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 ... -  《-》

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 ... -  《-》

Influence of ethanol adaptation on Salmonella enterica serovar Enteritidis survival in acidic environments and expression of acid tolerance-related genes.

Cross-protection to environmental stresses by ethanol adaptation in Salmonella poses a great threat to food safety because it can undermine food processing interventions. The ability of Salmonella enterica serovar Enteritidis (S. Enteritidis) to develop acid resistance following ethanol adaptation (5% ethanol for 1 h) was evaluated in this study. Ethanol-adapted S. Enteritidis mounted cross-tolerance to malic acid (a two-fold increase in minimum bactericidal concentration), but not to acetic, ascorbic, lactic, citric and hydrochloric acids. The population of S. Enteritidis in orange juice (pH 3.77) over a 48-h period was not significantly (p > 0.05) influenced by ethanol adaptation. However, an increased survival by 0.09-1.02 log CFU/ml was noted with ethanol-adapted cells of S. Enteritidis compared to non-adapted cells in apple juice (pH 3.57) stored at 25 °C (p < 0.05), but not at 4 °C. RT-qPCR revealed upregulation of two acid tolerance-related genes, rpoS (encoding σS) and SEN1564A (encoding an acid shock protein), following ethanol adaptation. The relative expression level of the acid resistance gene hdeB did not change. The resistance phenotypes and transcriptional profiles of S. Enteritidis suggest some involvement of rpoS and SEN1564A in the ethanol-induced acid tolerance mechanism.

He S ，Cui Y ，Qin X ，Zhang F ，Shi C ，Paoli GC ，Shi X ... -  《-》

Ethanol adaptation induces direct protection and cross-protection against freezing stress in Salmonella enterica serovar Enteritidis.

Salmonella enterica serovar Enteritidis (Salm. Enteritidis) encounters mild ethanol stress during its life cycle. However, adaptation to a stressful condition may affect bacterial resistance to subsequent stresses. Hence, this work was undertaken to investigate the influences of ethanol adaptation on stress tolerance of Salm. Enteritidis. Salmonella Enteritidis was subjected to different ethanol adaptation treatments (2·5-10% ethanol for 1 h). Cellular morphology and tolerance to subsequent environmental stresses (15% ethanol, -20°C, 4°C, 50°C and 10% NaCl) were evaluated. It was found that 10% was the maximum ethanol concentration that allowed growth of the target bacteria. Ethanol adaptation did not cause cell-surface damage in Salm. Enteritidis as revealed by membrane permeability measurements and electron micrograph analysis. Salmonella Enteritidis adapted with 2·5-10% ethanol displayed an enhanced resistance to a 15%-ethanol challenge compared with an unchallenged control. The maximum ethanol resistance was observed when ethanol concentration used for ethanol adaptation was increased to 5·0%. Additionally, pre-adaptation to 5·0% ethanol cross-protected Salm. Enteritidis against -20°C, but not against 4°C, 50°C or 10% NaCl. Ethanol adaptation provided Salm. Enteritidis direct protection from a high level ethanol challenge and cross-protection from freezing, but not other stresses tested (low temperature, high salinity or high temperature). The results are valuable in developing adequate and efficient control measures for Salm. Enteritidis in foods.

He S ，Zhou X ，Shi C ，Shi X ... -  《-》

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 ... -  《-》