Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.     

Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.     

Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature

Inactivation of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice treated with ozone was evaluated. A five-strain mixture of E. coli O157:H7 or a five-serovar mixture of Salmonella was inoculated (7 log CFU/ml) into apple cider and orange juice. Ozone (0.9 g/h) was pumped into juices maintained at 4 degrees C, ambient temperature (approximately 20 degrees C), and 50 degrees C for up to 240 min, depending on organism, juice, and treatment temperature. Samples were withdrawn, diluted in 0.1% peptone water, and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on tryptic soy agar (TSA), sorbitol MacConkey agar, hemorrhagic coli agar, and modified eosin methylene blue agar; recovery of Salmonella was compared on TSA, bismuth sulfite agar, and xylose lysine tergitol 4 (XLT4) agar. After treatment at 50 degrees C, E. coli O157:H7 populations were undetectable (limit of 1.0 log CFU/ml; a minimum 6.0-log CFU/ml reduction) after 45 min in apple cider and 75 min in orange juice. At 50 degrees C, Salmonella was reduced by 4.8 log CFU/ml (apple cider) and was undetectable in orange juice after 15 min. E. coli O157:H7 at 4 degrees C was reduced by 4.8 log CFU/ml in apple cider and by 5.4 log CFU/ml in orange juice. Salmonella was reduced by 4.5 log CFU/ml (apple cider) and 4.2 log CFU/ml (orange juice) at 4 degrees C. Treatment at ambient temperature resulted in population reductions of less than 5.0 log CFU/ml. Recovery of E. coli O157:H7 and Salmonella on selective media was substantially lower than recovery on TSA, indicating development of sublethal injury. Ozone treatment of apple cider and orange juice at 4 degrees C or in combination with mild heating (50 degrees C) may provide an alternative to thermal pasteurization for reduction of E. coli O157:H7 and Salmonella in apple cider and orange juice.     

Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials

Brine solution injection of beef contaminated with Escherichia coli O157:H7 on its surface may lead to internalization of pathogen cells and/or cross-contamination of the brine, which when recirculated, may serve as a source of new product contamination. This study evaluated survival of E. coli O157:H7 in brines formulated without or with antimicrobials. The brines were formulated in sterile distilled water (simulating the composition of freshly prepared brines) or in a nonsterile 3% meat homogenate (simulating the composition of recirculating brines) at concentrations used to moisture-enhance meat to 110% of initial weight, as follows: sodium chloride (NaCl, 5.5%) + sodium tripolyphosphate (STP, 2.75%), NaCl + sodium pyrophosphate (2.75%), or NaCl + STP combined with potassium lactate (PL, 22%), sodium diacetate (SD, 1.65%), PL + SD, lactic acid (3.3%), acetic acid (3.3%), citric acid (3.3%), nisin (0.0165%) + ethylenediamine tetraacetic acid (EDTA, 200 mM), pediocin (11000 AU/mL) + EDTA, sodium metasilicate (2.2%), cetylpyridinium chloride (CPC, 5.5%), or hops beta acids (0.0055%). The brines were inoculated (3 to 4 log CFU/mL) with rifampicin-resistant E. coli O157:H7 (8-strain composite) and stored at 4 or 15 °C (24 to 48 h). Immediate (0 h) pathogen reductions (P < 0.05) of 1.8 to ≥ 2.4 log CFU/mL were observed in brines containing CPC or sodium metasilicate. Furthermore, brines formulated with lactic acid, acetic acid, citric acid, nisin + EDTA, pediocin + EDTA, CPC, sodium metasilicate, or hops beta acids had reductions (P < 0.05) in pathogen levels during storage; however, the extent of pathogen reduction (0.4 to > 2.4 log CFU/mL) depended on the antimicrobial, brine type, and storage temperature and time. These data should be useful in development or improvement of brine formulations for control of E. coli O157:H7 in moisture-enhanced meat products. PRACTICAL APPLICATION: Results of this study should be useful to the meat industry for developing or modifying brine formulations to reduce the risk of E. coli O157:H7 in moisture-enhanced meat products.     

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.     

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.     

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.     

Survival of Escherichia coli O157:H7 in ground-beef patties during storage at 2, -2, 15 and then -2 degrees C, and -20 degrees C

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (-20 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.     

Inhibition of Salmonella enterica and Escherichia coli O157:H7 on roasted turkey by edible whey protein coatings incorporating the lactoperoxidase system

The effects of whey protein isolate (WPI) coatings incorporating a lactoperoxidase system (LPOS) on the inhibition of Salmonella enterica and Escherichia coli O157:H7 on roasted turkey were studied by testing the initial inhibition as well as the inhibition during storage. The initial antimicrobial effects of WPI coatings incorporating LPOS (LPOS-WPI coatings) were examined with various inoculation levels and LPOS concentrations. LPOS-WPI coatings with 7 and 4% of LPOS demonstrated initial 3- and 2-log CFU/g reductions of S. enterica and E. coli O157:H7, respectively. The antimicrobial effect was observed regardless of whether the turkey was inoculated before or after coating. Storage studies were conducted for 42 days at 4 and 10 degrees C with S. enterica (6.0 log CFU/g)- or E. coli O157:H7 (5.6 log CFU/g)-inoculated sliced turkey. LPOS concentrations for the storage studies of S. enterica and E. coli O157:H7 were 5 and 3% (wt/wt), respectively, in the coating solution and in an LPOS solution for spreading. LPOS-WPI coatings inhibited the growth of both S. enterica and E. coli O157:H7 in turkey at both 4 and 10 degrees C for 42 days. The inhibition was more pronounced when the coating was formed on the surface of the turkey prior to inoculation than when the coating was formed on the inoculated surface. More effective inhibition of S. enterica and E. coli O157:H7 was observed with the LPOS-WPI coatings than with the LPOS solution-spreading treatment. LPOS-WPI coatings also retarded the growth of total aerobes during storage.     

Comparison of methods for detection and isolation of cold- and freeze-stressed Escherichia coli O157:H7 in raw ground beef

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at < or =0.5 and < or =2 CFU/g, and samples were then enriched immediately or were stored at 4 degrees C for 72 h or at -20 degrees C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42 degrees C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35 degrees C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42 degrees C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35 degrees C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+n at 35 degrees C, 3.7 times more likely with an initial inoculum of < or = 2.0 CFU/g than with < or = 0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.     

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.     

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.     

Antibacterial effect of lactoferricin B on Escherichia coli O157:H7 in ground beef.

The antibacterial activity of lactoferricin B on enterohemorrhagic Escherichia coli O157:H7 in 1% peptone medium and ground beef was studied at 4 and 10 degrees C. In 1% peptone medium, 50 and 100 microg of lactoferricin B per ml reduced E. coli O157:H7 populations by approximately 0.7 and 2.0 log CFU/ml, respectively. Studies comparing the antibacterial effect of lactoferricin B on E. coli O157:H7 in 1% peptone at pH 5.5 and 7.2 did not reveal any significant difference (P > 0.5) at the two pH values. Lactoferricin B (100 microg/g) reduced E. coli O157:H7 population in ground beef by about 0.8 log CFU/g (P < 0.05). No significant difference (P > 0.5) was observed in the total plate count between treatment and control ground beef samples stored at 4 and 10 degrees C. The antibacterial effect of lactoferricin B on E. coli O157:H7 observed in this study is not of sufficient magnitude to merit its use in ground beef for controlling the pathogen.     

Reduction in levels of Escherichia coli O157:H7 in apple cider by pulsed electric fields.

Many studies have demonstrated that high voltage pulsed electric field (PEF) treatment has lethal effects on microorganisms including Escherichia coli O157:H7; however, the survival of this pathogen through the PEF treatment is not fully understood. Fresh apple cider samples inoculated with E. coli O157:H7 strain EC920026 were treated with 10, 20, and 30 instant charge reversal pulses at electric field strengths of 60, 70, and 80 kV/cm, at 20, 30, and 42 degrees C. To accurately evaluate the lethality of apple cider processing steps, counts were determined on tryptic soy agar (TSA) and sorbitol MacConkey agar (SMA) to estimate the number of injured and uninjured E. coli O157:H7 cells after PEF treatment. Cell death increased significantly with increased temperatures and electric field strengths. A maximum of 5.35-log10 CFU/ml (P < 0.05) reduction in cell population was achieved in samples treated with 30 pulses and 80 kV/cm at 42 degrees C. Cell injury measured by the difference between TSA and SMA counts was found to be insignificant (P > 0.05). Under extreme conditions, a 5.91-log10 CFU/ml reduction in cell population was accomplished when treating samples with 10 pulses and 90 kV/cm at 42 degrees C. PEF treatment, when combined with the addition of cinnamon or nisin, triggered cell death, resulting in a reduction in E. coli O157:H7 count of 6 to 8 log10 CFU/ml. Overall, the combination of PEF and heat treatment was demonstrated to be an effective pasteurization technique by sufficiently reducing the number of viable E. coli O157:H7 cells in fresh apple cider to meet U.S. Federal Drug Administration recommendations.     

Transfer of Escherichia coli O157:H7 to romaine lettuce due to contact water from melting ice

Ice can be used to chill romaine lettuce and maintain relative humidity during transportation. Escherichia coli O157:H7 may contaminate water used for ice. The objective of this study was to determine the potential for E. coli O157:H7 contamination of romaine lettuce from either ice contaminated with the pathogen or by transfer from lettuce surfaces via melting ice. In experiment 1, lettuce was spot inoculated with E. coli O157:H7 and chilled with ice prepared from uncontaminated tap water. In experiment 2, water inoculated with this pathogen was frozen and used to ice lettuce. Three heads of lettuce were stacked in each container and stored at 4 or 20 degrees C. After the ice melted, E. coli O157:H7 attachment to and recovery from the lettuce leaves were determined. For experiment 1, the population of E. coli O157:H7 attached to inoculated sites averaged 3.8 and 5.5 CFU/cm2 at 4 and 20 degrees C, respectively. Most of the uninoculated sites became contaminated with the pathogen due to ice melt. For experiment 2, 3.5 to 3.8 log CFU E. coli O157:H7 per cm2 was attached to the top leaf on the first head. After rinsing with chlorinated water (200 microg/ml), E. coli O157:H7 remained on the surface of the top head (1.8 to 2.0 log CFU/cm2). There was no difference in numbers of E. coli O157:H7 recovered from each sampling site at 4 and 20 degrees C. Results show that E. coli O157:H7 can be transferred onto other produce layers in shipping containers from melted ice made of contaminated water and from contaminated to uncontaminated leaf surfaces.     

Survival of a five-strain cocktail of Escherichia coli O157:H7 during the 60-day aging period of cheddar cheese made from unpasteurized milk

The U.S. Food and Drug Administration Standard of Identity for Cheddar cheeses requires pasteurization of the milk, or as an alternative treatment, a minimum 60-day aging at > or =2 degrees C for cheeses made from unpasteurized milk, to reduce the number of viable pathogens that may be present to an acceptable risk. The objective of this study was to investigate the adequacy of the 60-day minimum aging to reduce the numbers of viable pathogens and evaluate milk subpasteurization heat treatment as a process to improve the safety of Cheddar cheeses made from unpasteurized milk. Cheddar cheese was made from unpasteurized milk inoculated with 10(1) to 10(5) CFU/ml of a five-strain cocktail of acid-tolerant Escherichia coli O157:H7. Samples were collected during the cheese manufacturing process. After pressing, the cheese blocks were packaged into plastic bags, vacuum sealed, and aged at 7 degrees C. After 1 week, the cheese blocks were cut into smaller-size uniform pieces and then vacuum sealed in clear plastic pouches. Samples were plated and enumerated for E. coli O157:H7. Populations of E. coli O157:H7 increased during the cheese-making operations. Population of E. coli O157:H7 in cheese aged for 60 and 120 days at 7 degrees C decreased less than 1 and 2 log, respectively. These studies confirm previous reports that show 60-day aging is inadequate to eliminate E. coli O157:H7 during cheese ripening. Subpasteurization heat-treatment runs were conducted at 148 degrees F (64.4 degrees C) for 17.5 s on milk inoculated with E. coli O157:H7 at 10(5) CFU/ml. These heat-treatment runs resulted in a 5-log E. coli O157: H7 reduction.     

Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation

Lactobacillus reuteri strain 12002 was used for reuterin production in the two-step fermentation process. A batch culture fermentation was used to produce a maximum biomass of L. reuteri. Then cells were harvested, resuspended in a glycerol-water solution, and anaerobically incubated to produce reuterin. The lyophilized supernatants (approximately 4000 activity units (AU) of reuterin per ml) were diluted in distilled water for decontamination and preservation trials. The MIC values of reuterin for Escherichia coli O157:H7 and Listeria monocytogenes were 4 and 8 AU/ml, respectively. In meat decontamination experiments, the surface of cooked pork was inoculated with either L. monocytogenes or E. coli O157:H7 at a level of approximately log10 5 CFU/cm2, incubated for 30 min at 7 degrees C, and decontaminated by exposure to reuterin (500 AU/ml). The bactericidal effect of reuterin was analyzed 15 s and 24 h after exposure at 7 degrees C. After 15 s of exposure to reuterin, viable numbers decreased by 0.45 and 0.3 log10 CFU/cm2 for E. coli O157:H7 and L. monocytogenes, respectively. After 24 h the numbers decreased by 2.7 log10 CFU/cm2 for E. coli O157:H7 and by 0.63 log10 CFU/cm2 for L. monocytogenes. In the same experiment, the combined effect of reuterin and lactic acid was also investigated. Adding lactic acid (5%, vol/vol) to reuterin significantly enhanced (P < or = 0.05) the efficacy of reuterin. No additional effect (P < or = 0.05) was found when ethanol (40%) was added to the mixture of reuterin and lactic acid. To evaluate the preservative effect of reuterin during meat storage, reuterin was added to raw ground pork contaminated with E. coli O157:H7 or L. monocytogenes. Reuterin at a concentration of 100 AU/g resulted in a 5.0-log10 reduction of the viability of E. coli O157:H7 after 1 day of storage at 7 degrees C. Reuterin at a concentration of 250 AU/g reduced the number of the viable cells of L. monocytogenes by log10 3.0 cycles after 1 week of storage at 7 degrees C.     

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.     

Use of steam condensing at subatmospheric pressures to reduce Escherichia coli O157:H7 numbers on bovine hide.

This study used a laboratory-scale apparatus to apply subatmospheric steam to bovine hide pieces inoculated with Escherichia coli O157:H7 in maximum recovery diluent (MRD) and in high-liquid content and low-liquid content fecal suspensions (HLC fecal and LLC fecal, respectively). The survival of the organism in fecal clods, which were stored for 24 days in a desiccated state, was assessed. Inoculated fecal clods were also treated with subatmospheric steam. Steam treatment at 80 +/- 2 degrees C for 20 s reduced E. coli O157:H7 concentrations on hide inoculated to initial concentrations of approximately 7 log10 CFU/g by 5.46 (MRD inoculum), 4.17 (HLC fecal inoculum), and 5.99 (LLC fecal inoculum) log10 CFU/g. The reductions achieved in samples inoculated with LLC feces were larger than in samples inoculated with HLC feces (P < 0.05). Treatment at 80 +/- 2 degrees C for 10 s resulted in significantly smaller reductions (P < 0.05) on hide pieces of 2.54 (MRD), 1.94 (HLC fecal), and 2.15 (LLC fecal) log10 CFU/g. There were no significant differences among the reductions observed in all inoculum types in samples treated for 10 s. E. coli O157:H7 inoculated in fecal clods to 7.78 log10 CFU/g and stored at 4 or 15 degrees C survived for at least 24 days. Steam treatment (20 s) of 3-day-old clods reduced surviving E. coli O157:H7 numbers from 4.20 log10 CFU/g to below the limit of detection of the assay used (1.20 log10 CFU/g). This study shows that steam condensing at or below 80 +/- 2 degrees C can reduce E. coli O157:H7 when present on bovine hide, reducing the risk of cross contamination to the carcass during slaughter and dressing.     

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.