Effects of acid adaptation of Escherichia coli O157:H7 on efficacy of acetic acid spray washes to decontaminate beef carcass tissue

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4 degrees C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.     

Effects of Acid Adaptation of Escherichia coli O157:H7 on Efficacy of Acetic Acid Spray Washes To Decontaminate Beef Carcass Tissue

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4°C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

Survival of Escherichia coli O157:H7 in broth and processed salami as influenced by pH, water activity, and temperature and suitability of media for its recovery.

The survival of unheated and heat-stressed (52 degrees C, 30 min) cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) adjusted to various pHs (6.0, 5.4, and 4.8) with lactic acid and various water activities (a(w)s) (0.99, 0.95, and 0.90) with NaCl and incubated at 5, 20, 30, and 37 degrees C was studied. The performance of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), and modified eosin methylene blue agar in supporting colony development of incubated cells was determined. Unheated cells of E. coli O157:H7 grew to population densities of 10(8) to 10(9) CFU ml-1 in TSB (pHs 6.0 and 5.4) at an a(w) of 0.99. Regardless of the pH and a(w) of TSB, survival of E. coli O157:H7 was better at 5 degrees C than at 20 or 30 degrees C. At 30 degrees C, inactivation or inhibition of growth was enhanced by reduction of the a(w) and pH. A decrease in the a(w) (0.99 to 0.90) of TSB in which the cells were heated at 52 degrees C for 30 min resulted in a 1.5-log10 reduction in the number of E. coli O157:H7 cells recovered on TSA; pH did not significantly affect the viability of cells. Recovery was significantly reduced on MSMA when cells were heated in TSB with reduced pH or a(w) for an increased length of time. With the exception of TSB (a(w), 0.90) incubated at 37 degrees C, heat-stressed cells survived for 24 h in recovery broth. TSB (a(w), 0.99) at pH 6.0 or 5.4 supported growth of E. coli O157:H7 cells at 20 or 37 degrees C, but higher numbers of heated cells survived at 5 or 20 degrees C than at 37 degrees C. The ability of unheated and heat-stressed E. coli O157:H7 cells to survive or grow as affected by the a(w) of processed salami was investigated. Decreases of about 1 to 2 log10 CFU g-1 occurred soon after inoculation of salami (pHs 4.86 and 4.63 at a(w)s of 0.95 and 0.90, respectively). Regardless of the physiological condition of the cells before inoculation into processed salami at an a(w) of either 0.95 or 0.90, decreases in populations occurred during storage at 5 or 20 degrees C for 32 days. If present at < or = 100 CFU g-1, E. coli O157:H7 would unlikely survive storage at 5 degrees C for 32 days. However, contamination of salami with E. coli O157:H7 at 10(4) to 10(5) CFU g-1 after processing would pose a health risk to consumers for more than 32 days if storage were at 5 degrees C. Regardless of the treatment conditions, performance of the media tested for the recovery of E. coli O157:H7 cells followed the order TSA > modified eosin methylene blue agar > MSMA.     

Thermal tolerance of acid-adapted and unadapted Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice

AIMS: A study was performed to determine D values of acid-adapted and unadapted cells of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice. METHODS AND RESULTS: Salmonella enterica serotype Poona, S. enterica serotype Saphra, two strains of E. coli O157:H7, and two strains of L. monocytogenes were grown in tryptic soy broth (TSB) and TSB supplemented with 1% glucose for 24 h at 37 degrees C. Decimal reduction times (D values) of cells suspended in unpasteurized cantaloupe juice and watermelon juice were determined. Acid-adapted cells of Salmonella and E. coli O157:H7, but not L. monocytogenes, had increased thermal tolerance compared with cells that were not acid-adapted. There was no correlation between soluble solids content of the two types of juice and thermal resistance. CONCLUSIONS: Growth of Salmonella and E. coli O157:H7 in cantaloupe juice, watermelon juice, or other acidic milieu, either in preharvest or postharvest environments, may result in cross protection to heat. The pasteurization conditions necessary to achieve elimination of pathogens from these juices would consequently have to be more severe if cells are habituated to acidic environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Insights from this study provide guidance to developing pasteurization processes to eliminate Salmonella, E. coli O157:H7, and L. monocytogenes in cantaloupe juice and watermelon juice.     

Survival and growth of Escherichia coli O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature.

A study was undertaken to determine the fate of Escherichia coli O157:H7 in ground, roasted beef as influenced by the combined effects of pH, acidulants, temperature, and time. There was essentially no change in the viable population of E. coli O157:H7 when beef salads (pH 5.40 to 6.07) containing up to 40% mayonnaise were incubated at 5 degrees C for up to 72 h. At 21 and 30 degrees C, significant (P < or = 0.05) increases in populations of the organism occurred in salads containing 16 to 32% mayonnaise (pH 5.94 to 5.55) between 10 and 24 h of incubation. Death was more rapid as the pH of acidified beef slurries incubated at 5 degrees C was decreased from 5.98 to 4.70. E. coli O157:H7 grew in control slurries (pH 5.98) and in slurries containing citric and lactic acids (pHs 5.00 and 5.40) incubated at 21 degrees C for 24 h; decreases occurred in slurries acidified to pHs 4.70, 5.00, and 5.40 with acetic acid or pH 4.70 with citric or lactic acid. At 30 degrees C, populations decreased in slurries acidified to pHs 4.70 and 5.00 with acetic acid. Citric and lactic acids failed to prevent significant increases in populations in slurries at pH 4.70 to 5.40 between 10 and 24 h of incubation. The order of effectiveness of acidulants in inhibiting growth was acetic acid > lactic acid > or = citric acid. The same order was observed for inactivation of E. coli O157:H7 in acidified (pH 5.00) beef slurry heated at 54 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.     

Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.     

Survival or growth of Escherichia coli O157:H7 in a model system of fresh meat decontamination runoff waste fluids and its resistance to subsequent lactic acid stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 +/- 0.1) or in water (pH 7.2 +/- 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25 degrees C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4 degrees C and lowest at 25 degrees C. The pathogen survived without growth in water washings at 4 and 10 degrees C, while it grew by 0.8 to 2.7 log cycles at 15 and 25 degrees C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10 degrees C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25 degrees C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15 degrees C > 10 degrees C > 4 degrees C, while at 25 degrees C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15 degrees C may maintain a higher acid resistance than when acid habituated at 4 degrees C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Evaluation of the pH-dependent,stationary-phase acid tolerance in Listeria monocytogenes and Salmonella Typhimurium DT104 induced by culturing in media with 1% glucose: a comparative study with Escherichia coli O157:H7

AIMS: To comparatively evaluate the adaptive stationary-phase acid tolerance response (ATR) in food-borne pathogens induced by culturing in glucose-containing media, as affected by strain variability and antibiotic resistance, growth temperature, challenge pH and type of acidulant. METHODS AND RESULTS: Antibiotic resistant or sensitive strains of Listeria monocytogenes, Salmonella including S. Typhimurium DT104, and Escherichia coli O157:H7 were cultured (30 degrees C for 24 h; 10 degrees C for up to 14 days) in trypticase soya broth with yeast extract (TSBYE) with 1% or without glucose to induce or prevent acid adaptation, respectively. Cultures were subsequently exposed to pH 3.5 or 3.7 with lactic or acetic acid at 25 degrees C for 120 min. Acid-adapted cultures were more acid tolerant than nonadapted cultures, particularly those of L. monocytogenes and Salmonella. No consistent, positive or negative, influence of antibiotic resistance on the pH-inducible ATR or acid resistance (AR) was observed. Compared with 30 degrees C cultures, growth and acid adaptation of L. monocytogenes and S. Typhimurium DT104 at 10 degrees C markedly reduced their ATR and AR in stationary phase. E. coli O157:H7 had the greatest AR, relying less on acid adaptation. A 0.2 unit difference in challenge pH (3.5-3.7) caused great variations in survival of acid-adapted and nonadapted cells. CONCLUSIONS: Culturing L. monocytogenes and Salmonella to stationary phase in media with 1% glucose induces a pH-dependent ATR and enhances their survival to organic acids; thus, this method is suitable for producing acid-adapted cultures for use in food challenge studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial pathogens may become acid-adapted in foods containing glucose or other fermentable carbohydrates. Low storage temperatures may substantially decrease the stationary-phase ATR of L. monocytogenes and S. Typhimurium DT104, but their effect on ATR of E. coli O157:H7 appears to be far less dramatic.     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY OF HEAT-STRESSED ESCHERICHIA COLI O157:H7

Tryptone soya agar (TSA) and three selective media, BCM^1M O157:H7(+) agar (BCM), modified eosin methylene blue agar (MEMB), and sorbitol MacConkey agar (SMAC) were evaluated for recovery of two strains of E. coli O157:H7 (salami and cider isolates) heated at 56, 58, and 60C for up to 60 min in tryptone soya broth (TSB). TSA and MEMB were equally effective at recovery of heat-stressed (56, 58, and 60C) E . coli O 157:H7 and superior to SMAC and BCM (P 0.05). When heated at 56 and 58C, recovery of E. coli O157:H7 on MEMB and TSA was not significantly different (P > 0.05); recovery was poorer on SMAC, followed by BCM (P 0.05). There was no significant difference in recovery of E. coli O157:H7 on BCM and SMAC when strains were heated at 60C (P > 0.05).     

Ethanol-mediated variations in cellular fatty acid composition and protein profiles of two genotypically different strains of Escherichia coli O157:H7

Two strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB, pH 7.1) supplemented with 0, 2.5, 5.0, 7.5, and 10% ethanol at 30 degrees C for up to 54 h. Growth rates in TSB supplemented with 0, 2.5, and 5.0% ethanol decreased with an increase in ethanol concentration. Growth was not observed in TSB supplemented with 7.5 or 10% ethanol. The pH of TSB containing 5.0% ethanol decreased to 5.8 within 12 h and then increased to 7.0 at 54 h. The ethanol content in TSB supplemented with 2.5 or 5.0% ethanol did not change substantially during the first 36 h of incubation but decreased slightly thereafter, indicating utilization or degradation of ethanol by both strains. Glucose was depleted in TSB supplemented with 0, 2.5, or 5.0% ethanol within 12 h. Cells grown under ethanol stress contained a higher amount of fatty acids. With the exceptions of cis-oleic acid and nonadecanoic acid, larger amounts of fatty acid were present in stationary-phase cells of the two strains grown in TSB supplemented with 5.0% ethanol for 30 h than in cells grown in TSB without ethanol for 22 h. The trans-oleic acid content was 10-fold higher in the cells grown in TSB with 5.0% ethanol than those grown in TSB without ethanol. In contrast, cis-oleic acid was not detected in ethanol-stressed cells but was present at concentrations of 0.32 and 0.36 mg/g of cells of the two strains grown in TSB without ethanol. Protein content was higher in ethanol-stressed cells than in nonstressed cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles varied qualitatively as affected by the strain and the presence of ethanol in TSB. An ethanol-mediated protein (28 kDa) was observed in the ethanol-stressed cells but not in control cells. It is concluded that the two test strains of E. coli O157:H7 underwent phenotypic modifications in cellular fatty acid composition and protein profiles in response to ethanol stress. The potential for cross protection against subsequent stresses applied in food preservation technologies as a result of these changes is under investigation.     

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.     

Survival or Growth of Escherichia coli O157:H7 in a Model System of Fresh Meat Decontamination Runoff Waste Fluids and Its Resistance to Subsequent Lactic Acid Stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 ± 0.1) or in water (pH 7.2 ± 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25°C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4°C and lowest at 25°C. The pathogen survived without growth in water washings at 4 and 10°C, while it grew by 0.8 to 2.7 log cycles at 15 and 25°C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10°C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25°C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15°C > 10°C > 4°C, while at 25°C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15°C may maintain a higher acid resistance than when acid habituated at 4°C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice

Outbreaks of diarrhoea and haemolytic uraemic syndrome have been associated with the consumption of apple cider and apple juice. The organism implicated in these outbreaks has been Escherichia coli O157:H7, indicating the resistance of the serotype to acidic pH. On comparing the growth of this serotype with a control strain of E. coli, it was found that strain O157:H7 grew well in trypticase soy broth at pH levels ranging from 2.0 to 9.0, while control strains failed to grow at pH levels below 4.0 and above 9.0. The growth of both strains were inhibited by adding 0.05% of either benzoic acid or sorbic acid. Similarly, O157:H7 grew well in both natural (unpasteurized) as well as in pasteurized apple juice and the growth was inhibited by adding 0.1% of either benzoic acid or sorbic acid. Control strains of E. coli failed to grow in either types of apple juice. The possible sources of contamination of natural apple juice with O157:H7 serotype are discussed.     

The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica

The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype O : 9, two of serotype O : 3, and one each of serotypes O : 8 and O : 5, 27. In a culture medium acidified with HC1 to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20°, 10°, 7° and 4°C for 21 d was in the ranges 4.18–4.36, 4.26–4.50, 4.36–4.83 and 4.42–4.80, respectively. The minimum pH for growth was also determined in media that contained 17, 33 and 50 mmol/1 acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/1 citric acid adjusted to pH values between 41 and 4.9 at intervals of approximately 0.2 unit, and 22, 44, and 111 mmol/1 lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic > lactic > citric and the minimum inhibitory concentrations were greater at higher temperatures.     

Ethanol-Mediated Variations in Cellular Fatty Acid Composition and Protein Profiles of Two Genotypically Different Strains of Escherichia coli O157:H7

Two strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB, pH 7.1) supplemented with 0, 2.5, 5.0, 7.5, and 10% ethanol at 30°C for up to 54 h. Growth rates in TSB supplemented with 0, 2.5, and 5.0% ethanol decreased with an increase in ethanol concentration. Growth was not observed in TSB supplemented with 7.5 or 10% ethanol. The pH of TSB containing 5.0% ethanol decreased to 5.8 within 12 h and then increased to 7.0 at 54 h. The ethanol content in TSB supplemented with 2.5 or 5.0% ethanol did not change substantially during the first 36 h of incubation but decreased slightly thereafter, indicating utilization or degradation of ethanol by both strains. Glucose was depleted in TSB supplemented with 0, 2.5, or 5.0% ethanol within 12 h. Cells grown under ethanol stress contained a higher amount of fatty acids. With the exceptions of cis-oleic acid and nonadecanoic acid, larger amounts of fatty acid were present in stationary-phase cells of the two strains grown in TSB supplemented with 5.0% ethanol for 30 h than in cells grown in TSB without ethanol for 22 h. The trans-oleic acid content was 10-fold higher in the cells grown in TSB with 5.0% ethanol than those grown in TSB without ethanol. In contrast, cis-oleic acid was not detected in ethanol-stressed cells but was present at concentrations of 0.32 and 0.36 mg/g of cells of the two strains grown in TSB without ethanol. Protein content was higher in ethanol-stressed cells than in nonstressed cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles varied qualitatively as affected by the strain and the presence of ethanol in TSB. An ethanol-mediated protein (28 kDa) was observed in the ethanol-stressed cells but not in control cells. It is concluded that the two test strains of E. coli O157:H7 underwent phenotypic modifications in cellular fatty acid composition and protein profiles in response to ethanol stress. The potential for cross protection against subsequent stresses applied in food preservation technologies as a result of these changes is under investigation.     

Comparison of the glutamate-, arginine- and lysine-dependent acid resistance systems in Escherichia coli O157:H7

AIMS: The objective of this study was to investigate the effect of growing conditions on the glutamate-, arginine- and lysine-dependent acid resistance (AR) systems of Escherichia coli O157:H7. METHODS AND RESULTS: Seven E. coli O157:H7 strains were grown in five different media at neutral or acidic pH under aerobic or anaerobic conditions, and the survival rate after acid shocks (pH 2.0, 1 h, 37 degrees C) in the presence of glutamate, arginine and lysine was determined. Six strains induced the glutamate-dependent AR at stationary phase, and maximal survival were observed (> or =10%) when grown in pH 5- Luria-Bertani media with glucose (LBG) and in pH 4.5-anaerobic media. The arginine- and lysine-dependent systems were also present, but were only induced if cells had grown in LBG. For strain ATCC 43895, the minimum glutamate concentration that resulted in at least 10% survival rate was 10 micromol l(-1), but it required at least 10-fold more arginine and lysine. CONCLUSIONS: The lysine-dependent AR system could be as important as the arginine-mediated one, but the contribution of both systems to E. coli O157:H7 overall AR response might be minor compared with the glutamate-dependent system. SIGNIFICANCE AND IMPACT OF THE STUDY: Under typical environmental conditions, the glutamate-dependent AR system might be solely responsible for protecting cells against acidic pH.     

Combined effect of mild heat and acetic acid treatment for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an asparagus puree

AIMS: This study was conducted to validate combined heat and acid treatments for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an acidified brine containing, or pickled, asparagus model food. METHODS AND RESULTS: A mixture of three strains of E. coli O157:H7, L. monocytogenes and S. typhimurium were inoculated onto pickled asparagus samples. Combinations of various concentrations of acetic acid [0%, 0.25%, 0.5%, 0.75%, 1%, 1.5% and 2% (v/v)] and various temperatures (40 degrees C, 50 degrees C, 60 degrees C and 75 degrees C) were investigated. Following treatment, asparagus samples were stored at room temperature and enumerated at 0, 0.5, 1, 2 and 3 days. Heat and acetic acid treatments were synergistic. The inhibitory effects of these combined treatments on the tested foodborne pathogens were also effective during storage. Loss of green colour in the pickled asparagus significantly increased with increasing concentrations of acetic acid. CONCLUSIONS: Using a combination of mild heat and acetic acid treatments can successfully control E. coli O157:H7, L. monocytogenes and S. typhimurium in pickled asparagus, combinations of heat and acid are synergistic and effective treatments can be selected to reduce adverse effect on colour which occur during product storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heating plus acetic acid treatment are synergistic, so combined treatments can be developed, which would lower the temperature and amount of acetic acid required for minimally processed vegetables while maintaining pathogen control.     

Recovery of E. coli O157 strains after exposure to acidification at pH 2

Aims: Rapid detection and selective isolation of E. coli O157:H7 strains have been difficult owing to the potential interference from background microflora present in high background food matrices. To help selectively isolate E. coli O157H7 strains, a useful plating technique that involved acidifying the cultures to pH 2 was evaluated with a large number of E. coli O157:H7 strains to ensure response to treatment was consistent across strains. Methods and Results: Escherichia coli O157, 46 strains including ATCC 35150, were acidified to pH 2 following enrichment and plated onto Tryptic Soy Agar + 0·6% Yeast Extract (TSA‐YE) and Sorbitol MacConkey Agar + cefixime and tellurite (CT‐SMAC). Samples were enumerated and modest decreases in plate counts were observed on TSA‐YE media, with a greater reduction observed on CT‐SMAC. Conclusions: The acid‐resistant character of E. coli O157:H7 is a consistent trait and may be used for improved isolation of the organism from mixed cultures. Significance and Impact of the Study: There was little difference observed between the commonly used laboratory strain E. coli O157:H7 35150 and 45 other strains of E. coli O157 when subjected to acidifying conditions prior to plating, demonstrating that an acid rinse procedure was equally effective across a wide variety of E. coli O157 strains and broadly applicable for isolating unknown strains from food samples.