Ming-Lun Chiang

The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988.

Enterobacter sakazakii is an emerging opportunistic pathogen associated with life-threatening illnesses in infants, with infant formula serving as the principal mode of transmission. In the present study, C. sakazakii (formely E. sakazakii) BCRC 13988 was subjected to various heat shock treatments (42-48 degrees C for 5-15 min). Its subsequent survival at 51 degrees C and the leakage of intracellular materials was investigated. It was found that 47 degrees C was the maximum growth temperature of the test organism. In addition, heat shock enhanced the thermal tolerance of C. sakazakii BCRC 13988. Within heat shock temperatures between 42 and 47 degrees C, the thermal tolerance enhancing effect increased as the length or temperature of the heat shock treatment was increased. However, increasing the heat shock temperature to 48 degrees C reduced the thermal tolerance enhancing effect. Among the various heat shocked cells examined, the 47 degrees C-15 min-heat shocked C. sakazakii exhibited the highest thermal tolerance. Moreover, electron micrograph analysis showed that heat shock treatment caused damage and disruption in C. sakazakii cells. There was a significant increase (P<0.05) in the leakage of nucleic acid and protein in the supernatant of the heat shocked cell suspension that increased as the temperature and duration of heat shock increased.

Ethanol shock changes the fatty acid profile and survival behavior of Vibrio parahaemolyticus in various stress conditions.

Vibrio parahaemolyticus 690, a clinical strain, was subjected to ethanol shock in the presence of 5% ethanol for a period of 30 and 60 min. Survival behaviors of the ethanol shocked and control cells of V. parahaemolyticus in the presence of H(2)O(2) (20 ppm), crystal violet (3 ppm), NaCl (20%), and low pH solution (pH 4.4) containing various organic acids including lactic acid, acetic acid, citric acid and tartaric acid (25 mM) were compared. In addition, the effects of ethanol shock on the fatty acid profile and recovery of V. parahaemolyticus on tryptic soy agar (TSA) plus various amounts of NaCl were also investigated. After ethanol shock, it was found that the proportion of vaccenic acid (18:1) increased, while the proportion of palmitic acid (16:0) and ratio of saturated fatty acid to unsaturated fatty acid decreased in cells of V. parahaemolyticus. The recovery of the ethanol-shocked cells on TSA plus 6.0% or 7.5% NaCl was significantly less than the control cells. Furthermore, ethanol shock increased the survival of V. parahaemolyticus in the presence of H(2)O(2), while made the test organism less resistant to crystal violet, high NaCl and organic acids. The degree of decreased acid tolerance observed on the ethanol-shocked cells of test organism varied with the organic acid examined. Finally, ethanol shock for 60 min exhibited either a higher or similar degree of effect on the test organism (depending on the type of stress encountered) than did ethanol shock for 30 min. Data obtained from the present study does provide useful information that is indispensable when control measure of V. parahaemolyticus is to be performed efficiently and adequately.

The Effect of Heat Shock on the Response of Cronobacter sakazakii to Subsequent Lethal Stresses

Cronobacter sakazakii, formerly Enterobacter sakazakii, has been implicated in a severe form of neonatal meningitis. In this study, C. sakazakii BCRC 13988 was first exposed to heat-shock treatment at 47 degrees C for 15 min. The heat-shocked C. sakazakii was subjected to several lethal challenges including low temperature (3 degrees C and -20 degrees C), pH 3.3, 15% ethanol, high osmotic pressure (tryptic soy broth + 75% sorbitol, a(w) 0.81), and drying. It was found that heat shock significantly (p < 0.05) enhanced the resistance of C. sakazakii to all the lethal stresses examined. After 60 min of exposure to 15% ethanol, the survival of the heat-shocked cells was approximately 752 times that of the nonshocked cells. Compared with the nonshocked C. sakazakii, the heat-shocked cells exhibited a 322- and 1.6-fold increase in survival after 7 days of exposure to -20 degrees C and 3 degrees C, respectively. A 48-fold increase in the survival was noted with the heat-shocked cells after 6 h of exposure to dry air (relative humidity 37%) at 25 degrees C, showing a survival of 0.00107% which is approximately 50-fold that of the control. After 36 h of exposure to the high osmotic stress environment, the survival of the heat-shocked C. sakazakii was found to be approximately 119 times that of the control cells. Finally, an increased survival of approximately 72 times that of the control cells was observed with the heat-shocked C. sakazakii after 60 min of challenge at pH 3.3.

Survival of the acid-adapted Bacillus cereus in acidic environments

In this study, the acid tolerance of Bacillus cereus 1-4-1 after adaptation at pH 5.5 for 1, 2 and 4 h was first determined. The survival of acid-adapted and non-adapted cells of B. cereus in phosphate buffer solution (PBS pH 4.0) containing various organic acids such as acetic, propionic, citric, lactic or tartaric acid as well as in a commercial acidic beverage of mixed fruits and vegetables (pH 3.7) was then examined. Results revealed that acid adaptation time influenced the increased tolerance of B. cereus in PBS (pH 4.0). The 2 h-adapted cells exhibited the highest acid tolerance in PBS. The presence of chloramphenicol during the acid adaptation reduced the extent of increased acid tolerance. Acid adaptation was also found to enhance the tolerance of the test organism in the presence of the various organic acids tested. While the extent of increased acid tolerance varied with the organic acid examined. Acid-adapted B. cereus cells exhibited the largest extent of increased tolerance, showing an increased survival of ca. 1000 folds, in the propionic acid-containing PBS. Additionally, a higher survival percentage was noted with the acid-adapted than the non-adapted cells of B. cereus in the acidic beverage stored at 4 or 25 degrees C.

Protein expression in Vibrio parahaemolyticus 690 subjected to sublethal heat and ethanol shock treatments.

Cells of Vibrio parahaemolyticus 690 were subjected either to heat shock at 42 degrees C for 45 min or to ethanol shock in the presence of 5% ethanol for 60 min. The protein profiles of the unstressed and stressed V. parahaemolyticus cells were compared. Additionally, the induction of DnaK- and GroEL-like proteins in the unstressed and stressed cells of V. parahaemolyticus was also examined. Analysis with one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that three proteins with molecular masses of 93, 77, and 58 kDa were induced by both heat shock and ethanol shock. The protein patterns revealed by two-dimensional electrophoresis were more detailed than those revealed by one-dimensional SDS-PAGE. It was found that heat shock and ethanol shock affected the expression of a total of 28 proteins. Among them, four proteins with molecular masses of 94, 32.1, 26.7, and 25.7 kDa were enhanced by both heat shock and ethanol shock. Furthermore, immunoblot analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the test organism, with the heat-shocked and ethanol-shocked cells producing a GroEL-like protein in a larger quantity than the unstressed cells. However, DnaK-like protein was not detectable in either the unstressed or the stressed cells.

Survival of Vibrio parahaemolyticus under environmental stresses as influenced by growth phase and pre-adaptation treatment.

In this study, the susceptibility of Vibrio parahaemolyticus in different growth phases after exposure to lethal stresses including 47 degrees C and 8% ethanol was first investigated. The effect of a cultures growth phase on both the heat and ethanol shock response of V. parahaemolyticus was then examined. It was found that cells of V. parahaemolyticus in the mid-exponential phase, regardless of adaptation, were most susceptible to environmental stresses, while cells in the stationary phase were least susceptible to the lethal stresses examined. Adaptation with heat shock at 42 degrees C for 45 min or ethanol shock with 5% ethanol for 60 min induced an increased resistance of V. parahaemolyticus to subsequent lethal stresses at 47 degrees C and 8% ethanol. While the adaptation treatments resulted in a reduced resistance of the test organism to pH 4.4 and 20% NaCl. Generally, the extent of changes in the resistance of V. parahaemolyticus to lethal stresses between the adapted and control cells was found to be growth phase dependent. Compared with the respective control cells, the adapted late-exponential phase cells exhibited the greatest extent of change, while the adapted stationary phase cells showed the least change in their resistance to the lethal stresses examined.

The effect of acid adaptation on the susceptibility of Bacillus cereus to the stresses of temperature and H2O2 as well as enterotoxin production.

In the present study, Bacillus cereus 1-4-1, which is capable of causing diarrheal syndrome, was subjected to acid adaptation at pH 5.5 for 2 hours. The effect of acid adaptation on the survival of B. cereus subjected to subsequent lethal challenges at both low (4 degrees and -18 degrees C) and high temperatures (49 degrees C) as well as in the presence of 5 mM H(2)O(2) was investigated. Additionally, enterotoxin production by B. cereus as influenced by acid adaptation was examined. Results revealed that acid adaptation increased the survival of B. cereus during storage at -18 degrees C while a decreased survival was noted for the acid-adapted cells during storage at 4 degrees C. In addition, the acid-adapted cells were less susceptible to high temperature than the nonadapted cells. On the other hand, acid adaptation did not change the susceptibility of test organism to H(2)O(2). It was also found that acid adaptation time affected the enterotoxin production of B. cereus cells. The 1- to 2-hour acid-adapted cells exhibited a reduced level of enterotoxin production while cells acid adapted for 4 hours and the nonadapted cells showed no difference in the level of enterotoxin production. Besides, the acid-adapted cells showed a reduced lag period for growth and enterotoxin production when they were grown in tryptic soy broth.