Studies on the growth of Escherichia coli O157:H7 strains at 45.5 degrees C

The objectives of the present report were to examine the ability of 18 strains of Escherichia coli O157:H7 to grow in EC broth at 42.4, 43.5, 44.5, and 45.5 degrees C, and to document the incidence of phenotypic variants present in low numbers that are capable of growth at 45.5 degrees C in EC broth. Among the 18 strains of E. coli O157:H7 studied, only 3 were capable of producing turbid growth with gas formation in EC broth at 45.5 degrees C with 1 x 10(2) initial CFU/ml. Higher initial densities of CFU resulted in turbid growth and gas formation in EC broth at 45.5 degrees C with all strains. The presence of bile salts #3 in EC broth was found to be inhibitory at 45.5 degrees C. All 18 strains were found to be capable of growth at 45.5 degrees C in nonselective media. The ability of at least one sensitive strain to grow in EC broth at 45.5 degrees C was found to be dependent on the initial number of CFU/ml. Prior growth of cells of a sensitive strain in EC broth at 45.5 degrees C from a cell density of 2.0 x 10(7) to 8.0 x 10(7) CFU/ml followed by removal of cells and reinoculation at a cell density of 2.0 x 10(6) CFU/ml resulted in growth at 45.5 degrees C that did not occur without such conditioning of the inhibitory medium. These results indicate that the ability of most strains of E. coli O157:H7 to grow in EC broth at 45.5 degrees C is dependent on the initial density of CFU and that at low densities of CFU the ability to initiate growth is dependent on either low numbers of phenotypic variants tolerant to the presence of bile salts #3 in EC broth at 45.5 degrees C or to conditioning of the medium with prior elevated numbers of cells.     

Application of caffeine, 1,3,7-trimethylxanthine,to control Escherichia coli O157:H7

The objective of this research was to determine the effectiveness of caffeine on inactivation of Escherichia coli O157:H7 in brain heart infusion (BHI) broth. Overnight samples of five E. coli O157:H7 strains of (E0019, F4546, H1730, 944 and Cider) were used in this study. These strains were individually inoculated at an initial inoculum level of 2 log CFU/ml into BHI broth containing caffeine at different concentrations (0.00%, 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, and 2.00%). Samples were then incubated at 37 °C for 24 h. Bacterial growth was monitored at different time intervals by measuring turbidity at 610 nm using a spectrophotometer. Results revealed that the addition of caffeine inhibited the growth of E. coli O157:H7. Significant growth inhibition was observed with concentration levels of 0.50% and higher. These results indicate that caffeine has potential as an antimicrobial agent for the treatment of E. coli O157:H7 infection and should be investigated further as a food additive to increase biosafety of consumable food products.     

pH-dependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli in the presence of various acidulants

The effect of acidulant identity on the pH-dependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli was studied. Nine strains of E. coli (seven O157:H7, one O111:H-, and one biotype 1 reference strain) were cultured individually for 18 h at 37 degrees C in tryptic soy broth (TSB) plus 1% dextrose and in TSB without dextrose to yield acid resistance induced and noninduced stationary-phase cells, respectively. These cultures were then inoculated into brain heart infusion broth (BHI) supplemented with 0.5% citric, malic, lactic, or acetic acid and adjusted to pH 3.0 with HCl. The BHI tubes were incubated at 37 degrees C for up to 7 h and samples were removed after 0, 2, 5, and 7 h and plated for counting CFU on BHI agar and MacConkey agar (MA). The results were compared to data previously obtained with HCl only. Acid resistance varied substantially among the isolates, being dependent on the strain, the acidulant, and the induction of pH-dependent acid resistance. Hydrochloric acid was consistently the least damaging to cells; lactic acid was the most detrimental. The relative activity of the other acids was strain dependent. Inducing pH-dependent acid resistance increased the already substantial acid tolerance of stationary-phase E. coli. The extent of injury also varied with acid and strain, with as much as a 5-log-cycle differential between BHI agar and MA CFU counts. The accurate determination of the survival of enterohemorrhagic E. coli in acidic foods must take into account the biological variability of the microorganism with respect to its acid resistance and its ability to enhance survival through the induction of physiological stress responses.     

Fate of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH values and temperatures

Survival and growth of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH levels (pH 3.4 to 6.8) and temperatures were studied. Sterile strawberry juice (pH 3.6) and acidified trypticase soy broth (TSB) media (pH 3.4 to 6.8) were inoculated with approximately 6.7 log CFU/ml E. coli O157:H7 or 7.3 log CFU/ ml L. monocytogenes, incubated for 3 days at 4 and 37 degrees C. Bacterial levels were determined after 2 h, 1 day, and 3 days using surface plating nonselectively on tryptic soy agar and selectively on sorbitol MacConkey agar for E. coli O157:H7 or modified Oxford agar for L. monocytogenes. A spectrophotometer (660 nm) was also used to study growth inhibition of L. monocytogenes in different TSB and strawberry juice media (pH 3.4 to 7.3). E. coli O157:H7 survived well at pH values of 3.4 to 6.8 at 4 degrees C, but the number of injured cells increased as pH decreased and incubation time increased. At 37 degrees C, E. coli O157:H7 was inactivated at pH of < or = 3.6 but could grow at pH 4.7. L. monocytogenes was quickly injured at pH of < or = 4.7 within 2 h of storage at 4 degrees C and then was slightly and gradually inactivated as storage time increased. L. monocytogenes survived well at pH 6.8 at 4 degrees C and grew well at 37 degrees C. Growth of L. monocytogenes at 37 degrees C was inhibited in TSB by 1% citric acid and 0.5% malic acids at pH 3.4 or by 50% strawberry juice at pH 4.7. Bacterial injury and inactivation appeared to be induced by the acids in strawberry juice. The acids, pH value, temperature, and time were important factors for bacterial survival, inactivation, and growth in the media tested.     

Escherichia coli O157: H7 Growth in Laboratory Media as Affected by Phenolic Antioxidants

Five strains of Escherichia coli O157:H7 with ATCC 11775 E. coli were grown in brain heart infusion (BHI) broth (pH 5.8, adjusted with citric acid) and treated with butylated hydroxyanisole (BHA), butylated hy-droxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate (PG) individually or combined. Additives ranged from 100–400 ppm with inocula levels between 5 and 104 CFU/mL in tissue culture plates or in flasks; samples were incubated at 4°C or 37°C for 24 hr. Additive antimicrobial efficacy varied with inoculum level and incubation temperature. BHA at <200 ppm was bactericidal on all strains. Poly-hydroxyl additives (TBHQ, PG) were less effective at 4°C. BHA-BHT combinations were synergistic at 4°C.     

A model study of Escherichia coli O157:H7 survival in fermented dry sausages--influence of inoculum preparation, inoculation procedure, and selected process parameters

The influence of inoculum preparation, inoculation level, and inoculation procedure on Escherichia coli O157:H7 inactivation during the manufacture of fermented sausage was evaluated in a model study. Prior growth in glucose-enriched tryptone soya broth, which provided exposure to mildly acidic conditions (pH 4.8), had no effect on the later survival of E. coli O157: H7 strains 5-1 and ATCC 43894 under extremely acidic conditions (pH 2), but the same strains became sensitive to acidity after 7 days of incubation on the surface of refrigerated beef (as per the normal contamination route from slaughter to further processing). In subsequent sausage production trials, the extent of destruction observed for E. coli O157:H7 strains F-90, 5-1, and ATCC 43894 inoculated directly into the meat batter was unchanged when the inoculation level was decreased from 7.3 to 4.7 log CFU/g, but the level of inactivation was ca. 1 log higher when the surfaces of beef cuts, rather than the batter, were inoculated 7 days prior to processing. Regardless of processing conditions (fermentation to a pH of < or = 5.0 at 24 or 37 degrees C, drying at 14 degrees C to a water activity [a(w)] value of 0.91 or 0.79), strains F-90, 5-1, and ATCC 43894 showed similar survival capacities during the manufacture of sausage. A approximately 2-log reduction in pathogen numbers was generally obtained after samples were dried to an a(w) of 0.91, irrespective of fermentation temperature. The addition of a 5-day predrying holding stage at the fermentation temperature significantly (P < 0.05) increased pathogen inactivation when fermentation was carried out at 37 degrees C (but not when it was carried out at 24 degrees C). However, significant pathogen reductions (4 to 5 log CFU/g) were achieved only for extensively dried products (a(w) = 0.79).     

Effects of cold storage and heat–acid shocks on growth and verotoxin 2 production ofEscherichia coliO157:H7

Escherichia coliO157:H7 was cold-stored (4°C) either in nutritious menstruum [buffered Brain Heart Infusion (BHI) broth] or with starvation (buffered saline) at pH 7.0 or 5.5. Cultures grown in BHI broth at 37°C for 24h served as non-cold-stored controls. After 4-weeks cold storage, bacterial cells were shocked by heat (45°C for 5min) and acid (pH 2.5 for 30min at 37°C) and subsequently moved to optimal conditions (BHI broth of pH 7.4 incubated at 37°C). The results showed: (a) both lag-phase duration and growth rate of this pathogen at 37°C significantly increased after cold-storage with starvation, but not after cold storage in the nutritious menstruum; (b) combined heat–acid shocks increased growth rates at 37°C of both previously cold-stored and non-cold-stored bacterial cells; (c) final concentrations of verotoxin produced by bacterial cells at 37°C were not affected by previous cold storage in the nutritious menstruum; (d) verotoxin production by bacterial cells at 37°C increased after cold storage with starvation, and heat–acid shocks further enhanced that production. Further research is needed to evaluate the food safety implications of these results, i.e. whether cells ofE. coliO157:H7 originating from nutrient-poor/lower-pH environments may be more harmful to humans than those from nutrient-rich/higher-pH foods.     

Survival differences of Escherichia coli O157:H7 strains in apples of three varieties stored at various temperatures

Differences in survival and growth among five different Escherichia coli O157:H7 strains in three apple varieties were determined at various temperatures. Jonathan, Golden Delicious, and Red Delicious apples were wounded and inoculated with E coli O157:H7 strains C7929 (apple cider isolate), 301C (chicken isolate), 204P (pork isolate), 933 (beef isolate), and 43890 (human isolate) at an initial level of 6 to 7 log CFU/g. The inoculated apples were stored at a constant temperature of 37, 25, 8, or 4 degrees C or at 37 degrees C for 24 h and then at 4 degrees C, and bacterial counts were determined every week for 28 days. By day 28, for Jonathan apples at 25 degrees C, the apple isolate counts were significantly higher than the chicken and human isolate counts. At 4 degrees C for 28 days, the human isolate inoculated into Jonathan, Golden Delicious, and Red Delicious apples was present in significantly smaller numbers than the other strains. The apple isolate survived significantly better at 4 degrees C, yielding the highest number of viable cells. By days 21 and 28, for apples stored at 37 degrees C for the first 24 h and then at 4 degrees C, the counts of viable E. coli O157:H7 apple and human isolates were 6.8 and 5.8 log CFU/g at the site of the wound, whereas for apples kept at 4 degrees C for the duration of storage, the respective counts were 5.6 and 1.5 log CFU/g. Our study shows that E. coli O157:H7 strains responded differentially to their ability to survive in these three apple varieties at 25 or 4 degrees C and produced higher viable counts when apples were temperature abused at 37 degrees C for 24 h and then stored at 4 degrees C for 27 days.     

Survival of Escherichia coli O157:H7 in traditional African yoghurt fermentation

Growth and survival of a nontoxigenic strain of Escherichia coli O157:H7 (ATCC 43888) was determined in traditionally fermented pasteurized milk. Preheated milk was inoculated with 1% (v/v) of a mixed culture of Lactobacillus delbrueckii ssp. bulgaricus (NCIMB 11778) and Streptococcus salivarius ssp. thermophilus (NCIMB 110368) and incubated at 25, 30, 37 or 43 degrees C for 24 h. E. coli O157:H7 (10(5) CFU/ml) were introduced into the milk pre- and post-fermentation. Fermented milk samples were subsequently stored at either 4 degrees C (refrigerator temperature) or 25 degrees C (to mimic African ambient temperature) for 5 days. After 24 h of fermentation, the pH of the samples fermented at the higher temperatures of 37-43 degrees C decreased from 6.8 to 4.4-4.0 ( +/- 0.2) whereas at the lower temperature of 25 degrees C, the pH decreased to pH 5.0 +/- 0.1. During this period, viable counts for E. coli O157:H7 increased from 10(5) to 10(8) - 10(9) CFU/ml except in milk fermented at 43 degrees C wherein viability declined to 10(4) CFU/ml. In fermented (25-30 degrees C) milk stored at 4 degrees C for 5 days, E. coli O157:H7 viability decreased from 10(8-9) to 10(6-7) CFU/ml whereas milk fermented at 43 degrees C resulted in loss of detectable cells. In contrast, storage of fermented milk samples at 25 degrees C for 5 days eventually resulted in complete loss of viability irrespective of fermentation temperature. Stationary phase E. coli O157:H7 inoculated post-fermentation (25 and 43 degrees C) survived during 4 degrees C storage, but not 25 degrees C storage. Fermentation temperature and subsequent storage temperature are critical to the growth and survival of E. coli O157:H7 in traditional fermented products involving yoghurt starter cultures.     

Fate of Escherichia coli O157:H7 in coleslaw during storage

An outbreak of Escherichia coli O157:H7 infection associated with the consumption of coleslaw in several units of a restaurant chain prompted a study to determine the fate of the pathogen in two commercial coleslaw preparations (pH 4.3 and 4.5) held at 4, 11, and 21 degrees C for 3 days. At an initial population of 5.3 log10 CFU/g of coleslaw, E. coli O157:H7 did not grow in either coleslaw stored at the three temperatures. Rather, the population of E. coli O157:H7 decreased by 0.1 to 0.5 log10 CFU/g within 3 days. The greatest reduction (0.4 and 0.5 log10 CFU/g) in population occurred at 21 degrees C, whereas only slight decreases (0.1 to 0.2 log10 CFU/g) occurred at 4 and 11 degrees C. A pH of 4.3 to 4.5 of coleslaw had little effect on reducing E. coli O157:H7 populations. Results suggest that the tolerance of E. coli O157:H7 to acid pH, not temperature abuse, is a major factor influencing the pathogen's fate in restaurant-prepared coleslaw.     

High-pressure resistance variation of Escherichia coli O157:H7 strains and Salmonella serovars in tryptic soy broth, distilled water, and fruit juice

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7 and five serovars of Salmonella enterica was investigated in tryptic soy broth, sterile distilled water, and commercially sterile orange juice (for Salmonella) and apple cider (for E. coli). Samples were subjected to high-pressure processing treatment at 300 and 550 MPa for 2 min at 6 degrees C. Samples were plated onto tryptic soy agar directly after pressurization and after being held for 24 h at 4 degrees C. At 300 MPa, little effect was seen on E. coli O157:H7 strains, while Salmonella serovars varied in resistance, showing reductions between 0.26 and 3.95 log CFU/ml. At 550 MPa, E. coli O157:H7 strains exhibited a range of reductions (0.28 to 4.39 log CFU/ml), while most Salmonella populations decreased beyond the detection limit (> 5-log CFU/ml reduction). The most resistant strains tested were E. coli E009 and Salmonella Agona. Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water. E. coli O157:H7 cultures held for 24 h at 4 degrees C after treatment at 550 MPa showed a significant log decrease as compared with cultures directly after treatment (P < or = 0.05), while Salmonella serovars did not show this significant decrease (P > 0.05). All Salmonella serovars tested in orange juice treated at 550 MPa for 2 min at 6 degrees C and held for 24 h showed a > 5-log decrease, while E. coli O157:H7 strains require a higher pressure, higher temperature, longer pressurization, or a chemical additive to achieve a 5-log decrease.     

Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages

Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival. Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose. Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates. Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values). Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested. Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested. Extensive strain-to-strain variation was observed for all parameters tested. Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively. Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures. The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.     

Growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes in egg products held at different temperatures

Growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes in steamed eggs and scrambled eggs held at different temperatures (5, 18, 22, 37, 55, and 60 degrees C) were investigated in the present study. Among the holding temperatures tested, both pathogens multiplied best at 37 degrees C followed by 22, 18, and 5 degrees C. In general, E. coli O157:H7 grew better in the egg products than L. monocytogenes did at all the storage temperatures tested except at 5 degrees C. E. coli O157:H7 did not grow in steamed eggs and scrambled eggs held at 5 degrees C. L. monocytogenes showed a slight population increase of approximately 0.6 to 0.9 log CFU/g in these egg products at the end of the 36-h storage period at 5 degrees C. The population of both pathogens detected in the egg products was affected by the initial population, holding temperature, and length of the holding period. It was also noted that L. monocytogenes was more susceptible than E. coli O157:H7 in steamed eggs held at 60 degrees C. After holding at 60 degrees C for 1 h, no detectable viable cells of L. monocytogenes with a population reduction of 5.4 log CFU/g was observed in steamed eggs, whereas a lower population reduction of only approximately 0.5 log CFU/ml was noted for E. coli O157:H7.     

Validation of a manufacturing process for fermented, semidry Turkish soudjouk to control Escherichia coli O157:H7

Two soudjouk batters were prepared from ground beef (20% fat) and nonmeat ingredients and inoculated with a five-strain mixture of Escherichia coli O157:H7 to yield an initial inoculum of 7.65 log10 CFU/g. One batter contained a commercial-starter culture mixture (approximately 8.0 log10 CFU/g) and dextrose (1.5%), while the other batter relied upon a natural fermentation with no added carbohydrate. Following mixing, sausage batters were held at 4 degrees C for 24 h prior to stuffing into natural beef round casings. Stuffed soudjouk sticks were fermented and dried at 24 degrees C with 90 to 95% relative humidity (RH) for 3 days and then at 22 degrees C with 80 to 85% RH until achieving a product moisture level of approximately 40%. After fermentation and drying with an airflow of 1 to 1.5 m/s, the sticks were either not cooked or cooked to an instantaneous internal temperature of 54.4 degrees C (130 degrees F) and held for 0, 30, or 60 min. The sticks were then vacuum packaged and stored at either 4 or 21 degrees C. For each of three trials, three sticks for each treatment/batter were analyzed for numbers of E. coli O157:H7 after inoculation, after fermentation, after cooking, and after storage for 7, 14, 21, and 28 days. Reductions in numbers of E. coli O157:H7 after fermentation and drying for sticks fermented by the starter culture (pH 4.6) and for sticks naturally fermented (pH 5.5) were 1.96 and 0.28 log10 CFU/g, respectively. However, cooking soudjouk sticks produced with a starter culture and holding at 54.4 degrees C for 0, 30, or 60 min reduced pathogen numbers from an initial level after fermentation and drying of 5.69 log10 CFU/g to below a detectable level by either direct plating (<1.0 log10 CFU/g) or by enrichment. In contrast, cooking soudjouk sticks produced without an added starter culture decreased pathogen numbers from an initial level after fermentation and drying of 7.37 to 5.65 log10 CFU/g (54.4 degrees C, no hold), 5.04 log10 CFU/g (54.4 degrees C, 30 min hold), and 4.67 log10 CFU/g (54.4 degrees C, 60 min hold). In general, numbers of E. coli O157:H7 within both groups of soudjouk sticks decreased faster during storage at 21 degrees C compared to 4 degrees C. After 28 days of storage, total reductions in pathogen numbers in soudjouk sticks produced using a starter culture but that were not subsequently cooked were 7.65 and 3.93 log10 CFU/g at 21 and 4 degrees C, respectively. For naturally fermented soudjouk, total reductions varied from 4.47 to 0.45 log10 CFU/g, depending on the cooking time and storage temperature. These data provide guidelines for manufacturers of dry sausage of ethnic origin, including soudjouk, to assess the safety of their processes for control of E. coli O157:H7.     

Effect of pH on survival, thermotolerance, and verotoxin production of Escherichia coli O157:H7 during simulated fermentation and storage

Heat treatment is increasingly being introduced to fermented meat processing, since the acid tolerance properties of Escherichia coli O157:H7 can permit this organism to survive traditional processing procedures. This study investigated the effect of growth pH and fermentation on the thermotolerance at 55 degrees C of E. coli O157:H7 in a model fermented meat system. E. coli O157:H7 (strain 380-94) was grown at pH 5.6 or 7.4 (18 h at 37 degrees C), fermented to pH 4.8 or 4.4 in brain heart infusion broth, and stored for 96 h. Cells grown at pH 5.6 had higher D values at 55 degrees C (D55 degrees C) than cells grown at pH 7.4 (P < 0.001). Cells fermented to pH 4.8 had higher D55 degrees C than those fermented to pH 4.4 (P < 0.001). Cells fermented to pH 4.8 demonstrated an increase in D55 degrees C during storage (P < 0.001), whereas cells fermented to pH 4.4 showed a decrease in D55 degrees C during the same period (P < 0.001). The effect of growth pH on verotoxin production by E. coli O157:H7 was assessed using the verotoxin assay. Cells grown at pH 5.6 had lower verotoxin production then cells grown at pH 7.4. This effect was not sustained over storage. These results indicate that a lower growth pH can confer cross-protection against heat. This has implications for the production of acidic foods, such as fermented meat, during which a heating step may be used to improve product safety.     

Inactivation of Escherichia coli O157: H7 and Listeria monocytogenes by PR-26, a synthetic antibacterial peptide.

This study reports the antibacterial effect of PR-26, a synthetic peptide derived from the first 26 amino acid sequence of PR-39, an antimicrobial peptide isolated from porcine neutrophils. A three-strain mixture of Escherichia coli O157:H7 or Listeria monocytogenes of approximately 10(8) CFU was inoculated to a final concentration of 10(7) CFU/ml in 1% peptone water (pH 7.0), containing 50 or 75 microg/ml of PR-26, and incubated at 37 degrees C for 0, 6, 12, and 24 h; at 24 degrees C for 0, 12, 24, and 36 h; or at 10 or 4 degrees C for 0, 24, 72, and 120 h. Control samples included 1% peptone water inoculated with each pathogen mixture but containing no PR-26. The surviving population of each pathogen at each sampling time was determined by plating on tryptic soy agar with incubation at 37 degrees C for 24 h. At 37 degrees C, PR-26 decreased E. coli O157:H7 and L. monocytogenes populations by >5.0 log CFU/ml at 12 h, with complete inactivation at 24 h. At 24 degrees C, PR-26 reduced E. coli O157:H7 and L. monocytogenes by approximately 3.5, 4.0, and 4.5 log CFU/ml at the end of 12-, 24-, and 36-h incubations, respectively. At 4 and 10 degrees C, the inhibitory effect of PR-26 on E. coli O157:H7 and L. monocytogenes was significantly lower (P < 0.05) than that at 37 and 24 degrees C: a 2- to 3-log CFU/ml reduction was observed at 120-h incubation. Results indicate that PR-26 could potentially be used as an antimicrobial agent, but applications in appropriate foods need to be validated.     

Survival and growth of Escherichia coli O157:H7 in roast beef and salami after exposure to an alkaline cleaner

Survival and growth of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 after exposure to an alkaline cleaner for 2 min and inoculating into roast beef (pH 6.3) and hard salami (pH 4.9) at low (0.003 to 0.52 CFU/g) and high (0.69 to 31.5 CFU/g) populations were determined. Roast beef was stored at 4 and 12 degrees C; salami was stored at 4, 12, and 20 degrees C. At 4 degrees C, untreated cells of both strains showed greater reductions in populations in salami than in roast beef during a 21-day storage period. Populations of treated and untreated cells recovered from roast beef and salami stored at 4 degrees C on tryptic soy agar were significantly (P < or = 0.05) higher than on sorbitol MacConkey agar, indicating that a portion of the cells was injured. Treated and untreated cells grew in roast beef at 12 degrees C. Growth of treated cells of the FRIK 816-3 strain in roast beef at 12 degrees C was significantly slower than that of the EDL 933 strain. Populations of both strains decreased at different rates in salami stored at different temperatures (20 degrees C > 12 degrees C > 4 degrees C). E. coli O157:H7 strain EDL 933 grew more rapidly at 20 degrees C in a slurry (pH 5.97) prepared from stored salami (17 days at 20 degrees C) on which Penicillium chrysogenum had grown than in a slurry (5.23) prepared from salami showing no mold growth. Within 2 to 3 days, populations were ca. 3 log CFU/ml higher in slurry made from infected salami than in control salami. Results indicate that treatment of E. coli O157: H7 with an alkaline cleaner for 2 min does not impair resuscitation and growth of surviving cells in roast beef at 12 degrees C. Cross protection of cells exposed to an alkaline cleaner against subsequent stress conditions imposed by roast beef and salami stored at 4 degrees C was not evident in either of the test strains.     

Acid tolerance of acid-adapted and nonadapted Escherichia coli O157:H7 following habituation (10 degrees C) in fresh beef decontamination runoff fluids of different pH values

This study evaluated survival of Escherichia coli O157:H7 strain ATCC 43895 during exposure to pH 3.5 following its habituation for 2 or 7 days at 10 degrees in fresh beef decontamination waste runoff fluid mixtures (washings) containing 0, 0.02, or 0.2% of lactic or acetic acids. Meat washings and sterile water (control) were initially inoculated with approximately 5 log CFU/ml of acid- and nonadapted E. coli O157:H7 cells cultured (30 degrees C, 24 h) in broth with and without 1% glucose, respectively. After 2 days, E. coli O157:H7 survivors from acetate washings (pH 3.7 to 4.7) survived at pH 3.5 better than E. coli O157:H7 survivors from lactate washings (pH 3.1 to 4.6), especially when the original inoculum was acid adapted. Also, although E. coli O157:H7 habituated in sterile water for 2 days survived well at pH 3.5, the corresponding survivors from nonacid water meat washings (pH 6.8) were rapidly killed at pH 3.5, irrespective of acid adaptation. After 7 days, E. coli O157:H7 survivors from acetate washings (pH 3.6 to 4.7) continued to resist pH 3.5, whereas those from lactate washings died off. This loss of acid tolerance by E. coli O157:H7 was due to either its low survival in 0.2% lactate washings (pH 3.1) or its acid sensitization in 0.02% lactate washings, in which a Pseudomonas-like natural flora showed extensive growth (> 8 log CFU/ml) and the pH increased to 6.5 to 6.6. Acid-adapted E. coli O157:H7 populations habituated in water washings (pH 7.1 to 7.3) for 7 days continued to be acid sensitive, whereas nonadapted populations increased their acid tolerance, a response merely correlated with their slight (< 1 log) growth at 10 degrees C. These results indicate that the expression of high acid tolerance by acid-adapted E. coli O157:H7 can be maintained or enhanced in acid-diluted meat decontamination waste runoff fluids of pH levels that could permit long-term survival at 10 degrees C. Previous acid adaptation, however, could reduce the growth potential of E. coli O157:H7 at 10 degrees C in nonacid waste fluids of high pH and enriched in natural flora. These conditions might further induce an acid sensitization to stationary E. coli O157:H7 cells.     

Effects of pH and agitation on the growth of Listeria monocytogenes Scott A in brain heart infusion broth containing combined potassium lactate and sodium diacetate during storage at 4 or 10 degrees C

The objective of this study was to compare the effects of pH on the growth kinetics of Listeria monocytogenes Scott A in static and agitated broths stored at 4 and 10 degrees C with and without a combination of 1.85% potassium lactate (PL) and 0.13% sodium diacetate (SDA) (3.3% of a 60% commercial solution, PURASAL P Opti.Form 4). The pH of brain heart infusion broth without (control) or with 1.85% PL + 0.13% SDA was adjusted to 5.5, 6.0, 6.5, and 7.5. L. monocytogenes Scott A was inoculated (at 10(2) CFU/ml) into pH-adjusted broth, which was stored at 4 or 10 degrees C with or without agitation. At pH 5.5, a listeriostatic effect was observed for the broth containing 1.85% PL + 0.13% SDA at 4 and 10 degrees C both with and without agitation. At pH 6.0, 1.85% PL + 0.13% SDA fully controlled the growth of L. monocytogenes Scott A in static broth at 4 degrees C for up to 20 days and significantly slowed the growth of the pathogen in agitated broth. At 10 degrees C, the growth of L. monocytogenes Scott A was significantly reduced by 1.85% PL + 0.13% SDA in agitated and unagitated broths. At pH 6.5, 1.85% PL + 0.13% SDA significantly suppressed the growth of L. monocytogenes Scott A at both 4 degrees C (P < 0.001) and 10 degrees C (P < 0.01). At pH 7.5, 1.85% PL + 0.13% SDA had a limited effect on the growth of L. monocytogenes Scott A in broth stored at 4 and 10 degrees C. At 4 degrees C, agitation decreased the lag time and increased the growth rate of L. monocytogenes Scott A at all tested pHs. A similar but less obvious trend was observed for broths stored at 10 degrees C. These results indicate that lactate-diacetate combinations effectively acted with low pH and temperature to inhibit the growth of L. monocytogenes Scott A.     

Growth of Escherichia coli O157:H7 in raw ground beef stored at 10 degrees C and the influence of competitive bacterial flora,strain variation,and fat level

Pure-culture broth-based models of the growth of Escherichia coli O157:H7 have been used to estimate its behavior in ground beef, even though these models have not been adequately validated for this food product. This situation limits accurate estimates of the behavior of E. coli O157:H7 in ground beef and introduces uncertainties in risk assessments. In the present study, the growth of single and multiple strains of E. coli O157:H7 were measured in retail ground beef stored at 10 degrees C for up to 12 days, and the results were compared with estimates generated by the U.S. Department of Agriculture's Pathogen Modeling Program (PMP; version 5.1). At pH 5.9, the PMP predicted a maximum population density (MPD) of 9.13 log10 CFU/g, an exponential growth rate (EGR) of 0.052 log10 CFU/h, and a lag time of 56.3 h. Similar parameter values were observed for sterilized ground beef; however, no lag phase was observed. In contrast, the mean MPD and EGR for retail ground beef were 5.09 log10 CFU/g and 0.019 log10 CFU/h, respectively, and no lag phase was observed. Both the EGR and the MPD increased with decreasing fat levels. There was low variation in the MPD and EGR parameters for the nine E. coli O157:H7 ground beef isolates. Two isolates of competitive native flora were separately added to sterilized ground beef, and the EGR and MPD decreased as the ratio of competitive flora to E. coli O157:H7 increased. For one strain, at ratios of 1:1, 10:1, and 100:1, the EGRs were 0.033, 0.025, and 0.018 log10 CFU/h, respectively, and the MPDs were 6.14, 5.08, and 4.84 log10 CFU/g, respectively. These results demonstrate that existing broth-based models for E coli O157:H7 must be validated for food and that models should consider the effects of the food matrix, the competitive microflora, and potential pathogen strain variation.