cultures, previous adaptation to single stresses (acid or salt stress) increased the heat resistance of Salmonella Enteritidis, resulting in increased D-values, whereas the combination of acid and salt stress reduced heat tolerance; acid stress played a more critical role in mediating this effect than salt concentration. To elucidate the related mechanisms, the expression levels of heat shock sigma factors (rpoH) and heat shock genes (dnaK and groEL) were analyzed and found to be associated with the heat resistance of Salmonella Enteritidis. The refrigeration period was negatively correlated (P < 0.01) with the D-value (r = -0.505) and with the transcript levels of rpoH (r = -0.654), dnaK (r = -0.652), and groEL (r = -0.645). Our findings demonstrated that acid-salt combined preservation techniques and subsequent refrigeration may prevent S. Enteritidis survival in heat-pasteurized food products caused by cross-protection of acid or salt adapted cells.

Kang IB ，Kim DH ，Jeong D ，Park JH ，Seo KH ... -  《-》

Growth temperature alters Salmonella Enteritidis heat/acid resistance, membrane lipid composition and stress/virulence related gene expression.

The influence of growth temperature (10, 25, 37, and 42 °C) on the survival of Salmonella Enteritidis in simulated gastric fluid (SGF; pH=2.0) and during heat treatment (54, 56, 58, and 60 °C), on the membrane fatty acid composition, as well as on stress-/virulence-related gene expression was studied. Cells incubated at temperatures lower or higher than 37 °C did not increase their acid resistance, with the maximum D-value of 3.07 min in cells grown at 37 °C; while those incubated at higher temperature increased their heat resistance, with the maximum D60 °C-values of 1.4 min in cells grown at 42 °C. A decrease in the ratio of unsaturated to saturated fatty acids was observed as the growth temperature increased. Compared to the control cells grown at 37 °C, the expression of rpoS was 16.5- and 14.4-fold higher in cells cultivated at 10 and 25 °C, respectively; while the expression of rpoH was 2.9-fold higher in those cultivated at 42 °C. The increased expression of stress response gene rpoH and the decreased ratio of unsaturated to saturated fatty acids correlated with the greater heat resistance of bacteria grown at 42 °C; while the decreased expression of stress response gene rpoS at 42 °C might contribute to the decrease in acid resistance. Virulence related genes-spvR, hilA, avrA-were induced in cells cultivated at 42 °C, except sefA which was induced in the control cells. This study indicates that environmental temperature may affect the virulence potential of S. Enteritidis, thus temperature should be well controlled during food storage.

Yang Y ，Khoo WJ ，Zheng Q ，Chung HJ ，Yuk HG ... -  《-》

Sublethal concentrations of undissociated acetic acid may not always stimulate acid resistance in Salmonella enterica sub. enterica serovar Enteritidis Phage Type 4: Implications of challenge substrate associated factors.

Gavriil A ，Thanasoulia A ，Skandamis PN 《PLoS One》

Membrane lipid composition and stress/virulence related gene expression of Salmonella Enteritidis cells adapted to lactic acid and trisodium phosphate and their resistance to lethal heat and acid stress.

This study evaluated the acid and heat resistance of Salmonella Enteritidis in simulated gastric fluid (pH 2.0) and during thermal treatment (54-60 °C), respectively, after adaptation to lactic acid (LA) or trisodium phosphate (TSP) at various pHs (pH 5.3-9.0). The changes in membrane lipid composition and expression levels of RpoS and RpoH were examined to elucidate their roles in bacterial stress resistance. Transcriptional profile of several virulence-related genes was also analyzed. Results showed that LA-adapted cells at pH 5.3 and 6.3 had higher acid and heat resistance than control cells and cells adapted to TSP at pH 8.3 and 9.0. LA-adapted cells had the lowest ratio of unsaturated to saturated fatty acids, indicating that they might possess a less fluid membrane. It was observed that the expression levels of RpoH and RpoS were upregulated in TSP-adapted cells but not in LA-adapted cells. Thus, these results indicate that the increased acid and heat resistance of LA-adapted S. Enteritidis was possibly due to the decreased membrane fluidity instead of the upregulation of RpoS and RpoH. About 6.0, 2.1, and 2.46-fold upregulation of spvR, avrA, and hilA were observed in cells adapted to TSP at pH 9.0, except sefA that had its highest expression level in the control cells, indicating that the expression of these virulence genes highly depends on environmental conditions. This is the first study to show that the alteration in the cytoplasmic membrane rather than RpoS and RpoH plays a more crucial role in conferring greater acid and heat resistance on LA-adapted S. Enteritidis, thus providing a better understanding on the bacterial stress response to acidic conditions.

Yang Y ，Kadim MI ，Khoo WJ ，Zheng Q ，Setyawati MI ，Shin YJ ，Lee SC ，Yuk HG ... -  《-》

Response to Acid Adaptation in Salmonella enterica Serovar Enteritidis.

Acid adaptation in Salmonella Enteritidis was characterized by phenotypic and gene-expression analyses. S. Enteritidis cells at log-phase and stationary-phase were kept at pH 4.5 to 6.0 for 1 to 4 hours. All treatments induced various levels of acid tolerance response that were dependent on pH, exposure time and growth phase. This acid adaptation resulted in tolerance to 50 °C and 8% NaCl regardless of the growth phase. However, the tolerance of log-phase and stationary-phase cells to low temperatures (4 and -20 °C) was increased and decreased, respectively. RT-qPCR analysis revealed that genes involved in tolerance to acid (SEN1564A and cfa), heat (rpoH, uspB, and htrA), salt (proP, proV, and osmW), and cold (cspA, cspC, and csdA) stress were generally upregulated after acid adaptation. These results provide an initial insight into mechanisms of acid adaptation and induced cross protection in S. Enteritidis. PRACTICAL APPLICATION: Stress tolerance acquisition resulting from acid adaptation in foodborne pathogens poses a great threat to food safety. The current work showed that acid adaptation induced direct tolerance and cross-tolerance to high temperature, low temperature, and salt in Salmonella Enteritidis, possibly due to the upregulation of stress tolerance-related genes. These results provide key insights into acid adaptation mechanisms and efficient control of S. Enteritidis.

Ye B ，He S ，Zhou X ，Cui Y ，Zhou M ，Shi X ... -  《-》