Growth history influences starvation-induced expression of uspA, grpE, and rpoS and subsequent cryotolerance in Escherichia coli O157:H7

In this study, we investigated the effect of starvation on cryotolerance of Escherichia coli O157:H7 grown in tryptic soy broth (TSB) and Luria-Bertani broth (LB). Starved cells (cells suspended in water at 37 degrees C for 6 h) and control cells (cells in TSB or LB) were frozen at -18 degrees C for up to 240 h in their respective growth media. The E. coli grown in TSB showed a greater starvation effect (the difference in percent survival of starved and control cells) and cryotolerance. The starved E. coli grown in TSB showed a 30% increase in their ability to survive frozen storage for 24 h at -18 degrees C. The corresponding increase in survival for LB-grown E. coli was only 3.8%. Cryotolerance induced by starvation of TSB- and LB-grown E. coli was correlated with the expression of genes involved in general stress response pathways, such as uspA, grpE, and rpoS. The expression of uspA, grpE, and rpoS was quantified by measuring the green fluorescence generated from autofluorescent E. coli harboring puspA::gfp, pgrpE::gfp, and prpoS::gfp gene fusions. The results obtained in this study indicate that uspA, grpE, and rpoS were induced on starvation when E. coli was grown in TSB, and their expression correlated well with subsequent induction of cryotolerance developed at -18 degrees C. In contrast, cells grown in LB and subsequently exposed to starvation conditions showed no increase in expression of uspA, grpE, or rpoS, and, as expected, these cells did not exhibit increased cryotolerance at -18 degrees C. Knowledge of molecular mechanisms involved in cross-protection might make it possible to devise strategies to limit their effects and lead to ways to predict the survival of foodborne pathogens in stressful environments.     

Induced expression of the heat shock protein genes uspA and grpE during starvation at low temperatures and their influence on thermal resistance of Escherichia coli O157:H7

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37 degrees C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52 degrees C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37 degrees C, for 24 h at 10 degrees C, and for 2 days at 5 degrees C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5 degrees C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37 degrees C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.     

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.     

Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce

Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.     

uspA of Shigella sonnei

One of the strategies that bacteria utilize to combat environmental stress is to synthesize stress-responding proteins. In Escherichia coli, adverse environmental factors, such as starvation, heat, and the presence of acid, oxidants, heavy metals, and antibiotics, trigger the expression of the universal stress protein (USP). The gene of the USP, uspA, in E. coli K-12 and E. coli O157:H7 has been identified and sequenced. In this study, the nucleotide sequence of uspA in a strain of Shigella sonnei implicated in the 1998 parsley-related outbreak of shigellosis was determined. Within an 800-bp region sequenced, there were 17 bp mismatches between the uspA of S. sonnei and that of E. coli K-12. Among the 17 mismatched nucleotides, 8 were within the structure gene of uspA. A total of 12 bp variations were identified between the uspA of S. sonnei and that of E. coli O157:H7, of which 5 bp were internal to the coding region of uspA. However, unlike the mismatches between the uspA of E. coli K-12 and the same gene of E. coli O157:H7 and S. sonnei that resulted in a single amino acid substitution and changed an alanine to an arginine at position 140, the mismatches between S. sonnei and E. coli O157:H7 were silent and did not result in any amino acid substitution.     

Protective effect of exopolysaccharide colanic acid of Escherichia coli O157:H7 to osmotic and oxidative stress

Many strains of Escherichia coli O157:H7 produce, under stress, an exopolysaccharide (EPS) comprised of colanic acid (CA) and form mucoid colonies on minimal glucose agar (MGA) at ambient temperature. Previous research conducted in our laboratory involving a CA-proficient (W6-13) and a CA-deficient (M4020; wcaD::Ekan(r)) strain of E. coli O157:H7 revealed that CA conferred acid and heat tolerance to E. coli O157:H7. Cells covered with CA were more persistent during acid (pH 4.5, 5.5, and 6.5) and heat (55 and 60 degrees C) treatment. The goal of this research was to study the effect of CA on the fate of E. coli O157:H7 under osmotic and oxidative stress. Cells of W6-13 and M4020 were exposed to various concentrations of NaCl (0.5, 1.5, and 2.5 M) and H2O2 (0, 10, and 20 mM) in minimal glucose broth (MGB) at 22 degrees C. Viable counts of E. coli O157:H7 were determined within 48 h of the osmotic stress and 3 h of the oxidative stress. The results suggest that cells of E. coli O157:H7 deficient in CA production are more susceptible than its wild-type parent to NaCl ( P< 0.05) and H2O2 (P< or = 0.05). This indicates that CA plays a role in protecting E. coli O157:H7 from osmotic and oxidative stress.     

Validation of time and temperature values as critical limits for the control of Escherichia coli O157:H7 during the production of fresh ground beef

In order to provide beef processors with valuable data to validate critical limits set for temperature during grinding, a study was conducted to determine Escherichia coli o157:H7 growth at various temperatures in raw ground beef. Fresh ground beef samples were inoculated with a cocktail mixture of streptomycin-resistant E. coli O157:H7 to facilitate recovery in the presence of background flora. Samples were held at 4.4, 7.2, and 10 degrees C, and at room temperature (22.2 to 23.3 degrees C) to mimic typical processing and holding temperatures observed in meat processing environments. E. coli O157:H7 counts were determined by direct plating onto tryptic soy agar with streptomycin (1,000 microg/ml), at 2-h intervals over 12 h for samples held at room temperature. Samples held under refrigeration temperatures were sampled at 4, 8, 12, 24, 48, and 72 h. Less than one log of E. coli O157:H7 growth was observed at 48 h for samples held at 10 degrees C. Samples held at 4.4 and 7.2 degrees C showed less than one log of E. coli O157:H7 growth at 72 h. Samples held at room temperature showed no significant increase in E. coli O157:H7 counts for the first 6 h, but increased significantly afterwards. These results illustrate that meat processors can utilize a variety of time and temperature combinations as critical limits in their hazard analysis critical control point plans to minimize E. coli O157:H7 growth during the production and storage of ground beef.     

Acid adaptation and temperature effect on the survival of E. coli O157:H7 in acidic fruit juice and lactic fermented milk product.

In this study, two strains of Escherichia coli O157:H7, (ATCC 43889 and ATCC 43895) were acid adapted at pH 5.0 in tryptic soy broth (TSB) for 4 h. Commercial products of mango juice (pH 3.2), asparagus juice (pH 3.6), Yakult--a diluted milk fermented drink (pH 3.6), and low-fat yoghurt (pH 3.9) were inoculated with acid-adapted or nonadapted cells of E. coli O157:H7. Survival of the inoculated E. coli O157:H7 in these commercial food products during storage at 25 or 7 degrees C was examined. It was found that although survival of the acid-adapted and nonadapted E. coli O157:H7 ATCC 43895 in asparagus juice during storage at 7 degrees C did not show marked difference, in general, acid adaptation and low temperature enhanced the survival of E. coli O157:H7 in both the commercial fruit juices tested. On the contrary, acid adaptation reduced the survival of both the strains of the test organism in Yakult and low-fat yoghurt stored at 7 degrees C. Besides, E. coli O157:H7 ATCC 43895 survived longer than ATCC 43889 in all the products examined, regardless of the storage temperature and acid adaptation.     

The survival of Escherichia coli O157:H7 in the presence of Penicillium expansum and Glomerella cingulata in wounds on apple surfaces

The survival of Escherichia coli O157:H7 in the presence of one of two plant pathogens, Penicillium expansum and Glomerella cingulata, in wounds on apples was observed during 14 days storage at room temperature (RT) and at 4 degrees C. The aim of this work was to determine if changes in apple physiology caused by the proliferation of fungal decay organisms would foster the survival of E. coli O157:H7. Trials were performed where (A) plant pathogens (4 log10 spores) were added to apple wounds 4 days before the wounds were inoculated with E. coli O157:H7 (3 log10 CFU g(-1) apple) (both RT and 4 degrees C storage), (B) plant pathogens and E. coli O157:H7 were added on the same day (both RT and 4 degrees C storage), and (C) E. coli O157:H7 was added 2 days (RT storage) and 4 days (4 degrees C storage) before plant pathogens. In all trials E. coli O157:H7 levels generally declined to <1 log10 at 4 degrees C storage, and in the presence of P. expansum at 4 degrees C or RT. However, in the presence of G. cingulata at RT E. coli O157:H7 numbers increased from 3.18 to 4.03 log10 CFU g(-1) in the apple wound during trial A, from 3.26 to 6.31 log10 CFU g(-1) during trial B, and from 3.22 to 6.81 log10 CFU g(-1) during trial C. This effect is probably a consequence of the attendant rise in pH from 4.1 to approximately 6.8, observed with the proliferation of G. cingulata rot. Control apples (inoculated with E. coli O157:H7 only) were contaminated with opportunistic decay organisms at RT during trials A and B, leading to E. coli O157:H7 death. However, E. coli O157:H7 in control apples in trial C, where no contamination occurred, increased from 3.22 to 5.97 log10 CFU g(-1). The fact that E. coli O157:H7 can proliferate in areas of decay and/or injury on fruit highlights the hazards associated with the use of such fruit in the production of unpasteurized juice.     

Identification of Escherichia coli O157:H7 meat processing indicators for fresh meat through comparison of the effects of selected antimicrobial interventions

Fresh meat products can become contaminated with the pathogen Escherichia coli O157:H7 during the slaughter process; therefore, an E. coli O157:H7 indicator to verify the effectiveness of process controls in slaughter establishments would be extremely useful. The hides of 20 beef cattle were sampled, and 113 bacterial isolates were obtained. Thirteen of these isolates representing four genera, Escherichia, Enterobacter, Providencia, and Serratia, were selected based on growth and biochemical characteristics similar to those of five clinical strains of E. coli O157:H7. The temperature sensitivity was determined for the individual isolates and the five E. coli O157:H7 strains at 55 and 65 degrees C. D65-values for all 13 isolates were not significantly different from D65-values of the E. coli O157:H7 strains. E. coli isolates were the only isolates whose D55-values were not significantly different from those of the E. coli O157:H7 strains. E. coli isolates P3 and P68 were more resistant to the effects of 55 degrees C than were the other E. coli isolates but were not significantly different from E. coli O157:H7 WS 3331 (P > 0.05). The remaining E. coli isolates (P1, P8, and P14) were not significantly different from E. coli O157:H7 strains ATCC 35150, ATCC 43894, ATCC 43895, and WS 3062 (P > 0.05). Prerigor lean and adipose beef carcass tissue was artificially contaminated with stationary-phase cultures of the five E. coli beef cattle isolates or a cocktail of five E. coli O157:H7 strains in a fecal inoculum. Each tissue sample was processed with the following microbial interventions: 90 degrees C water; 90 degrees C water followed by 55 degrees C 2% lactic acid; 90 degrees C water followed by 20 degrees C 2% lactic acid; 20 degrees C water followed by 20 degrees C 2% lactic acid; 20 degrees C water followed by 20 degrees C 20 ppm chlorine; and 20 degrees C water followed by 20 degrees C 10% trisodium phosphate. The appropriateness of the E. coli isolates as potential E. coli O157:H7 indicators was dependent upon the microbial intervention utilized. For all microbial intervention methods applied irrespective of tissue type, the mean log reductions of at least two E. coli isolates were not significantly different from the mean log reduction of the E. coli O157:H7 cocktail (P > 0.05). Because of the frequent employment of multiple microbial interventions in the cattle industry, no single isolate can realistically represent the effectiveness of all microbial interventions for reduction of E. coil O157:H7. Thus, the use of a combination of E. coli isolates may be required to accurately predict the effectiveness of microbial intervention methods on the reduction of E. coli O157:H7 in beef carcass tissue.     

Effects of pH,temperature, and pre-pulsed electric field treatment on pulsed electric field and heat inactivation of Escherichia coli O157:H7

This investigation was undertaken to study the inactivation of Escherichia coli O157:H7 by pulsed electric field (PEF) treatment and heat treatment after exposure to different stresses. E. coli O157:H7 cells exposed to different pHs (3.6, 5.2, and 7.0 for 6 h). different temperatures (4, 35, and 40 degrees C for 6 h), and different pre-PEF treatments (10, 15, and 20 kV/cm) were treated with PEFs (20, 25, and 30 kV/cm) or heat (60 degrees C for 3 min). The results of these experiments demonstrated that a pH of 3.6 and temperatures of 4 and 40 degrees C caused significant decreases in the inactivation of E. coli O157:H7 by PEF treatment and heat treatment (P < 0.05). Pre-PEF treatments, pHs of 5.2 and 7.0, and a temperature of 35 degrees C, on the other hand, did not result in any resistance of E. coli O157:H7 cells to inactivation by PEF treatment and heat treatment (P > 0.05).     

Survival of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in and on vacuum packaged Lebanon bologna stored at 3.6 and 13.0 degrees C.

Escherichia coli O157:H7, Salmonella Typhimurium, or Listeria monocytogenes was spread onto the surface of Lebanon bologna luncheon slices using sterile glass rods. The inoculated slices were stacked and vacuum packaged. The packages were stored at 3.6 or 13 degrees C. The foodborne pathogens. E. coli O157:H7, Salmonella Typhimurium, or L. monocytogenes were reduced in Lebanon bologna during storage at 3.6 or 13 degrees C. The higher storage temperature (13.0 degrees C) resulted in significantly faster destruction of E. coli O157:H7 and L. monocytogenes, compared to storage at refrigeration temperature (3.6 degrees C) (P < 0.005). E. coli O157:H7 was the most resistant to destruction among the three foodborne pathogens. A linear destruction of E. coli O157:H7 occurred only after an initial lag period. Storage temperature did not have a significant effect on the rate of destruction of Salmonella Typhimurium. Foodborne pathogens inoculated prior to fermentation did not show any enhanced survival compared to control cells (inoculated after fermentation) during storage of the Lebanon bologna at 3.6 degrees C.     

Evaluation of increased incubation temperature and cefixime-tellurite treatment for the isolation of Escherichia coli O157:H7 from minced beef

To improve enrichment and isolation of Escherichia coli O157:H7, this study evaluated increased incubation temperature and cefixime-tellurite (CT) on five strains of each of the following bacteria, E. coli, Hafnia alvei, Enterobacter spp., Citrobacter freundii and E. coli O157:H7, and two strains of E. coli O157:nH7. These were grown in pure culture in LST broth with varying cefixime-tellurite concentrations. A range of incubation temperatures from 37 to 46 degrees C was investigated for the inhibition of cohabitant microorganisms. Minced beef, spiked with E. coli O157:H7 and cohabitant microorganisms was investigated. Increased incubation temperature (42 degrees C) and treatment with half of the prescribed amount of cefixime-tellurite by BAM for SMAC agar in enrichment step were the most effective in selectively growing E. coli O157:H7. The results show that E. coli O157:H7 is more resistant to these two conditions than the other cohabitant bacteria.     

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.     

Thermal resistance parameters for pathogens in white grape juice concentrate

The heat resistance of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes that were in stationary phase, had been exposed to high osmotic pressure, or were acid adapted was evaluated in white grape juice concentrate (58 degrees Brix, pH 3.3). The most heat-resistant cells of all three pathogens were those exposed to high osmotic pressure or in stationary phase. Unlike in single-strength juices, in concentrate the acid-adapted cells for all three pathogens were less heat resistant than were cells in the other physiological states. E. coli O157:H7 had the highest heat resistance for all temperatures tested (e.g., D62 degrees C = 1.8 +/- 0.3 min, with a z-value of 9.9 +/- 0.6 degrees C). L. monocytogenes exposed to high osmotic pressure had the highest z-value (12.3 +/- 1.2 degrees C), although its D-values for all temperatures tested were lower (e.g., D62 degrees C = 0.93 +/- 0.1 min) than those for E. coli O157:H7. Salmonella was the most sensitive of the pathogens under all conditions. Based on the results obtained in this study, one example of a heat treatment that will inactivate 5 log units of all three pathogens in white grape juice concentrate was calculated as 1.5 min at 71.1 degrees C (z = 10.3 degrees C). Validation studies confirmed the predicted D71 degrees C for E. coli O157:H7 exposed to high osmotic pressure.     

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.     

Penetration of Escherichia coli O157:H7 into lettuce as influenced by modified atmosphere and temperature.

The effects of temperature and atmospheric oxygen concentration on the respiration rate of iceberg lettuce and Escherichia coli O157:H7 cells attachment to and penetration into damaged lettuce tissues were evaluated. Respiration rate of lettuce decreased as the temperature was reduced from 37 to 10 degrees C. Reducing the temperature further to 4 degrees C did not affect the respiration rate of lettuce. Respiration rate was also reduced by lowering the atmospheric oxygen concentration. Lettuce was submerged in E. coli O157:H7 inoculum at 4, 10, 22, or 37 degrees C under 21 or 2.7% oxygen. Attachment and penetration of E. coli O157:H7 were not related to the respiration rate. The greatest numbers of E. coli O157:H7 cells attached to damaged lettuce tissues at 22 degrees C at both oxygen concentrations. More cells were attached under 21% oxygen than under 2.7% oxygen at each temperature, but this difference was small. Penetration of E. coli O157:H7 into lettuce tissue was determined by immunostaining with a fluorescein isothiocyanate-labeled antibody. Under 21% oxygen, E. coli O157:H7 cells showed greatest penetration when lettuce was held at 4 degrees C, compared to 10, 22. or 37 degrees C, and were detected at an average of 101 microm below the surfaces of cut tissues. However, under 2.7% oxygen, there were no differences in degree of penetration among four incubation temperatures. The degree of E. coli O157:H7 penetration into lettuce tissue at 4 or 22 degrees C was greater under 21% oxygen than under 2.7% oxygen; however, no difference was observed at 37 degrees C. Conditions that promote pathogen penetration into tissue could decrease the effectiveness of decontamination treatments.     

Response surface modeling for the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annuum L.) by chlorine dioxide gas treatments

The effects of chlorine dioxide (ClO2) gas concentration (0.1 to 0.5 mg/liter), relative humidity (RH) (55 to 95%), treatment time (7 to 135 min), and temperature (5 to 25 degrees C) on inactivation of Escherichia coli O157:H7 on green peppers were studied using response surface methods. A four-factor, central, composite, rotatable design was used. The microbial log reduction was measured as a response. A direct membrane-surface-plating method with tryptic soy agar and sorbitol MacConkey agar was used to resuscitate and enumerate ClO2-treated E. coli O157:H7 cells. The statistical analysis and the predictive model developed in this study suggest that ClO2 gas concentration, treatment time, RH, and temperature all significantly (P < 0.01) increased the inactivation of E. coli O157:H7. ClO2 gas concentration was the most important factor, whereas temperature was the least significant. The interaction between ClO2 gas concentration and RH indicated a synergistic effect. The predictive model was validated, and it could be used to determine effective ClO2 gas treatments to achieve a 5-log reduction of E. coli O157:H7 on green peppers.     

Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection

Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157: H7 for 24+/-1 h at 4 degrees C. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.     

Antibacterial effect of monocaprylin on Escherichia coli O157:H7 in apple juice

The antibacterial effect of low concentrations of monocaprylin on Escherichia coli O157:H7 in apple juice was investigated. Apple juice alone (control) or containing 2.5 mM (0.055%) or 5 mM monocaprylin was inoculated with a five-strain mixture of E. coli O157:H7 at approximately 6.0 log CFU/ml. The juice samples were stored at 23 or 4 degrees C for 14 or 21 days, respectively, and the population of E. coli O157:H7 was determined on tryptic soy agar plates supplemented with 0.6% yeast extract. At both storage temperatures, the population of E. coli O157:H7 in monocaprylin-supplemented juice samples was significantly lower (P < 0.05) than that in the control samples. The concentration of monocaprylin and the storage temperature had a significant effect on the inactivation of E. coli O157:H7 in apple juice. Monocaprylin at 5 mM was significantly more effective than 2.5 mM monocaprylin for killing E. coli O157:H7 in apple juice. Inactivation of E. coli O157:H7 by monocaprylin was more pronounced in juice stored at 23 degrees C than in the refrigerated samples. Results of this study indicated that monocaprylin is effective for killing E. coli O157:H7 in apple juice, but detailed sensory studies are needed to determine the organoleptic properties of apple juice containing monocaprylin.