Survival of Escherichia coli O157:H7 in Apple Cider as Affected by Dimethyl Dicarbonate, Sodium Bisulfite, and Sodium Benzoate

Survival of Escherichia coli O157:H7 in apple cider containing no preservatives, 0.025% dimethyl dicarbonate (DMDC), 0.045% sodium benzoate (SB), 0.0046% sodium bisulfite (NaS; 65.5% sulfur dioxide), or a combination of NaS and SB (NaS/SB) and stored at 4, 10, and 25°C was evaluated. E. coli O157:H7 survived for up to 18 days at 4,10, and 25°C in unpreserved apple cider. At 4 and 10°C, DMDC was most efficient at inactivating E. coli O157:H7, generally followed by NaS/SB SB, and NaS (p<0.05). E coli O157:H7 was more resistant to preservatives at 4°C than at 25°C (P < 0.05). E. coli O157:H7 was sublethally injured in cider containing preservatives, and to a lesser extent, in unpreserved cider. Generally, injury was more pronounced in cider containing DMDC, followed by NaS/SB, SB, and NaS (p<0.05).     

Inactivation of Escherichia coli with Power Ultrasound in Apple Cider

The use of acoustic energy to secure apple cider safety was explored. Inactivation tests were performed with Escherichia coli K12 at 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C with and without ultrasound, followed by a validation test with E. coli O157:H7 at 60 °C. The cell morphology was observed with environmental scanning electron microscopy for samples treated at 40 °C and 60 °C. Physical quality attributes of the apple cider (pH, titratable acidity, °Brix, turbidity, and color) were compared for treated samples. The inactivation tests showed that sonication increased E. coli K12 cell destruction by 5.3‐log, 5.0‐log, and 0.1‐log cycles at 40 °C, 50 °C, and 60 °C, respectively. The additional destruction due to sonication was more pronounced at sublethal temperatures. At the lethal temperature of 60 °C, the rate of death by ultrasound was not significantly different compared with the thermal‐alone treatment. The inactivation of E. coli K12 with heat was described by 1st‐order kinetics, especially at 50 °C and 60 °C. For ultrasound treatments, concave upward survival curves were observed, which had a shape factor in the range of 0.547 to 0.720 for a Weibull distribution model. Extensive damage for ultrasound treated E. coli K12 cells, including cell perforation, was observed. Perforation is a unique phenomenon found on ultrasound‐treated cells that could be caused by liquid jets generated by cavitation. Titratable acidity, pH, and °Brix of the cider were not affected by ultrasound treatment. Minor changes in color and turbidity for ultrasound treated samples, especially for sonication at 40 °C for 17.7 min, were observed.     

Fate of Escherichia coliO157: H7 in Chinese-style sausage subjected to different packaging and storage conditions

In this study, Chinese-style sausages were subjected to air, vacuum or nitrogen packaging and stored at either 5 or 25°C. The survival characteristics of Escherichia coli O157: H7 during the storage period were determined. Results revealed that, when stored at 5°C, the number of viable E coli O157: H7 in sausages decreased slowly as the storage period extended, regardless of packaging methods. E coli O157: H7 in sausages decreased from an initial population of ca 5·97 log CFU g⁻¹ to ca 4·42–4·81 log CFU g⁻¹ after 40 days of storage at 5°C. It was also found that viable cells of E coli O157: H7 declined more rapidly in sausage stored at 25°C than at 5°C. No viable E coli O157: H7 was detected in either vacuum-packed or nitrogen-packed sausage after 40 days of storage at 25°C. On the other hand, the population of E coli O157: H7 reduced to non-detectable levels in air-packed sausages after 20 days of storage. Refrigerated storage and vacuum or nitrogen packaging provided conditions that slowed down the death rate of E coli O157: H7 in sausage. Furthermore, it was noted that, among the curing agents tested, NaCl exerted the most significant lethal effect on E coli O157: H7 in sausage during the storage period. © 1998 Society of Chemical Industry.     

Recovery of heat-stressed E. coli O157:H7 from ground beef and survival of E. coli O157:H7 in refrigerated and frozen ground beef and in fermented sausage kept at 7°C and 22°C

A study was undertaken to obtain information on survival of Escherichia coli O157:H7 in ground beef subjected to heat treatment, refrigeration and freezing and on survival of E. coli O157:H7 in fermented sausage kept at 7°C and 22°C. For the challenge test, a mixture of E. coli O157:H7 strains (EH 321, EH 385, EH 302) was used and enumeration was performed on an isolation medium suitable for recovery of stressed organisms: modified Levine's eosin methylene blue agar (mEMB). Heat resistance of E. coli O157:H7 decreased after pre-incubation at a reduced temperature.Escherichia coli O157:H7 was more susceptible to heat inactivation after storage at 7°C and die-off was even more enhanced if cultures were frozen prior to heat inactivation. The enhanced reduction of the pathogen at 56°C after prior storage under refrigeration was confirmed in a test with inoculated ground beef.Escherichia coli O157:H7 was able to survive in ground beef at 7°C for 11 days and at −18°C for 35 days showing maximal one log reduction during the storage period. Thus, ground beef contaminated with E. coli O157:H7 will remain a hazard even if the ground beef is held at low or freezing temperatures. At both 7°C and 22°C, a gradual reduction of E. coli O157:H7 was noticed in fermented sausage over the 35 days storage period resulting in a 2 log decrease of the high inoculum (10⁶cfu 25 g⁻¹). For the low inoculum (10³cfu 25 g⁻¹) a 2·5 log reduction was obtained in 7 and 28 days storage at respectively 22 and 7°C. Application of good hygienic practices and implementation of HACCP in the beef industry are important tools in the control of E. coli O157:H7.     

Escherichia coli O157:H7 facilitates the penetration of Staphylococcus aureus into table eggs

Abstract: Fresh eggshells collected from a local farm were subjected to different levels of surface contamination with feces containing different levels (3 to 5 log₁₀) of Escherichia coli O157:H7 or Staphylococcus aureus and incubated at 3 different temperatures (10, 25, and 32 °C). The penetration rates of contaminating bacteria were followed throughout the incubation period by tracing bacterial presence in shell, shell membranes, albumen, and yolk. The study revealed the ability of both E. coli O157:H7 and enterotoxigenic S. aureus to grow on shell in feces, penetrate the shell, and move and multiply within egg contents at different rates and periods depending on bacterial type and incubation conditions. High temperatures (25 and 32 °C) increased penetration rate, whereas storage at 10 °C decreased significantly the rate of penetration. High levels of contamination with E. coli O157:H7 also shortened the time needed for the penetration process. Results showed that when eggshells were contaminated with both organisms simultaneously, the penetration of E. coli O157:H7 preceded that of S. aureus and facilitated the invasion of the latter bacteria.     

Addition of fumaric acid and sodium benzoate as an alternative method to achieve a 5-log reduction of Escherichia coli O157:H7 populations in apple cider

A study was conducted to develop a preservative treatment capable of the Food and Drug Administration-mandated 5-log reduction of Escherichia coli O157:H7 populations in apple cider. Unpreserved apple cider was treated with generally recognized as safe acidulants and preservatives before inoculation with E. coli O157:H7 in test tubes and subjected to mild heat treatments (25, 35, and 45 degrees C) followed by refrigerated storage (4 degrees C). Fumaric acid had significant (P < 0.05) bactericidal effect when added to cider at 0.10% (wt/vol) and adjusted to pH 3.3, but citric and malic acid had no effect. Strong linear correlation (R2 = 0.96) between increasing undissociated fumaric acid concentrations and increasing log reductions of E. coli O157:H7 in apple cider indicated the undissociated acid to be the bactericidal form. The treatment that achieved the 5-log reduction in three commercial ciders was the addition of fumaric acid (0.15%, wt/vol) and sodium benzoate (0.05%, wt/vol) followed by holding at 25 degrees C for 6 h before 24 h of refrigeration at 4 degrees C. Subsequent experiments revealed that the same preservatives added to cider in flasks resulted in a more than 5-log reduction in less than 5 and 2 h when held at 25 and 35 degrees C, respectively. The treatment also significantly (P < 0.05) reduced total aerobic counts in commercial ciders to populations less than those of pasteurized and raw ciders from the same source (after 5 and 21 days of refrigerated storage at 4 degrees C, respectively). Sensory evaluation of the same ciders revealed that consumers found the preservative-treated cider to be acceptable.     

Inhibition of Shiga Toxin 2 (Stx2) in Apple Juices and its Resistance to Pasteurization

Abstract: In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P < 0.05) in the densities or molecular weights between Stx2 in either PBS or apple juices. These results suggest that Stx2 may be reversibly bound to small molecular weight constituents in the juice. The Stx2 toxin was not inactivated on exposure to heat programs (63 °C for 30 min, 72 °C for 15 s, 89 °C for 1 s) commonly used to pasteurize apple juice, but lost all activity when exposed to 100 °C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin. Practical Application: This study explores the possibility of using naturally occurring antioxidative substances, in this case high phenolic apples juices, to inactivate Shiga toxin (Stx2) produced by E. coli O157:H7 in contaminated foods.     

Fate of Escherichia coli O157: H7 in Chinese-Style Sausage During the Drying Step of the Manufacturing Process as Affected by the Drying Condition and Curing Agent

In this study, Chinese-style sausage was subjected to three different air-blast drying conditions commonly employed during the manufacturing process. The fate of Escherichia coli O157: H7 during the drying period was determined and compared. The effect of curing agents on the survival of E coli O157: H7 was also identified. Results showed that populations of E coli O157: H7 decreased ca 1.51 Log CFU g^-1 in sausage containing curing agents after a 6-h drying period at 50°C. However, the number of viable cells of E coli O157: H7 increased slightly in sausage without curing agents. When subjected to air-blast drying at 55°C for 6 h or at 55°C for 2·5 h and then 60°C for 3·5 h, a reduction in the number of viable cells of E coli O157: H7 was observed in sausage with or without curing agents. The reduction was more significant in sausage containing curing agents than in those without curing agents. No viable E coli O157: H7 was detected after 6 h of drying in samples containing curing agents, while the control samples still contained a viable E coli O157: H7 population of ca 2·65–4·36 Log CFU g^-1. After drying the sausage at 55°C for 4 h, inactivation of E coli O157: H7 increased in the presence of 30·00 g kg^-1 sodium chloride or 1·50 g kg^-1 sodium sorbate. On the other hand, the presence of 0·07–0·15 g kg^-1 sodium nitrite did not increase the inactivation of E coli O157: H7 compared to that in the control. © 1997 SCI.     

Kinetics of ultraviolet light inactivation of Escherichia coli O157:H7 in liquid foods

The effects of pH, depth of food medium and ultraviolet (UV) light dose on the inactivation of Escherichia coli O157:H7 in UV‐opaque products such as apple juice (pH 3.5) and egg white (pH 9.1) were investigated. The applied UV dose ranged from 0 to 6.5 mW min cm^?2, while the depths of the medium were 1, 3.5, 5 and 10 mm. The pH of the medium did not affect the inactivation of E coli O157:H7, since similar inactivation characteristics were obtained for both apple juice and liquid egg white. As expected, decreasing the depth of the medium increased the inactivation of E coli O157:H7. More than a 5‐log reduction was obtained when the fluid depth and UV dose were 1 mm and 6.5 mW min cm^?2 respectively. However, less than a 1‐log reduction was obtained when the fluid depth was 10 mm. A two‐phase kinetic model was used to model the inactivation of E coli O157:H7. This model indicated that at higher fluid depths the inactivation rate was controlled by the second, slower inactivation phase, resulting in a lower overall inactivation. The visual appearance of the treated apple juice and egg white did not show any discolouration changes during 4 weeks of storage at ambient temperature (25 °C). Copyright © 2003 Society of Chemical Industry     

Fate of Escherichia coli in apple and reduction of its growth using the postharvest biocontrol agent Candida sake CPA‐1

BACKGROUND: Generally, acidic fruits and fruit juices are considered ‘safe’ from a microbiological point of view. However, some outbreaks of foodborne illnesses have been linked to the consumption of unpasteurised cider. The aim of this work was to study the survival of Escherichia coli in apple juice, wounds and flesh and on apple surfaces at different temperatures and to determine the effect of the fungal biocontrol agent Candida sake CPA‐1 against the colonisation of apple by E. coli. RESULTS: Trials were conducted with a mixture of five strains of E. coli isolated from apples. E. coli was unable to grow in apple juice at 5, 15 and 25 °C but survived. At 10 °C and above, E. coli thrived in fresh‐cut apple and wounds. At 5 °C it survived in apple wounds after 27 days of storage and after 21 days in fresh‐cut apples. When E. coli was inoculated in apple wounds together with the yeast antagonist C. sake, its growth was reduced by approximately 1 log cfu wound^?1 at 25 °C. At 5 °C no effect of the biocontrol agent was observed. CONCLUSION: Despite the low pH of apple, a rapid increase in the bacterial population is possible if the temperature is not kept low enough. The biocontrol agent C. sake, developed to prevent fruit decay during storage, could also reduce E. coli growth in wounded apples at abusive temperatures. This would represent an additional benefit of using this biocontrol agent when applied to control postharvest diseases. Copyright © 2009 Society of Chemical Industry     

Behavior of Escherichia coli O157:H7 and Listeria monocytogenes in Tryptic soy broth subjected to various low temperature treatments

The inactivation and injury of Escherichia coli O157:H7 and Listeria monocytogenes in Tryptic soy broth stored at −5, −18 and −28°C were studied. Regardless of storage temperature, viable populations of E. coli O157:H7 and L. monocytogenes determined with TSA (uninjured and injured cells) or TSAB (uninjured cells), decreased as the storage time increased. However, the least surviving population of both test organisms was noted when stored at −18°C followed by those stored at −28 and −5°C. The viable populations of E. coli O157:H7 determined either with TSA or TSAB, was reduced most drastically during the first day of storage then decreased slowly thereafter. Viable populations of L. monocytogenes declined slightly and gradually during the entire storage period. Furthermore, E. coli O157:H7 was found more susceptible to the freezing storage than L. monocytogenes. After 21-day storage at −18°C, population reduction of E. coli O157:H7 determined with TSA was ca 1.72 log CFU/ml. On the other hand, a population reduction of only 0.64 log CFU/ml was noted with L. monocytogenes. Besides, the surviving population of E. coli O157:H7 contained a larger proportion of injured cells than L. monocytogenes.     

Response of Escherichia coli O157:H7 in apple and orange juices by hydrostatic pressure treatment with rapid decompression

The bactericidal efficiency of hydrostatic pressure treatment combined with a slow decompression (SD; about 30 s) or a rapid decompression (RD; about 2 ms) against clinically isolated Escherichia coli O157:H7 was investigated in apple juice, orange juice and McIlvaine buffers having the same pH values of the juices used. Effects of the SD and RD treatments on survivability of E. coli O157:H7 cells during storage at 4°C in the juices were also investigated. The RD treatment showed higher inactivation effect than the SD treatment in both the juices and buffers. Untreated E. coli O157:H7 cells were not inactivated during storage for 5 days; however, post-treatment storage after both the SD and RD treatments reduced survivability of E. coli O157:H7 cells in the juices. The degree of the reduction was higher in the cells subjected to the RD treatment than to the SD treatment.     

Survival and growth of foodborne pathogens in minimally processed vegetables at 4 and 15 °C

Abstract: We conducted this study to investigate the survival and growth of pathogens on fresh vegetables stored at 4 and 15 °C. Vegetables (romaine lettuce, iceberg lettuce, perilla leaves, and sprouts) were inoculated with 4 pathogens (Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157:H7) and stored at 2 different temperatures for different periods of time (3, 6, 9, 12, and 15 d at 4 °C and 1, 2, 3, 5, and 7 d at 15 °C). Populations of the 4 pathogens tended to increase on all vegetables stored at 15 °C for 7 d. Populations of E. coli O157:H7 and S. Typhimurium increased significantly, by approximately 2 log₁₀CFU/g, on loose and head lettuce stored at 15 °C for 1 d. No significant differences were observed in the growth of different pathogens on vegetables stored at 4 °C for 15 d. E. coli O157:H7 did not survive on sprouts stored at 15 or 4 °C. The survival and growth of food pathogens on fresh vegetables were very different depending on the pathogen type and storage temperature. Practical Application: Survivals and growth of pathogens on various vegetables at 4 and 15 °C were observed in this study. Survivals and growth of pathogens on vegetables were different depending on the pathogen type and storage temperature. Therefore, vegetables should be stored under refrigerated conditions (below 4 °C) prior to consumption. This recommendation may vary depending on the type of vegetable.     

Antimicrobial Activity of Vanillin and Mixtures with Cinnamon and Clove Essential Oils in Controlling <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in Milk

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of vanillin against Listeria monocytogenes Scott A and Escherichia coli O157:H7 was determined in tripticase soy broth (TSB), pH 7 and 6, incubated at 35 °C/24 h and in semi-skim milk incubated at 35 °C/24 h and 7 °C/14 days. The influence of the fat content of milk on the antimicrobial activity of vanillin was tested in whole and skim milk incubated at 7 °C/14 days. Mixtures of clove and cinnamon with vanillin were also evaluated in semi skim milk incubated at 7 °C. The MICs for L. monocytogenes were 3,000 ppm in TSB (pH 7) and 2,800 ppm in TSB (pH 6). The MICs for E. coli O157:H7 were 2,800 ppm in TSB (pH 7) and 2,400 ppm in TSB (pH 6). The MBCs in TSB were 8,000 ppm for L. monocytogenes and 6,000 ppm for E. coli O157:H7. The pH values assayed did not influence significantly the MIC or MBC in TSB. The MICs in semi-skim milk for L. monocytogenes and E. coli O157:H7 were 4,000 and 3,000 ppm at 35 °C/24 h, and 2,500 and 1,000 ppm at 7 °C/7 days, respectively. The MBCs were 20,000 ppm for L. monocytogenes and 11,000 ppm for E. coli O157:H7. High incubation temperatures did not affect the MBC but increased the MIC of the vanillin in milk. This effect could be attributed to the increased membrane fluidity and to the membrane perturbing activity of vanillin at low temperatures. The fat in milk reduced significantly the antimicrobial activity of vanillin, probably due to effect protective of the fat molecules. Mixtures of clove and cinnamon leaves inhibited the growth of L. monocytogenes in a similar way that vanillin alone but had a synergistic effect on the E. coli O157:H7. Mixtures of cinnamon bark and vanillin had always a synergistic effect and some of the combination assayed showed bactericidal activity on the population of L. monocytogenes and E. coli O 157:H7.     

Thermal Inactivation of Escherichia coli O157:H7 in Strawberry Puree and Its Effect on Anthocyanins and Color

Raw whole strawberries, if contaminated with pathogens, such as Escherichia coli O157:H7, must be pasteurized prior to consumption. Therefore, the objective of this research was to investigate the thermal inactivation kinetics of E. coli O157:H7 in strawberry puree (SP), and evaluate the changes in anthocyanins and color, and the survival of yeasts and molds (YM) after thermal processing. Inoculated with a 5‐strain cocktail, fresh SP, with or without added sugar (20 and 40 °Brix), was heated at 50, 52, 54, 57.5, 60, and 62.5 °C to determine the thermal resistance of E. coli O157:H7. In raw SP, the average D‐values of E. coli O157:H7 were 909.1, 454.6, 212.8, 46.1, and 20.2 s at 50, 52, 54, 57.5, and 60 °C, respectively, with a z‐value of 5.9 °C. While linearly decreasing with temperature, the log D‐values of E. coli O157:H7 increased slightly with sugar concentration. The log degradation rates of anthocyanins increased linearly with temperature, but decreased slightly with sugar concentrations. These results suggest that sugar may provide some protection to both E. coli O157: H7 and anthocyanins in SP. The browning index was not affected by heating at 50 and 52 ºC at low sugar concentrations, but increased by an average of 1.28%, 2.21%, and 10.1% per min when SP was exposed to heating at 54, 57.5, and 60 °C, respectively. YM was also inactivated by heating. This study demonstrated that properly designed thermal processes can effectively inactivate E. coli O157:H7 and YM in contaminated SP, while minimizing the changes in anthocyanins and color.     

CONDITIONS AFFECTING AUTOINDUCER‐2‐BASED QUORUM‐SENSING OF ESCHERICHIA COLI O157:H7 ON FRESH BEEF

ABSTRACT

This study evaluated whether inoculated (none, 1, 5 log colony‐forming units [cfu]/cm²) Escherichia coli O157:H7 would result in detection of autoinducer (AI)‐2‐like activity on beef. Inoculated fresh beef, containing low (LNB) or high (HNB) initial levels of natural flora, was analyzed for bacterial populations and AI‐2‐like activity during aerobic or vacuum‐packaged storage (4, 10, 25C). As expected, no growth of E. coli O157:H7 was detected at 4C, while at 10C, growth was detected only on LNB samples stored aerobically; AI‐2‐like activity was minimal (P ≥ 0.05) at both temperatures. E. coli O157:H7 showed more growth in LNB than HNB, and in aerobically than vacuum‐packaged samples inoculated with 1 log cfu/cm² of the pathogen during storage at 25C. AI‐2‐like activity was generally higher in LNB than HNB samples stored aerobically at 25C, while no significant AI‐2‐like activity was detected in samples stored in vacuum packages. The results indicated that E. coli O157:H7 may exhibit AI‐2‐like activity on aerobically stored beef in the presence of lower initial levels of natural flora, and at temperatures allowing prolific growth of the pathogen. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be of importance in beef stored at low temperatures.

PRACTICAL APPLICATIONS

This study presents evidence that Escherichia coli O157:H7 showed autoinducer (AI)‐2 activity and involved in quorum‐sensing on fresh beefcontaining low initial levels of natural flora during aerobic storage at abusive storage temperatures. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be important in beef stored at recommended low temperatures.     

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.     

Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx ₁, stx ₂, and wzy _O157 genes in combination with probes and primers targeting either the fliC _h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx ₁, stx ₂, wzy _O157, and eae genes; however, using the assay targeting the stx ₁, stx ₂, wzy _O157, and fliC _h7 gene combination, a positive result was always obtained for the fliC _h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC _h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx ₁, stx ₂, eae, and wzy _O157 or the fliC _h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC _h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.     

Thermal Inactivation of Escherichia coli O157:H7 in Cooked Turkey Products

Survival of Escherichia coli O157:H7 when heated in commercial-type turkey products was determined. Thermal death times (TDT) were determined at 52–60°C in ground turkey with no additives, 3% fat; ground turkey with no additives, 11% fat; turkey ham batter, 11% fat; turkey frank batter, 17% fat; and turkey sausage batter, 31% fat. Mean D₅₂-values ranged from 44.9 to 116 min; D₅₅-values from 6.63 to 39.4 min; D₅₇-values from 2.20 to 11.7 min; D₆₀-values from 0.68 to 5.86 min. At all temperatures, survival of E. coli O157:H7 was greater in formulated products than in turkey meat with no additives. Greatest survival occurred in the turkey frank batter. Using our z-value data, times to provide a 5 D kill of E. coli O157:H7 in turkey franks cooked at 60°C, 65.6°C, or 71°C would be 26, 3.1, or 0.37 min, respectively.     

Acceptability of a multi-step intervention system to improve apple cider safety

A multi-step intervention system against Escherichia coli O157:H7 in apple cider was studied. The system consisted of adding 0.05% (w/v) each of sodium benzoate and potassium sorbate, holding cider at 35 °C for 6 h, followed by freezing and thawing the cider. This system was tested in triplicate 10 ml volumes of eight lots of cider throughout the 2000 season. For all ciders, E. coli O157:H7 numbers were reduced by ⩾3.0 log units, with ⩾5.0 log unit reductions for 6 of 8 ciders. For blends of early- and late-season (2000) ciders treated and then thawed in 3.5 l volumes (jugs), no survivors were detected (⩾3.3–4.3 log unit reductions). In consumer acceptance panels (one cider tasted per panel) each treated cider received a mean (n of 191–201) score of 5.5–6.1 (5=“like slightly”; 6=“like moderately”). In a direct comparison, the treated cider had a significantly (P<0.05) lower mean score (n=189) of 5.6 compared to heat-pasteurized cider (mean score of 6.1). Although the multi-step intervention system may provide sufficient lethality under certain conditions, its inconsistent lethality and lower consumer acceptability reduce its usefulness as an alternative to high-temperature short-time pasteurization.