Acid tolerance in Salmonella typhimurium induced by culturing in the presence of organic acids at different growth temperatures.

The influence of growth temperature and acidification of the culture medium up to pH 4.25 with acetic, citric, lactic and hydrochloric acids on the growth and subsequent acid resistance at pH 3.0 of Salmonella typhimurium CECT 443 was studied. The minimum pH value which allowed for S. typhimurium growth within the temperature range of 25-37 degrees C was 4.5 when the pH was reduced using citric and hydrochloric acids, and 5.4 and 6.4 when lactic acid and acetic acid were used, respectively. At high (45 degrees C) or low (10 degrees C) temperatures, the growth pH boundary was increased about 1 pH unit. The growth temperature markedly modified the acid resistance of the resulting cells. In all cases, D-values were lower for cells grown at 10 degrees C and significantly increased with increasing growth temperature up to 37 degrees C, at which D-values obtained were up to 10 times higher. Cells grown at 45 degrees C showed D-values similar to those found for cells grown at 25 degrees C. The growth of cells in acidified media, regardless of the pH value, caused an increase in their acid resistance at the four incubation temperatures, although the magnitude of the Acid Tolerance Response (ATR) observed depended on the growth temperature. Acid adapted cultures at 10 degrees C showed D-values ranging from 5.75 to 6.91 min, which turned out to be about 2 times higher than those corresponding to non-acid adapted cultures, while higher temperatures induced an increase in D-values of at least 3.5 times. Another finding was that, while at 10 and 45 degrees C no significant differences among the effect of the different acids tested in inducing an ATR were observed, when cells were grown at 25 and 37 degrees C citric acid generally turned out to be the acid which induced the strongest ATR. Results obtained in this study show that growth temperature is an important factor affecting S. typhimurium acid resistance and could contribute to find new strategies based on intelligent combinations of hurdles, which could prevent the development or survival of Salmonella spp. in foods. The fact that moderately low temperatures (10 degrees C) markedly decrease the acid resistance and increase the growth pH boundary of S. typhimurium suggests the convenience to control the temperature during food processing as a strategy to prevent the growth and survival of this pathogenic microorganism.

Alvarez-Ordóñez A ，Fernández A ，Bernardo A ，López M ... -  《-》

Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance.

The effects of growth temperature (in the range 10-45 degrees C) and acidification up to pH 4.5 of the culture medium (Brain Heart Infusion, BHI) with different organic acids (acetic, citric and lactic) and hydrochloric acid on membrane fatty acid composition and heat resistance of Salmonella typhimurium CECT 443 were studied. The heat resistance was maximal in cells grown at 45 degrees C (cells grown in non-acidified BHI showed a D58-value of 0.90 min) and decreased with decreasing growth temperature up to 10 degrees C (D58-value of 0.09 min). The growth of cells in acidified media caused an increase in their heat resistance. In general, acid adapted cells showed D-values of between 1.5 and 2 times higher than the corresponding for non-acid adapted control cells. This cross-protection response, which has important implications in food processing, was not dependent on the pH value and the acid used to acidify the growth medium. A membrane adaptation corresponding to an increase in the unsaturated to saturated fatty acids ratio (UFA/SFA) and membrane fluidity was observed at low growth temperature. Moreover, the acidification of the growth medium caused a decrease in UFA/SFA ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in cyc19 relative concentration. Thus, acid adapted cells showed CFA levels 1.5 times higher than non-acid adapted control cells. A significant proportion of unsaturated fatty acids were converted to their cyclopropane derivatives during acid adaptation. These changes in membrane fatty acid composition result in cells with decreased membrane fluidity. A clear relation between membrane fatty acid composition and heat resistance was observed. In general, D-values were maximum for cells with low UFA/SFA ratio, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting acid adapted cells from heat inactivation. However, changes observed in membrane fatty acid composition are not enough to explain the great thermotolerance of cells grown at 45 degrees C. Thus, other mechanisms, such as the synthesis of Heat Shock Proteins, could be responsible for this increase in the bacterial heat resistance.

Alvarez-Ordóñez A ，Fernández A ，López M ，Arenas R ，Bernardo A ... -  《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》

A comparative study of thermal and acid inactivation kinetics in fruit juices of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Senftenberg grown at acidic conditions.

Acid and heat inactivation in orange and apple juices of Salmonella enterica serovar Typhimurium Colección Española de Cultivos Tipo (i.e., Spanish Type Culture Collection) 443 (CECT 443) (Salmonella Typhimurium) and S. enterica serovar Senftenberg CECT 4384 (Salmonella Senftenberg) grown in buffered brain heart infusion (pH 7.0) and acidified brain heart infusion up to pH 4.5 with acetic, citric, lactic, and hydrochloric acids was evaluated. Acid adaptation induced an adaptive response that increased the subsequent resistance to extreme pH conditions (pH 2.5) and to heat, although the magnitude of these responses differed between the two isolates and fruit juices. The acid resistance in orange juice for acid-adapted cells (D-values of 28.3-34.5 min for Salmonella Senftenberg and 30.0-39.2 min for Salmonella Typhimurium) resulted to be about two to three times higher than that corresponding to non-acid-adapted cells. In apple juice, acid-adapted Salmonella Senftenberg cells survived better than those of Salmonella Typhimurium, obtaining mean D-values of 114.8 +/- 12.3 and 41.9 +/- 2.5 min, respectively. The thermotolerance of non-acid-adapted Salmonella Typhimurium in orange (D(58)-value: 0.028 min) and apple juices (D(58)-value: 0.10 min) was approximately double for acid-adapted cells. This cross-protection to heat was more strongly expressed in Salmonella Senftenberg. D(58)-values obtained for non-acid-adapted cells in orange (0.11 min) and apple juices (0.19 min) increased approximately 10 and 5 times, respectively, after their growth in acidified media. The conditions prevailing during bacterial growth and heat treatment did not significantly influence the z-values observed (6.0 +/- 0.3 degrees C for Salmonella Typhimurium and 7.0 +/- 0.3 degrees C for Salmonella Senftenberg). The enhanced acid resistance found for both isolates could enable them to survive for prolonged time periods in the gastrointestinal tract, increasing the risk of illness. Further, it should be taken into account that microbial growth in acidified media also induces a cross-protection response against heat that should also be considered for the design of pasteurization processes for acid foods.

Alvarez-Ordóñez A ，Fernández A ，Bernardo A ，López M ... -  《-》

Relationship between membrane fatty acid composition and heat resistance of acid and cold stressed Salmonella senftenberg CECT 4384.

This study evaluates the adaptative response to heat (63 degrees C) and the modifications in membrane fatty acid composition of Salmonella senftenberg after its growth in an acidified medium and after its exposure to combinations of acid and cold stresses. Cells were grown in Brain Heart Infusion (BHI) buffered at pH 7.0 and acidified up to pH 4.5 (fresh cultures) and kept at refrigeration temperature (4 degrees C) for 7 days (refrigerated cultures). The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, but combinations of sublethal stresses reduced S. senftenberg heat tolerance, specially when the growth medium pH was decreased. Acid-adapted cells showed D(63)-values ranging from 3.10 to 6.27 min, while non-acid-adapted cells showed D(63)-values of 1.07 min. As pH decreased, over the pH range studied (7.4-4.5), D(63)-values of the resulting cells increased. However, refrigerated acid-adapted cells showed lower D(63)-values, which ranged from 0.95 to 0.49 min. A linear relationship between the thermotolerance of S. senftenberg cells and the previous growth medium pH was found in both fresh and refrigerated cultures, which allowed us to predict changes in heat resistance of S. senftenberg that occur at any pH value within the range used in the present study in which most foodstuffs are included. Both acidification of the growth medium and refrigeration storage of cells induced modifications in membrane fatty acid composition, which were clearly linked to their heat resistance. Acid-adapted cells, regardless of the pH value of the growth medium, showed the lowest UFA/SFA ratio and a CFA content 1.5-2-fold higher than that observed for non-acid-adapted cells. On the other hand, the UFA/SFA ratio found for S. senftenberg cells exposed to a cold stress was 1.2-1.8-fold higher than that observed for non-refrigerated cultures. This increase in the UFA/SFA ratio was specially high for acid-adapted cells. The highest thermotolerance was observed for cells with low UFA/SFA ratio, and, consequently, having a low membrane fluidity. However, changes observed in CFA content did not explain the great heat sensitivity of refrigerated acid-adapted cells.

Alvarez-Ordóñez A ，Fernández A ，López M ，Bernardo A ... -  《-》

Adaptive acid tolerance response in Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Typhi.

The survival of bacteria in various environments depends on a number of protective responses including acid tolerance response (ATR). In this study, ATR phenomenon was compared in Salmonella enterica serovar Typhi 6 and Salmonella enterica serovar Typhimurium 98 under different culture conditions. Survival of the adapted culture (pre-acid shocked to pH 5.5) was significantly better (p < 0.05) as compared to control, unadapted culture after acid shock at pH 3.3. However, the ATR varied with the serovar, incubation temperature and the growth medium used (all p-values < 0.05). S. Typhi 6 failed to grow in pH 3.3 at 45 degrees C. The addition of tetracycline or chloramphenicol (1.0 microg ml(-1)) to adapted cultures during or after acid shock (pH 3.3) had no effect on ATR expression. In S. Typhimurium 98, growth was increased by 10% or greater in adapted culture (when grown at pH 3.3) as compared to growth observed with an unadapted culture (when grown at pH 7.3) on transfer to fresh growth medium at pH 7.3. A poor ATR observed in non-growing S. Typhimurium 98 suspensions clearly showed that ATR is an energy-consuming process. Storage of S. Typhimurium 98 cultures in pH 4.5 nutrient broth at 4 degrees C demonstrated that prolonged exposure to acidic conditions is more detrimental in comparison to the cultures stored at pH 7.3 at this temperature.

Tiwari RP ，Sachdeva N ，Hoondal GS ，Grewal JS ... -  《JOURNAL OF BASIC MICROBIOLOGY》