Impact of cold and cold-acid stress on poststress tolerance and virulence factor expression of Escherichia coli O157:H7

The effect of extended cold or cold-acid storage of Escherichia coli O157:H7 on subsequent acid tolerance, freeze-thaw survival, heat tolerance, and virulence factor (Shiga toxin, intimin, and hemolysin) expression was determined. Three E. coli O157:H7 strains were stressed at 4 degrees C in TSB or pH 5.5 TSB for 4 weeks. The acid (TSB [pH 2.0] or simulated gastric fluid [pH 1.5]) tolerance, freeze-thaw (-20 degrees C to 21 degrees C) survival, and heat (56 degrees C) tolerance of stressed cells were compared with those of control cells. The beta-galactosidase activities of stressed and control cells containing a lacZ gene fusion in the stx2, eaeA, or hlyA gene were determined following stress in TSB or pH 5.5 TSB at 37 degrees C and in the exponential and stationary phases. Cold and cold-acid stresses decreased acid tolerance (P < 0.05), with a larger decrease in acid tolerance being observed after cold stress than after cold-acid stress (P < 0.05). Cold stress increased freeze-thaw survival for all three strains (P < 0.05). Prior cold or cold-acid stress had no effect on virulence factor production (P > 0.05), although growth in acidic media (pH 5.5) enhanced eaeA and hlyA expression (P < 0.05). These results indicate that the prolonged storage of E. coli O157:H7 at 4 degrees C has substantial effects on freeze-thaw tolerance but does not affect subsequent virulence gene expression.     

Acid stress, starvation, and cold stress affect poststress behavior of Escherichia coli O157:H7 and nonpathogenic Escherichia coli.

The effects of acid shock, acid adaptation, starvation, and cold stress of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant (FRIK 816-3), and nonpathogenic E. coli (ATCC 25922) on poststress heat resistance and freeze-thaw resistance were investigated. Following stress, heat tolerance at 56 degrees C and freeze-thaw resistance at -20 to 21 degrees C were determined. Heat and freeze-thaw resistance of E. coli O157:H7 and nonpathogenic E. coli was enhanced after acid adaptation and starvation. Following cold stress, heat resistance of E. coli O157:H7 and nonpathogenic E. coli was decreased, while freeze-thaw resistance was increased. Heat and freeze-thaw resistance of the rpoS mutant was enhanced only after acid adaptation. Increased or decreased tolerance of acid-adapted, starved, or cold-stressed E. coli O157:H7 cells to heat or freeze-thaw processes should be considered when processing minimally processed or extended shelf-life foods.     

Growth and Survival Differences of Vibrio vulnificus and Vibrio parahaemolyticus Strains during Cold Storage

ABSTRACT:  Vibrio vulnificus  and  Vibrio parahaemolyticus  are the most common  Vibrio  species associated with seafood illness in the United States. Our study was conducted to determine if strain-to-strain differences exist in the growth and survival of 8 different  V. vulnificus  and  V. parahaemolyticus  strains at low temperatures. By day 10,  V. vulnificus  strain 515-4C2 had significantly higher counts ( P  < 0.05) (1.97 log CFU/g) compared with strains 3315, 1007, 29306 at 5 °C, which reached nondetectable levels. At 8 °C, strain 515-4C2 had significantly higher counts ( P  < 0.05) (2.23 log CFU/mL) compared with 1007, 33815, 541(O) 49C, which reached nondetectable levels. At 10 °C, only  V. vulnificus  strain 33815 reached nondetectable levels. At 5 °C,  V. parahaemolyticus  strain 541(O) 57C had the highest counts (5.28 log CFU/g) by day 10 while strain 33847 had significantly lower counts (3.46 log CFU/g). After 10 d at 8 °C,  V. parahaemolyticus  strain M350A had the highest counts (7.97 log CFU/mL) while strain 541(O) 57C had the lowest counts (4.80 log CFU/mL). At 10 °C,  V. parahaemolyticus  strain NY477 had significantly higher counts ( P  < 0.05) with 8.31 log CFU/mL compared with strain 33847, which had the lowest counts (6.77 log CFU/mL). Our research has shown that various  V. vulnificus  and  V. parahaemolyticus  strains vary in their ability to survive and grow at refrigeration temperatures.     

Conditions for a 5-log reduction of Vibrio vulnificus in oysters through high hydrostatic pressure treatment

Vibrio vulnificus is frequently associated with oysters, and since oysters are typically consumed raw on a half shell, they can pose a threat to public health due to ingestion of this pathogenic marine microorganism. Oysters should be processed to reduce the number of this pathogen. High pressure processing is gaining more and more acceptance among oyster processors due to its ability to shuck oysters while keeping the fresh-like characteristics of oysters. Nine strains of V. vulnificus were tested for their sensitivities to high pressure. The most pressure-resistant strain of V. vulnificus, MLT 403, was selected and used in the subsequent experiments to represent a worst case scenario for evaluation of the processing parameters for inactivation of V. vulnificus in oysters. To evaluate the effect of temperature on pressure inactivation of V. vulnificus, oyster meats were inoculated with V. vulnificus MLT 403 and incubated at room temperature for 24 h. Oyster meats were then blended and treated at 150 MPa for 4 min, and 200 MPa for 1 min. Pressure treatments were carried out at -2, 1, 5, 10, 20, 30, 40, and 45 degrees C. Cold temperatures (<20 degrees C) and slightly elevated temperatures (>30 degrees C) substantially increased pressure inactivation of V. vulnificus. For example, a 4-min treatment of 150 MPa at -2 and 40 degrees C reduced the counts of V. vulnificus by 4.7 and 2.8 log, respectively, while at 20 degrees C the same treatment only reduced counts by 0.5 log. Temperatures of -2 and 1 degrees C were used to determine the effect of pressure level, temperature, and treatment time on the inactivation of V. vulnificus infected to live oysters through feeding. To achieve a >5-log reduction in the counts of V. vulnificus in a relatively short treatment time (or=250 MPa at -2 or 1 degrees C.     

Recovery and detection of Vibrio vulnificus during cold storage

Different cultural techniques and molecular methods for the detection of Vibrio vulnificus during cold storage in a model broth system were compared. Two strains of V. vulnificus were grown to stationary phase and inoculated (10(6) CFU/mL) into tryptic soy broth with 2% sodium chloride (TSBN2) or artificial seawater (ASW), both pre-chilled to 5 degrees C. These were stored for 10 days, with sub-sampling conducted at time 0 and every 2 days thereafter. Each subsample was plated, by both pour and spread plate techniques, onto tryptic soy agar 2% sodium chloride (TSAN2) with or without catalase (400 or 600 U) or sodium pyruvate (80 or 160 mg) supplementation. Nucleic acids were extracted from subsamples and subjected to PCR and RT-PCR with hemolysin as the target. Higher recoveries of V. vulnificus were obtained with spread plating compared to pour plating (P<0.05). The addition of sodium pyruvate (80 mg) or catalase (400 U) significantly increased cell recovery (P<0.05). PCR amplification signals were stronger than RT-PCR signals at each timepoint, and results were generally consistent between TSAN2 and ASW for each strain. These results will aid in the design of optimum methods to recover and/or detect V. vulnificus cells subjected to sublethal stress that might be encountered in food processing and storage.     

冷诱导金黄色葡萄球菌环境胁迫耐受性及生物被膜控制

为研究金黄色葡萄球菌经冷诱导后对其他环境胁迫耐受性的变化,将耐冷冻能力较强的分离株S.aureus W3与标准菌株S.aureus CICC10201分别在4 ℃下冷胁迫5 h后,测定其在高温、酸碱、氯化钠、过氧化氢、酒精的胁迫下存活率变化。同时对分离株S.aureus W3的生物被膜形成及控制进行了研究。结果表明,经-20 ℃冷藏8周后,S.aureus W3的存活率显著高于S.aureus CICC10201(p<0.05)。经冷诱导后,S.aureus W3在45~60 ℃环境下存活率提升至18.5%。冷诱导使金黄色葡萄球菌对酸敏感性增强但对碱产生交叉抗性。冷诱导后的两株菌在氯化钠中的存活率均降低,S.aureus W3最低下降了24.09%。另外,冷诱导后金黄色葡萄球菌对酒精的耐受性增强。分离株S.aureus W3在冷诱导前后过氧化氢耐受性没有明显变化(p>0.05),但标准菌株S.aureus CICC10201敏感性增强,存活率显著下降(p<0.05)。分离株S.aureus W3较S.aureus CICC10201具有更强的生物被膜形成能力,运用1% 84消毒水能有效清除其生物被膜。本研究为冷冻食品中金黄色葡萄球菌的安全控制提供理论依据。     

Susceptibility of the heat-, acid-, and bile-adapted Vibrio vulnificus to lethal low-salinity stress

As a marine pathogenic bacterium that inhabits seawater or seafood, Vibrio vulnificus encounters low salinity and other stresses in the natural environment and during food processing. This investigation explores the cross-protective response of sublethal heat-, acid-, or bile-adapted V. vulnificus YJ03 against lethal low-salinity stress. Experimental results reveal that the acid (pH 4.4)- and heat (41 degrees C)-adapted V. vulnificus were not cross-protected against the lethal low-salinity challenge (0.04% NaCl). The bile (0.05%)-adapted exponential- and stationary-phase cells were cross-protected against low salinity, whereas low-salinity (0.12% NaCl)-adapted stationary cells were sensitized against 12% bile stress. Results of this study provide further insight into the interaction between low salinity and other common stresses in V. vulnificus.     

Use of mutant strain for evaluating processing strategies to inactivate Vibrio vulnificus in oysters.

Vibrio vulnificus is a ubiquitous marine bacterium frequently isolated from shellfish and associated with severe and often fatal disease in humans. Various control strategies to reduce the disease risk associated with V. vulnificus contamination in shellfish have been proposed. However, evaluating the efficacy of these control strategies is complicated because of the difficulty in distinguishing V. vulnificus from the high levels of background environmental Vibrio spp. The purpose of this research was to develop a model indicator V. vulnificus strain that could be readily differentiated from background microflora and used to facilitate the evaluation of processing efficacy. A spontaneous nalidixic acid-resistant strain of V. vulnificus (Vv-NA) was prepared from a wild-type parent (Vv-WT) using selective plating techniques. Vv-NA was very similar to Vv-WT with respect to biochemical characteristics, appearance on selective plating media, detection limits using most probable number and polymerase chain reaction, and growth rate. In comparative freeze inactivation studies on pure cultures, Vv-WT and Vv-NA had similar freeze inactivation profiles at -20 degrees C (conventional freezing), at -85 degrees C (cold blast freezing), and in liquid nitrogen (cryogenic freezing). In oyster homogenates artificially inoculated with Vv-NA, the organism was inactivated 95 to 99% after freezing, irrespective of freezing temperature. Thermal inactivation comparisons of pure cultures of Vv-WT and Vv-NA using the capillary tube method revealed statistically significant differences in D values at 47 degrees C (2.2 versus 3.0 min, respectively) and 50 degrees C (0.83 versus 0.56 min, respectively), but nearly identical values at 52 degrees C (0.21 versus 0.22 min, respectively). However, these D values were notably higher than those reported by other investigators and hence provided a conservative means by which to evaluate thermal inactivation. In oyster homogenates seeded with Vv-NA, D values of 1.3+/-0.09 min and 0.41+/-0.01 min were obtained at 46 degrees C and 48 degrees C, respectively. This study demonstrated that Vv-NA is readily enumerated and could be used as a surrogate for evaluating the degree of V. vulnificus inactivation provided by freezing and thermal treatments of oyster homogenates.     

Impact of acid on survival of Vibrio vulnificus and Vibrio vulnificus phage

Three strains of Vibrio vulnificus and V. vulnificus phages were tested for acid sensitivity at 21 degrees C. V. vulnificus strain 304 was more resistant to pH 4.0 than strains CVD-1 and A-9, whereas acid sensitivities of V. vulnificus strains at pH 3.0 and 2.0 were similar. V. vulnificus phage strain 110A-7 was more resistant to pH 4.0 than strain 153A-7, whereas acid sensitivities of phage strains at pH 3.5 and 3.0 were similar. Numbers of V. vulnificus and its phage were close to the limit of detection after 100 s at pH 2.0 and after 24 min at pH 3.0. Acid D-values at 21 degrees C decreased as pH decreased for both V. vulnificus and phages. D-values of phage strains at pH 3.5 were 10-fold greater than those of host strain at pH 4.0. D-values of phage strains were slightly greater than those of host strain at pH 3.0. These results suggest that V. vulnificus and its phage were very sensitive to pH of less than 3.0, although V. vulnificus phages were more resistant to acid than their host.     

Microbiological quality and safety of quahog clams, Mercenaria mercenaria, during refrigeration and at elevated storage temperatures

The effects of storage temperatures and times on the microbiological quality and safety of hard-shelled quahog clams (Mercenaria mercenaria) were examined. Samples were stored at four different incubation temperatures (3.3, 7.2, 10.0, and 12.8 degrees C) for a period of 3 weeks, following their harvest from summer growing waters (> or = 27 degrees C) and winter waters (< or = 4 degrees C). Clams were analyzed for two naturally occurring pathogens, Vibrio parahaemolyticus and Vibrio vulnificus. During the summer, V. parahaemolyticus was isolated from 56% of the stored samples, with the highest concentration, 6,100/g, occurring on day 12 at 12.8 degrees C. Also, during the summer, V. vulnificus was isolated from 11% of the stored samples, with the highest concentration of 1,500/g occurring on day 15 at 7.2 degrees C. No Vibrio spp. were detected during the winter. During summer storage, aerobic mesophilic counts on plate count agar (PCA) containing 2% NaCl ranged from 10(4) to 10(8) CFU/g, and during storage of the winter samples, aerobic mesophilic PCA (with added NaCl) counts ranged from <100 to 10(4) CFU/g. Comparatively, summer storage mesophilic counts on PCA containing no added NaCl ranged from <100 to 10(5) CFU/g, and for the winter samples the range was <100 to 10(2) CFU/g. Coliform and fecal coliform counts ranged from <0.3 to 61.1/g and <0.3 to 24.4/g, respectively. There was no statistical correlation between the length of storage or the temperature of incubation and the presence of V. parahaemolyticus, V. vulnificus, coliforms, or fecal coliforms. However, storage time and incubation temperature affected the PCA counts (P < or = 0.05) in quahog clams.     

Growth and stress resistance variation in culture broth among Listeria monocytogenes strains of various serotypes and origins

Twenty-five Listeria monocytogenes strains of various serotypes and sources, including clinical and food isolates associated with the same outbreaks, were characterized and compared based on growth rates and heat and acid death rates. Growth was monitored in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 4 and 30 degrees C for 32 days and 20 h, respectively. Heat and acid stress responses in TSBYE heated to 55 degrees C or acidified to pH 3.0 with lactic acid were evaluated for 240 or 120 min, respectively. Extensive variation in growth and stress resistance was observed among the tested strains. Growth rate differences were less evident at 30 than at 4 degrees C, where growth rates (log CFU per milliliter per day) ranged from 0.28 to 0.43. Thermal and acid death rates (log CFU per milliliter per minute) ranged from -0.023 to -0.052 and from -0.012 to -0.134, respectively. Serotype appeared to play a significant role (P < 0.05) only with respect to the heat resistance of the organism. Serotype 4b isolates as a group had lower heat resistance than did isolates representing all other serotypes combined. Although no clear origin-related (food versus clinical) trends were observed under the tested conditions, outbreak-related isolates of serotype 4b had lower acid death rates (higher acid resistance) (P < 0.05) than did the rest of the strains belonging to this serotype. Strain Scott A exhibited slow growth at 4 degrees C and low acid resistance, behavior that was distinct among both clinical and serotype 4b isolates. The results of this study highlight the risks associated with extrapolation to other strains of findings obtained with only one strain of L. monocytogenes. This information should be useful when test strains are to be selected for the evaluation of antimicrobial alternatives in ready-to-eat meat and other food products and when risk assessments are to be conducted.     

Effectiveness of icing as a postharvest treatment for control of Vibrio vulnificus and Vibrio parahaemolyticus in the eastern oyster (Crassostrea virginica)

The focus of this research was to investigate the efficacy of icing as a postharvest treatment for reduction of the levels of Vibrio vulnificus and Vibrio parahaemolyticus in commercial quantities of shellstock oysters. The experiments were conducted in June and August of 2006 and consisted of the following treatments: (i) on-board icing immediately after harvest; (ii) dockside icing approximately 1 to 2 h prior to shipment; and (iii) no icing (control). Changes in the levels of pathogenic Vibrio spp. during wholesale and retail handling for 2 weeks postharvest were also monitored. On-board icing achieved temperature reductions in all sacks in accordance with the National Shellfish Sanitation Program standard, but dockside icing did not meet this standard. Based on one-way analysis of variance, the only statistically significant relationship between Vibrio levels and treatment occurred for samples harvested in August; in this case, the levels of V. vulnificus in the noniced oysters were significantly higher (P < 0.05) than were the levels in the samples iced on-board. When analyzing counts over the 14-day storage period, using factorial analysis, there were statistically significant differences in V. vulnificus and V. parahaemolyticus levels by sample date and/or treatment (P < 0.05), but these relationships were not consistent. Treated (iced) oysters had significantly higher gaping (approximately 20%) after 1 week in cold storage than did noniced oysters (approximately 10%) and gaping increased significantly by day 14 of commercial storage. On-board and dockside icing did not predictably reduce the levels of V. vulnificus or V. parahaemolyticus in oysters, and icing negatively impacted oyster survival during subsequent cold storage.     

Impact of antibiotic stress on acid and heat tolerance and virulence factor expression of Escherichia coli O157:H7

This study was conducted to determine the effect of antibiotic stress on the virulence factor expression, simulated gastric fluid (SGF; pH 1.5) survival, and heat tolerance (56 degrees C) of Escherichia coli O157:H7. The MIC for three antibiotics (trimethoprim, ampicillin, and ofloxacin) was determined for two E. coli O157:H7 strains (ATCC 43895 [raw hamburger isolate] and ATCC 43890 [fecal isolate]) by the dilution series method. Subsequently, cells were stressed at the MIC of each antibiotic for 4 h, and poststress tolerance and virulence factor production were evaluated. Heat tolerance (56 degrees C) was determined by the capillary tube method, and SGF (pH 1.5) survival was used to assess acid tolerance. Virulence factor expression (stx, hlyA, and eaeA) was evaluated by the creation of lacZ gene fusions and then use of the Miller assay (a beta-galactosidase assay). Stressed and control cells were evaluated in triplicate. The MIC for trimethoprim was 0.26 mg/liter for both strains; for ampicillin, it was 2.05 mg/liter for both strains; and for ofloxacin, it was 0.0256 and 0.045 mg/liter for each strain. Heat tolerance and SGF survival following antibiotic stress decreased when compared with control cells (P < 0.05). Exposure to ofloxacin increased stx and eaeA expression (P < 0.05). Exposure to ampicillin or trimethoprim increased eaeA expression (P < 0.05). hly expression increased following trimethoprim stress (P < 0.05). Antibiotics can increase E. coli O157:H7 virulence factor production, but they do not produce a cross-protective response to heat or decreased pH.     

Effect of nutrient starvation on the resistance of Escherichia coli O157:H7 to subsequent heat stress

The resistance of three strains of Escherichia coli O157:H7 in their stationary growth phase to starvation (24 h in water at 37 degrees C) followed by a heat treatment (56 degrees C for up to 90 min) was determined. Starvation was found to increase significantly the resistance of two strains (NCTC 12079; eae+, VT1+, VT2+, and ATCC 43889 eae+, VT2+) but not the remaining strain (ATCC 43890 eae+, VT1+). Strain NCTC 12079 (only one tested) was shown to retain all of the three virulence factors after the two stresses. De novo protein synthesis was shown to be required for heat resistance. Evidence using an rpoS mutant indicated a central role for this gene in inducing heat resistance after a starvation stress. It is hoped that this work will contribute to more accurate risk assessments in certain food processing operations.     

低温贮藏条件下创伤弧菌和副溶血性弧菌失活模型的建立

为掌握对虾中创伤弧菌和副溶血性弧菌在5℃和一18~C低温贮藏条件下失活动力学特征,分别采用线性模型、Weibull模型、Logistic模型对创伤弧菌和副溶血性弧菌的失活曲线进行拟合。研究结果表明,在5~C条件下,创伤弧茵VvHB09的耐冷力较强;．18℃条件下,副溶血性弧菌ATCC17802和创伤弧菌VvSH09的耐冷力较强。线性模型比weibull模型、Logistic模型更适合拟合创伤弧菌和副溶血性弧菌的失活特征。     

Growth and recovery of selected gram-negative bacteria in reconditioned wastewater.

Previous reports indicate that Escherichia coli O157:H7, Salmonella spp., and Vibrio cholerae can grow in nutrient-limited, reconditioned wastewater over the temperature range of 4 to 46 degrees C when the biological oxygen demand of this water is <2, while its coliform growth response (CGR) is >2. In the current study, we investigated the growth response of Vibrio parahaemolyticus, Shigella spp., Vibrio vulnificus, and Pseudomonas aeruginosa in water samples with a CGR of >2 over the temperature range of 4 to 50 degrees C. Both the nonselective media, tryptic soy agar, and the selective media used to identify the pathogen were used for their recovery. The selective media were thiosulfate-citrate-bile-sucrose (TCBS), MacConkey agar (MAC), and Pseudomonas isolation agar (PIA) for the Vibrio, Shigella, and Pseudomonas spp., respectively. V. parahaemolyticus numbers declined rapidly after surviving for 6 days under the nutrient-limiting growth conditions. Shigella spp. did not grow but survived for >28 days at 4 to 25 degrees C. V. vulnificus grew over the narrow temperature range of 12 to 21 degrees C and survived for >21 days at the higher and lower temperature ranges. P. aeruginosa survived and grew during the 14-day test period at 13 to 35 degrees C. Recovery on the nonselective agar gave statistically (P > 0.05) higher numbers than the respective selective media commonly used for these pathogens. These results indicate that caution should be used in attempting direct recoveries using selective media of the four gram-negative bacteria species used in this study from the nutrient-limited water environment.     

Thermal resistance of heat-, cold-, and starvation-injured Salmonella in irradiated comminuted Turkey

To investigate the effects of sublethal stress on Salmonella thermal inactivation kinetics, an eight-strain Salmonella cocktail was subjected to heat shock (30 min at 54 degrees C), cold shock (2 h at 4 degrees C), and starvation stress (10 days in phosphate buffer at 4 degrees C), harvested by centrifugation, and inoculated into irradiated comminuted turkey. Immediately after stressing, the Salmonella cocktails contained 89.1% heat-injured, 44.7% cold-injured, and 67.7% starvation-injured cells, as determined by plating on selective and nonselective media. D60 degrees C-values for the heat-shocked cocktail (0.64 min on Trypticase soy agar containing 0.6% yeast extract [TSAYE], 0.35 min on xylose lysine desoxycholate [XLD] agar) were higher (P < 0.05) than those for the unshocked control (0.41 min on TSAYE, 0.17 min on XLD), whereas D60 degrees -values for the cold-shocked cocktail (0.38 min on TSAYE, 0.17 min on XLD) were not significantly different from those for the control. Starved cells had the same D60 degrees C-value on TSAYE as did the unshocked cocktail, but the D60 degrees C-value on XLD was significantly lower (0.14 min). Although starvation and cold shock were not thermally protective, heat shock increased thermal resistance, indicating that product history and the physiological state of the Salmonella cells should be considered when developing and validating thermal processes. D60 degrees C-values observed on selective media were significantly lower than those observed on nonselective media for all stress treatments and for the control. Therefore, nonselective culture media should be used to assess the response of microorganisms to a thermal challenge when developing performance standards for lethality.     

Characterization of the low-salinity stress in Vibrio vulnificus

Vibrio vulnificus is a marine pathogenic bacterium commonly found in seawater or seafood. This organism encounters low-salinity stress in its natural environment and during food processing. This study was designed to investigate the response of V. vulnificus YJ03 to lethal low salinity (0.04% NaCl) and its adaptation to sublethal salinity (0.12% NaCl with 20 amino acids added). A short period in the nonculturable state was induced by lethal low-salinity stress followed by cell death after 30 min of stress. Addition of 1 mM glycine betaine or 0.5 mM sucrose reduced the damage. Low-salinity adaptation was achieved in the exponential-phase cells but not in the stationary-phase cells. Significant protection against lethal low-salinity stress was attained when the cells were adapted for as little as 1.5 min. The adapted cells were significantly protected against lethal low salinity and 2.4% sodium sorbate but sensitized to the challenge of heat (52 degrees C) and acid (pH 3.2). Nonlethal low-salinity treatment of seafood should be avoided to prevent stress adaptation of V. vulnificus.