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.     

Influence of acid adaptation on the tolerance of Escherichia coli O157:H7 to some subsequent stresses

Three stains of Escherichia coli O157:H7, including ATCC 43889, ATCC 43895, and 933, were first subjected to acid adaptation at a pH of 5.0 for 4 h. Thermal tolerance at 52 degrees C and survival of the acid-adapted as well as the nonadapted cells of E. coli O157:H7 in the presence of 10% sodium chloride, 0.85% bile salt, or 15.0% ethanol were investigated. Results showed that the effect of acid adaptation on the survival of E. coli O157:H7 varied with the strains and types of subsequent stress. Acid adaptation caused an increase in the thermal tolerance of E. coli O157:H7 ATCC 43889 and ATCC 43895, but no significant difference in the thermal tolerance was noted between acid-adapted and nonadapted cells of E. coli O157:H7 933. Although the magnitude of increase varied with strains of test organisms, acid adaptation generally led to an increase in the tolerance of E. coli O157:H7 to sodium chloride. On the other hand, the susceptibility of acid-adapted cells of the three strains of E. coli O157:H7 tested did not show a significant difference from that of their nonadapted counterparts when stressed with bile salt. The acid-adapted cells of E. coli O157:H7 ATCC 43889 and ATCC 43895 were less tolerant than the nonadapted cells to ethanol, whereas the tolerance of adapted and nonadapted cells of E. coli O157:H7 933 showed no significant differences.     

Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids.

A study was done to determine if various organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted Escherichia coli O157:H7 cells. E. coli O157:H7 strain EO139, isolated from venison jerky, was grown in tryptic soy broth (TSB) and in TSB supplemented with 1% glucose (TSBG) for 18 h at 37 degrees C, then plated on tryptic soy agar (TSA) acidified with malic, citric, lactic, or acetic acid at pH 5.4, 5.1, 4.8, 4.5, 4.2, and 3.9. Regardless of whether cells were grown in TSB or TSBG, visible colonies were not formed when plated on TSA acidified with acetic, lactic, malic, or citric acids at pH values of < or =5.4, < or =4.5, < or =4.2, or < or =4.2, respectively. Cells not adapted to reduced pH did not form colonies on TSA acidified with lactic acid (pH 3.9) or acetic acid (pH 3.9 and 4.2); however, a portion of acid-adapted cells remained viable on TSA containing lactic acid (pH 3.9) or acetic acid (pH 4.2) and could be recovered in TSB. Inactivation of acid-adapted cells was less than that of unadapted cells in TSB acidified at pH 3.9 with citric, lactic, or acetic acid and at pH 3.4 with malic acid. Significantly (P< or =0.05) higher numbers of acid-adapted cells, compared with unadapted cells, were detected 12 h after inoculation of TSB acidified with acetic acid at pH 3.9; in TSB containing lactic acid (pH 3.9), the number of acid-adapted cells was higher than the number of unadapted cells after 5 h. In TSB acidified at pH 3.9 with citric acid or pH 3.4 with malic acid, significantly higher numbers of acid-adapted cells survived. This study shows that organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted E. coli O157:H7 cells, and acid-adapted cells are more tolerant than unadapted cells when subsequently exposed to reduced pH caused by these acids.     

Effect of trisodium phosphate adaptation on changes in membrane lipid composition, verotoxin secretion, and acid resistance of Escherichia coli O157:H7 in simulated gastric fluid

Escherichia coli O157:H7 (HEC), E. coli O157:H7 rpoS mutant (HEC-RM), and nonpathogenic E. coli (NPEC) were step-wise adapted to trisodium phosphate (TSP) by incubation in broths of increasing concentration, from 0% to 0.6%, at 37 degrees C for 24 h. After incubation at each concentration, each population was examined for acid resistance (D value) in simulated gastric fluid of pH 1.5, cell envelope membrane lipid composition, and intracellular and extracellular verotoxin concentrations. The ratio of cis-vaccenic acid (18:1omega7c) to palmitic acid (16:0) increased, indicating increased membrane fluidity with increasing TSP concentration up to 0.4%, but decreased at 0.6%. HEC and HEC-RM adapted at 0.4% TSP had the highest verotoxin concentrations of 1805 and 1879 ng/ml, respectively. In addition, with HEC the ratio of extracellular to intracellular verotoxin concentration decreased at higher TSP concentrations. In contrast, the ratio for HEC-RM increased at 0.4% TSP. HEC adapted to 0.4% TSP had the greatest survival in gastric fluid (58 min D value) among all treatments. For HEC, the increase in membrane fluidity was associated with increased acid resistance and extracellular verotoxin concentration for cells adapted to 0.4% TSP. In contrast, the increase in membrane fluidity was associated with decreased acid resistance of TSP adapted HEC-RM although the extracellular verotoxin concentration increased. Therefore, the deletion of the rpoS gene appeared to affect the changes in verotoxin concentration and acid resistance of TSP adapted E. coli O157:H7.     

Effect of acid adaptation on survival of Escherichia coli O157:H7 in meat decontamination washing fluids and potential effects of organic acid interventions on the microbial ecology of the meat plant environment.

The acid tolerance of Escherichia coli O157:H7 may be pH inducible. Correspondingly, organic acid meat decontamination washing fluids may enhance the establishment of acid-adapted E. coli O157:H7 strains in packing plants, especially in mixtures with water washings from meat that may be of sublethal pH. Acid-adapted and nonadapted cultures of a rifampin-resistant derivative of the acid-resistant E. coli O157:H7 strain ATCC 43895 were tested to evaluate their survival in meat-washing fluids over a wide pH range. The cultures were exposed (10(5) CFU/ml) to acidic (2% lactic acid. 2% acetic acid, or a mixture of the two with water washings at ratios of 1/1, 1/9, or 1/99 [vol/vol]) or nonacid (water) meat washings for up to 14 days at 4 or 10 degrees C storage. E. coli O157:H7 survived in water washings, but the low storage temperatures and predominant natural microbiota synergistically inhibited its growth. Compared with acid-adapted populations, nonadapted populations displayed greater potential for survival and a tendency to initiate growth in water meat washings at 10 degrees C. The pathogen survived in most of the acid washings throughout storage (14 days), sometimes with minimal population reductions. Overall. nonadapted populations declined faster than acid-adapted populations, while the declines increased as the acid concentration and temperature of storage increased and were more dramatic in lactate, compared to acetate, washings. Acid-containing washings were selective for growth of lactic acid bacteria and yeasts. indicating that organic acid treatments may alter the microbial ecology of meat plant environments and potentially that of the meat. These results should be considered when selecting decontamination technologies for meat.     

Increased acid tolerance of Escherichia coli O157:H7 as affected by acid adaptation time and conditions of acid challenge

Three strains of Escherichia coli O157:H7, ATCC 43889, 43895 and 933 were subjected to acid adaptation in Tryptic Soy Broth (pH 5.0) for 1, 2, 3, 4 and 6 h. Acid tolerance of the adapted cells was determined in subsequent acid challenge at pH 3.0, 4.0 and 5.0 (acidified with HCl) and in the presence of lactic, acetic or propionic acid. It was found that acid adaptation increased acid tolerance of the E. coli O157:H7 strains tested and was dependent on strain, acid adaptation time and pH of the challenge. Among the acid adaptation times tested, 4 h of adaptation enabled the test organism, regardless of strains, to exhibit the most pronounced acid adaptation response which was most marked at pH 3.0, followed by pH 4.0 and 5.0. The extent of increased acid tolerance varied with the strains of E. coli O157:H7 and challenge of organic acid. The 4-h acid-adapted cells of ATCC 43889 and 933 showed an increase in acid tolerance in the presence of lactic, acetic and propionic acids. An increase in tolerance was also noted with ATCC 43895 in the presence of acetic and lactic acid, but not in the presence of propionic acid.     

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.     

Acid tolerance of acid-adapted and nonacid-adapted Escherichia coli O157:H7 strains in beef decontamination runoff fluids or on beef tissue

This study assessed the acid tolerance response (ATR) of stationary phase, acid-adapted (tryptic soy broth [TSB]+1% glucose) or nonacid-adapted (glucose-free TSB) Escherichia coli O157:H7 strains (ATCC43889, ATCC43895, ATCC51658 and EO139), grown individually or in a mixed culture, prior to inoculation of beef or meat decontamination runoff (washings) fluids (acidic [pH 4.95] or nonacidic [pH 7.01]). The inoculated beef was left untreated or treated by dipping for 30s in hot water (75 degrees C) followed by 2% lactic acid (55 degrees C). Inoculated beef samples and washings were stored aerobically at 4 or 15 degrees C for 6d, and at set intervals (0, 2, and 6d) were exposed (for 0, 60, 120, and 180min) to pH 3.5 (adjusted with lactic acid) TSB plus 0.6% yeast extract. Overall, there were no significant (P0.05) differences in responses of cultures prepared as individual or mixed strains. Decontamination of meat did not affect the subsequent ATR of E. coli O157:H7 other than resulting in lower initial pathogen levels exposed to acidic conditions. In this study, E. coli O157:H7 appeared to become more tolerant to acid following incubation in acidic washings of sublethal pH (4.89-5.22) compared to nonacidic washings (pH 6.97-7.41) at 4 degrees C or in both types of washings incubated at 15 degrees C. The ATR of the pathogen inoculated into washings was enhanced when cells were previously acid-adapted and incubated at 4 degrees C. Similarly, the ATR on meat was increased by previous acid-adaptation of the inoculum in broth and enhanced by storage at 4 degrees C. Populations on treated meat were consistently lower than those on untreated meat during storage and following exposure to acid. Although on day-0 there were no significant (P0.05) differences in ATR between acid-adapted and nonacid-adapted populations on meat, acid-adapted cells displayed consistently higher resistance through day-6. This suggests that acid-adapted E. coli O157:H7 introduced on meat may become resistant to subsequent lactic acid exposure following storage at 4 degrees C.     

Stress response of acid-shocked Cronobacter sakazakii against subsequent acidic pH, mild heat, and organic acids

This study was conducted to determine the resistance of acid-shocked Cronobacter sakazakii to environmental stresses. C. sakazakii pre-exposed to various pH levels was treated with acid stress (pH 3.06), heat stress (55°C), and organic acid stress, respectively. Overall, higher D-values were obtained in samples pre-exposed to acidic pH conditions (pH 3.06, 4.00, and 5.02) compared to a control (pH 7.20) when the samples were subsequently stressed. For 0.1 M acetic acid, the D-values of nonadapted C. sakazakii ATCC 29004 and ATCC 29544 were 19.69 and 15.49 h, respectively, whereas the D-values of acid-shocked C. sakazakii ATCC 29004 and ATCC 29544 by pre-exposure to pH 4.0 were 34.59 and 24.25 h, respectively. Acid adaptation of C. sakazakii by preexposure to acidic pH can enhance the resistance of cells against subsequent environmental stresses such as acidic pH, heat, and organic acids.     

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.     

乳酸菌对肠出血性大肠杆菌O157:H7抑制作用的研究

为探讨乳酸菌对肠出血性大肠杆菌O157：H7 ATCC43895(E．Coli O157：H7)的抑制作用，在培养基上进行了研究。将E．Coli O157：H7与干酪乳杆菌干酪亚种、植物乳杆菌、发酵乳杆菌、乳酸乳球菌和瑞士乳杆菌同时接种在培养基中，E．Coli O157：H7的活性不受影响；将E．Coli O157：H7接种到培养了24h的乳酸茵培养液中，E．Coli O157：H7活性显下降。以乳酸调整的低pH值对E．Coli O157：H7有一定的杀灭作用。本研究表明：乳酸菌的代谢产物乳酸对E．Coli O157：H7有杀灭作用。     

Efficacy of selected acidulants in pureed green beans inoculated with pathogens (Escherichia coli O157:H7 and Listeria monocytogenes)

Studies were conducted to evaluate the combined effect of selected acidulants (acetic, citric, malic, and phosphoric acid) and heat on foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) in pureed green beans. To establish a consistent reference point for comparison, the molar concentrations of the acids remained constant while the acid-to-puree ratio, titratable acidity, and undissociated acid were either measured or calculated for a target acidified green beans at a pH of 3.8, 4.2, and 4.6. The D-values at 149 degrees F were used as the criteria for acid efficacy. Generally, acetic acid (puree, pH 3.8 and 4.2) represented the most effective acid with comparatively low D-values irrespective of the target microorganism. A 10-s heating at 149 degrees F inactivated approximately 10(6) CFU/ml of E. coli O157:H7 in pureed beans at pH 3.8. The efficacy of acetic acid is likely related to the elevated percent titratable acidity, undissociated acid, and acid-to-puree ratio. The effectiveness (which in this study represents the combined effect of acid and heat) of the remaining acids (citric, malic, and phosphoric) at puree pH values of 3.8 and 4.2 were statistically insignificant (alpha = 0.05). Surprisingly, acetic acid (puree, pH 4.6) appeared to be the least effective as compared to the other acids tested (citric, malic, and phosphoric) especially on E. coli O157:H7 cells, while L. monocytogenes had a similar resistance to all acids at puree pH 4.6. With the exception of citric acid (pH 3.8), acetic acid (pH 4.6), and malic acid (pH 3.8 and 4.6), which were statistically insignificant (P > 0.05), the D-values for L. monocytogenes were statistically different (P < or = 0.05) and higher than the D-values for E. coli under similar experimental conditions. A conservative process recommendation (referred to as the "safe harbor" process) was found sufficient and applicable to pureed green beans for the pH range studied.     

Inactivation of acid-adapted and nonadapted Escherichia coli O157:H7 during drying and storage of beef jerky treated with different marinades

The inactivation of both acid-adapted and unadapted Escherichia coli O157:H7 during the processing of beef jerky was studied. Following inoculation with the pathogen, beef slices were subjected to different predrying marinade treatments, dried at 60 degrees C for 10 h, and stored at 25 degrees C for 60 d. The predrying treatments evaluated were as follows: (i) no treatment (C), (ii) traditional marinade (TM), (iii) double-strength TM modified with added 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce with 5% ethanol (MM), (iv) dipping into 5% acetic acid for 10 min followed by application of TM (AATM), and (v) dipping into 1% Tween 20 for 15 min and then into 5% acetic acid for 10 min followed by TM (TWTM). Bacterial survivors were determined during drying and storage using tryptic soy agar with 0.1% pyruvate, modified eosin methylene blue agar, and sorbitol MacConkey agar. Results indicated that bacterial populations decreased during drying in the order of TWTM (4.9 to 6.7 log) > AATM > MM > C > or = TM (2.8 to 4.9 log) predrying treatments. Populations of acid-adapted E. coli O157:H7 decreased faster (P < 0.05) in AATM and TWTM than nonadapted cells during drying, whereas no significant difference was found in inactivation of acid-adapted and nonadapted inocula in C and TM samples. MM was more effective in inactivating the nonadapted than the adapted inoculum. Bacterial populations continued to decline during storage and dropped below the detection limit (-0.4 log10 CFU/cm2) as early as day 0 (after drying) or as late as day 60, depending on acid adaptation, predrying treatment, and agar medium. The results indicated that acid adaptation may not increase resistance to the hurdles involved in jerky processing and that use of additional antimicrobial chemicals or preservatives in jerky marination may improve the effectiveness of drying in inactivating E. coli O157:H7.     

Sensitivity of acid-adapted and acid-shocked Shigella flexneri to reduced pH achieved with acetic, lactic, and propionic acids.

Survival and growth characteristics of unadapted, acid-adapted, and acid-shocked Shigella flexneri 2a cells in acidified (pH 3.5 to 5.5) tryptic soy broth with 0.25% glucose (TSB) and tryptic soy agar (TSA) were determined. S. flexneri was grown at 37 degrees C for 18 h in tryptic soy broth without glucose (TSBNG) (unadapted) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted). Cells grown in TSBNG were acid shocked by adjusting 16-h cultures to pH 5.05 +/- 0.05 with lactic acid. Cells were then inoculated into TSB acidified with acetic, lactic, or propionic acids to pH 5.5, 4.5, or 3.5 and incubated at 37 degrees C for 6 h. The order of lethality at a given pH was lactic acid < acetic acid < propionic acid. Significantly (P < or = 0.05) higher numbers of acid-adapted cells, compared to acid-shocked and unadapted cells, were recovered from TSB acidified (pH 3.5) with lactic or acetic acids. None of the cells survived a 30-min exposure in TSB acidified with propionic acid to pH 3.5. When the three cell types were plated on TSA acidified with lactic, acetic, or propionic acids at pH < or = 4.5, < or = 5.5, and < or = 5.5, respectively, visible colonies were not detected. Viable unadapted, acid-adapted, and acid-shocked cells were, however, recovered from TSA acidified with all three acids at pH > or = 4.5. Acid-adapted and, to a lesser extent, acid-shocked cells survived at lower pH than did unadapted cells, indicating that prior exposure to mild acidic environment results in increased acid resistance. Survival of S. flexneri at a given pH was influenced by the type of acidulant used, a response characteristic exhibited by other gram-negative enteric pathogens.     

Influence of extended acid stressing in fresh beef decontamination runoff fluids on sanitizer resistance of acid-adapted Escherichia coli O157:H7 in biofilms

This study evaluated resistance to sanitizing solutions of Escherichia coli O157:H7 cells forming biofilms on stainless steel coupons exposed to inoculated meat decontamination runoff fluids (washings). A previously acid-adapted culture of a rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated in unsterilized or sterilized combined hot-water (85 degrees C) and cold-water (10 degrees C) (50/50 [vol/vol]) composite water (W) washings (pH 6.29 to 6.47) and in W washings mixed with 2% acetic acid (pH 4.60 to 4.71) or in 2% lactic acid W washings (pH 4.33 to 4.48) at a ratio of 1/99 (vol/vol). Stainless steel coupons (2 by 5 by 0.08 cm) were submerged in the inoculated washings and stored for up to 14 days at 15 degrees C. Survival of E. coli O157:H7 was determined after exposure (0 to 60 s for cells in suspension and 0 to 300 s for attached cells) to two commercial sanitizers (150 ppm peroxyacetic acid and 200 ppm quaternary ammonium compound) at 2, 7, and 14 days. E. coli O157:H7 attached more rapidly to coupons submerged in washings containing the natural flora than to those without. The attached cells were more resistant to the effects of the sanitizers than were the cells in suspension, and survival was highest in the presence of the natural flora. Attached cells in the presence of dilute acid washings were more sensitive to subsequent sanitizer treatments than were cells generated in the presence of W washings. Under the conditions of this study, cells of E. coli O157:H7 in W washings were more sensitive to acidic (peroxyacetic acid) than to alkaline (quaternary ammonium) sanitizers during storage. These results suggest that meat processing plants that apply no decontamination or that use only water washings of meat should consider using acidic sanitizers to enhance biofilm removal. Plants that apply both water and acidic washings may create a sublethal acid-stressing environment in the runoff fluids, sensitizing biofilm cells to subsequent sanitizing treatments.     

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.     

Absence of association of autoinducer-2-based quorum sensing with heat and acid resistance of Salmonella

This study used various approaches to investigate the potential association of autoinducer-2 (AI-2) presence with thermal and acid resistance of Salmonella cultures. Salmonella Thompson strains RM1987N (luxS-positive; AI-2 positive) and RM1987NLUX (luxS-negative; AI-2 negative) were exposed to 55 °C (6 h) in Luria-Bertani (LB) broth, while the luxS-negative S. Thompson strain and a Salmonella Typhimurium luxS-positive strain were exposed to 55 °C in AI-2-positive or -negative preconditioned (PC) media derived from S. Thompson and Escherichia coli O157:H7 luxS-positive and -negative strains. In addition, the luxS-negative S. Thompson strain was subjected to pH 3.5 PC media (35 °C, 6 h) with or without AI-2 activity, and acid-adapted or nonadapted S. Thompson strains were exposed to pH 3.0 LB broth (35 °C, 6 h). Surviving bacterial populations during exposure to 55 °C LB were not different between luxS-negative and -positive S. Thompson strains. In addition, heating at 55 °C of the luxS-negative S. Thompson strain in AI-2-positive and -negative PC media resulted in similar (P ≥ 0.05) survivor counts. Furthermore, surviving cell counts of S. Typhimurium (luxS-positive) in 55 °C AI-2-positive PC media were not different (P ≥ 0.05) than those in AI-2 negative PC media. No differences in surviving cell counts of the luxS-negative S. Thompson strain was found when exposed to pH 3.5 AI-2-positive and -negative PC media. Also, survivors of acid-adapted or nonadapted cells of luxS-negative and -positive S. Thompson strains were not different following exposure to pH 3.0 LB. The results indicated that, under the conditions of this study, AI-2-based quorum sensing did not appear to be associated with heat and acid resistance of Salmonella.     

Modeling the effect of inoculum size and acid adaptation on growth/no growth interface of Escherichia coli O157:H7

The objective of this study was to model with logistic regression the growth/no growth interface of different initial inoculation levels (10¹, 10³ and 10⁵ CFU/ml; study 1), or nonadapted vs acid-adapted (study 2) Escherichia coli O157:H7 as influenced by pH, NaCl concentration and incubation temperature. Study 1 was conducted with a mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in tryptic soy broth (TSB). Study 2 was conducted with the same mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in glucose-free TSB with 1% added glucose (final pH 4.83), or in diluted lactic acid meat decontamination runoff fluids (washings; final pH 4.92), or nonadapted cultures prepared in glucose-free TSB (final pH 6.45), or in water washings (final pH 6.87). Parameters included incubation temperature (10–35 °C), pH (3.52–7.32), and NaCl concentration (0–10% w/v). Growth responses were evaluated for 60 days turbidimetrically (610 nm) every 5 days in 160 (study 1) and 360 (study 2) combinations in quadruplicate samples, with a microplate reader. The lower the initial inoculum the higher were the minimum pH and a_w values permitting growth. Differences in the pH and a_w growth limits among inoculum concentrations increased at 15 and 10 °C. Acid-adapted cultures were able to grow at lower pH than nonadapted cultures, while at temperatures below 25 °C, growth initiation of nonadapted cultures stopped at higher a_w compared to acid-adapted cultures for the whole pH range of 3.52 to 7.32. A comparison with available data indicated that our model for acid-adapted E. coli O157:H7 in different environments may provide representative growth probabilities covering both nonadapted and stress-adapted contaminants.     

Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance

The effects of growth temperature (in the range 10-45 degrees C) and acidification up to pH 4.5 of the culture medium (Brain Heart Infusion, BHI) with different organic acids (acetic, citric and lactic) and hydrochloric acid on membrane fatty acid composition and heat resistance of Salmonella typhimurium CECT 443 were studied. The heat resistance was maximal in cells grown at 45 degrees C (cells grown in non-acidified BHI showed a D58-value of 0.90 min) and decreased with decreasing growth temperature up to 10 degrees C (D58-value of 0.09 min). The growth of cells in acidified media caused an increase in their heat resistance. In general, acid adapted cells showed D-values of between 1.5 and 2 times higher than the corresponding for non-acid adapted control cells. This cross-protection response, which has important implications in food processing, was not dependent on the pH value and the acid used to acidify the growth medium. A membrane adaptation corresponding to an increase in the unsaturated to saturated fatty acids ratio (UFA/SFA) and membrane fluidity was observed at low growth temperature. Moreover, the acidification of the growth medium caused a decrease in UFA/SFA ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in cyc19 relative concentration. Thus, acid adapted cells showed CFA levels 1.5 times higher than non-acid adapted control cells. A significant proportion of unsaturated fatty acids were converted to their cyclopropane derivatives during acid adaptation. These changes in membrane fatty acid composition result in cells with decreased membrane fluidity. A clear relation between membrane fatty acid composition and heat resistance was observed. In general, D-values were maximum for cells with low UFA/SFA ratio, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting acid adapted cells from heat inactivation. However, changes observed in membrane fatty acid composition are not enough to explain the great thermotolerance of cells grown at 45 degrees C. Thus, other mechanisms, such as the synthesis of Heat Shock Proteins, could be responsible for this increase in the bacterial heat resistance.