Cryotolerance of Escherichia coli 0157:H7 in Laboratory Media and Food

ABSTRACT: The present study compared the cryotolerance of E. coli 0157:H7 strains with nonpathogenic strains of E. coli. Cold shocked (exposed to 10°C for 6 h) and non-cold shocked (held at 20°C) cultures were frozen at -18°C for up to 240 h in brain heart infusion broth, apple juice, frozen yogurt, and ground beef. The E. coli 0157:H7 strains showed the greatest cold shock effect and cryotolerance. The cold shocked E. coli 0157:H7 strains showed a 25 to 35% increase in their ability to survive frozen storage for 24 h at -18°C compared to non-cold shocked cells. The corresponding value for non-O157 strains was only about 5%. The food matrix changed the cold shock response in all investigated strains. The largest cold shock effect was observed with broth cultures. Cryotolerance of E. coli was not observed in frozen yogurt and ground beef. The effect of prior cold shock was most pronounced in E. coli 0157:H7 strains after 24 h of freezing.     

Escherichia coli O157:H7 Biofilm Formation on Romaine Lettuce and Spinach Leaf Surfaces Reduces Efficacy of Irradiation and Sodium Hypochlorite Washes

Abstract: Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Biofilm-associated pathogens are relatively resistant to chemical treatments, but little is known about their response to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated with E. coli O157:H7 and stored at 4 °C for various times (0, 24, 48, 72 h) to allow biofilms to form. After each time, leaves were treated with either a 3-min wash with a sodium hypochlorite solution (0, 300, or 600 ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75, or 1 kGy). Viable bacteria were recovered and enumerated. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 1.3 log CFU/g for baby spinach and 1.8 log CFU/g for Romaine. Increasing time in storage prior to chemical treatment had no effect on spinach, and had an inconsistent effect on 600 ppm applied to Romaine. Allowing time for formation of biofilm-like aggregations reduced the efficacy of irradiation. D₁₀ values (the dose required for a 1 log reduction) significantly increased with increasing storage time, up to 48 h postinoculation. From 0 h of storage, D₁₀ increased from 0.19 kGy to a maximum of 0.40 to 0.43 kGy for Romaine and 0.52 to 0.54 kGy for spinach. SEM showed developing biofilms on both types of leaves during storage. Bacterial colonization of the stomata was extensive on spinach, but not on Romaine. These results indicate that the protection of bacteria on the leaf surface by biofilm formation and stomatal colonization can reduce the antimicrobial efficacy of irradiation on leafy green vegetables. Practical Application: Before incorporating irradiation into the overall GMP/GHP chain, a packer or processor of leafy green vegetables must determine at what stage of processing and shipping the irradiation should take place. As a penetrating process, irradiation is best applied as a postpackaging intervention. Time in refrigerated storage between packaging and processing may alter the antimicrobial efficacy of irradiation. Irradiation on a commercial scale should include efforts to minimize the time delay between final packaging and irradiation of leafy vegetables.     

Thermal Inactivation of Salmonella spp., Salmonella typhimurium DT104, and Escherichia coli 0157:H7 in Ground Beef

ABSTRACT: In 19.1% fat ground beef, Escherichia coli 0157:H7 was less heat- resistant at ≥58°C than the Salmonella typhimurium DT104 and Salmonella senftenberg , but at 55°C the D value was similar to DT104 strains and higher than an eight-strain Salmonella cocktail. Inactivation of E. coli 0157:H7 was more temperature-dependent than the cocktail and DT104 strains. E. coli and DT104 strains were more heat-resistant in beef containing 19% fat than 4.8% fat. The cocktail was more thermally stable in stationary as compared to log phase. Freezing of inoculated raw meat decreased heat resistance of the cocktail. The pathogenic strain, growth phase of the organism, state of the meat (fresh or frozen) and meat composition must be considered when designing protocols to verify thermal processes.     

Survival of Escherichia coli O157:H7 in the processing and post-processing stages of acidophilus yogurt

In this study the survival of Escherichia coli O157:H7 in the production process of acidophilus yogurt was examined and compared with traditional yogurt. Milk was inoculated with different doses of E. coli O157:H7 (10², 10⁴ and 10⁶ CFU mL^?1) and two kinds of yogurt were produced. Samples were taken for pH measurements and bacterial enumeration at 0, 3, 24, 48, and 72 h after inoculation. Escherichia coli O157:H7 was analysed by using a Most Probable Number technique and yogurt starter culture and Lactobacillus  acidophilus were counted by using classical culture methods. Elimination times of E. coli O157:H7 were determined for 10² CFU mL^?1 as 3 h, and for 10⁴ and 10⁶ CFU mL^?1 as 48 h in acidophilus yogurt. Elimination times in traditional yogurt were 48 h for 10² and 10⁴ CFU mL^?1 and 72 h for 10⁶ CFU mL^?1E. coli O157:H7. In conclusion, the elimination time of E. coli O157:H7 in acidophilus yogurt was shorter than in traditional yogurt during the processing and post‐processing stages.     

Survival of enterohaemorrhagic Escherichia coli O157:H7 strain in Turkish soudjouck during fermentation, drying and storage periods

Soudjouck (a kind of Turkish sausage) batter was inoculated with Escherichia coli O157:H7 at a level of 10⁵ colony-forming unit (CFUg) and kept overnight at 4°C. After stuffing the soudjouck batter into natural casing, fermentation was carried out at 24±2°C and 90–95% relative humidity (RH) for 3 days with subsequent drying at 22±2°C and 80–85% RH for 5 days. Then, half of soudjouck samples were vacuum-packed in polyethylene bags and the rest were kept open. All samples were stored at 4°C (55% RH) for 3 months. E. coli O157:H7 and lactic acid bacteria counts, moisture contents and pH values of the samples were determined during fermentation, drying and storage periods. Results showed that count of E. coli O157:H7 decreased by 3 log unit during fermentation and drying periods. It was observed that this pathogen survived longer in vacuum-packaged samples (more than 2 months) than non-vacuum samples (more than 1 month).     

Antibacterial activity against E. coli O157:H7, physical properties, and storage stability of novel carvacrol-containing edible tomato films

Edible films containing plant antimicrobials are gaining importance as potential treatment to extend product shelf life and reduce risk of pathogen growth on contaminated food surfaces. The main objective of the present study was to evaluate the antimicrobial activities, storage stabilities, and physical-chemical-mechanica1 properties of novel edible films made from tomatoes containing carvacrol, the main constituent of oregano oil. The antimicrobial activities against E. coli O157:H7 and the stability of carvacrol were evaluated during the preparation and storage of tomato-based films made by 2 different casting methods, continuous casting and batch casting. Antimicrobial assays of tomato films indicated that optimum antimicrobial effects occurred with carvacrol levels of approximately 0.75% added to tomato purees before film preparation. HPLC analysis of the films indicated that the carvacrol concentrations and bactericidal effect of the films remained unchanged over the storage period of up to 98 d at 5 and 25 degrees C. Carvacrol addition to the tomato puree used to prepare the films increased water vapor permeability of tomato films. The continuous method for casting of the films appears more suitable for large-scale use than the batch method. This 1st report on tomato-based edible antimicrobial tomato films suggests that these films have the potential to prevent adverse effects of contaminated food and promote human health associated with the consumption of tomatoes.     

Cold plasma reduction of Salmonella and Escherichia coli O157:H7 on almonds using ambient pressure gases

Contamination of raw nuts, including almonds, is a food safety concern. Cold plasma is a novel antimicrobial intervention that can eliminate foodborne pathogens. The objective of this work was to evaluate the efficacy of rapid cold plasma treatments in eliminating Salmonella and Escherichia coli O157:H7 from dry almonds. Three isolates of Salmonella (S. Anatum F4317, S. Stanley H0558, and S. Enteritidis PT30) and 3 isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894) were separately grown and spot-inoculated (10 μL) onto whole almonds and allowed to dry for 10 min. Inoculated almonds were treated with a cold plasma jet, with treatment variables evaluated in a factorial design for each isolate: time, distance, and feed gas. Treatment time was 0 s (control), 10 s, or 20 s. Distance from the emitter was 2, 4, or 6 cm. Feed gas was dry air or nitrogen. After treatment, the almonds were sampled using swabs. Survivors were enumerated on tryptic soy agar (TSA) plates. Cold plasma significantly reduced both pathogens on almonds. The greatest reduction observed was 1.34 log cfu/mL reduction of E. coli O157:H7 C9490 after 20 s treatment at 6 cm spacing. The interaction of treatment time with distance from plasma emitter head was complex, and isolate-dependent. Longer duration of treatment did not always result in enhanced reductions. In general, nitrogen as a feed gas resulted in a reduced antimicrobial efficacy compared to dry air. These results indicate that short pulses of atmospheric pressure cold plasma can significantly reduce Salmonella and E. coli O157:H7 on almonds.     

Comparative Growth ofEscherichia coli0157:H7, Spoilage Organisms and Shelf-Life of Shredded Iceberg Lettuce Stored under Modified Atmospheres

The objective of this work was to compare organoleptic and microbiological spoilage with the survival of Escherichia coli 0157:H7 in modified atmosphere (MA) stored shredded lettuce. The rates of growth of E coli 0157:H7, increase in aerobic plate counts (APC g⁻¹), and rates of visual spoilage of shredded lettuce held under air or MA at 13 and 22°C were compared. Samples were inoculated with nalidixic acid-resistant E coli 0157:H7 (ATCC 35150) and placed in a chamber which was continuously flushed with gas mixtures of 0/10/90, 3/0/97, 5/30/65, 20/0/80 (O₂/CO₂/N₂, v/v) and held at 13 or 22°C. APC growth was inhibited in 5/30/65 (O₂/CO₂/N₂) at 13°C compared to all other atmospheres which were not significantly different from each other. The growth rates for both E coli 0157:H7 and APC were greatest in air at 22°C. Carbon dioxide concentration had no significant effect on the growth of E coli 0157:H7 at either temperature. The shelf-life of shredded lettuce, as judged by appearance, was extended in atmospheres containing 30% CO₂ by approximately 300% compared to air. The APC were similar at the time when the shredded lettuce samples were judged unacceptable regardless of shelf-life. However, the extended shelf-life provided by the MA allowed E coli 0157:H7 to grow to higher numbers compared to air-held shredded lettuce.     

Inactivation of E. coli O157:H7 Using a Pulsed Nonthermal Plasma System

ABSTRACT: The use of elevated temperatures to pasteurize food products leads to a loss of food quality, which stimulates research into novel nonthermal processes for food preservation. Using nonthermal plasma discharges, applied directly into the food product, proved effective in reducing the number of Escherichia coli O157:H7 cells in apple juice, by up to 7 log units. The pulsed nonthermal plasma system was developed and used to study the effects of different process parameters on the inactivation. The process parameters considered were discharge voltage, pulse number, and pulse frequency. The apple juice used had a pH of 4 and a conductivity of 2.29 mS/cm.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI 0157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND COMPETITIVE POLYMERASE CHAIN REACTION

A protocol based on immunomagnetic separation (IMS) and competitive polymerase chain reaction (C-PCR) was developed for quantitative detection of Escherichia coli 0157:H7 in ground beef. Internal standard sequences were synthesized by PCR with hybrid primers composed in part of original primers for C-PCR. A single concentration of internal standards was used for C-PCR after calibration with known amounts of target DNA. After a 6 h enrichment in Tryptic Soy Broth (TSB), C-PCR enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with Shiga-like toxin 1 and 2 (SLT 1 and 2) primers respectively.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI 0157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND COMPETITIVE POLYMERASE CHAIN REACTION

ABSTRACT

A protocol based on immunomagnetic separation (IMS) and competitive polymerase chain reaction (C-PCR) was developed for quantitative detection of Escherichia coli 0157:H7 in ground beef. Internal standard sequences were synthesized by PCR with hybrid primers composed in part of original primers for C-PCR. A single concentration of internal standards was used for C-PCR after calibration with known amounts of target DNA. After a 6 h enrichment in Tryptic Soy Broth (TSB), C-PCR enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with Shiga-like toxin 1 and 2 (SLT 1 and 2) primers respectively.     

Impact of irradiation on the safety and quality of poultry and meat products: a review

For more than 100 years research on food irradiation has demonstrated that radiation will make food safer and improve the shelf life of irradiated foods. Using the current food safety technology, we may have reached the point of diminishing returns even though recent figures from the CDC show a significant drop in the number of foodborne illnesses. However, too many people continue to get sick and die from eating contaminated food. New and under utilized technologies such as food irradiation need to be re-examined to achieve new levels of safety for the food supply. Effects of irradiation on the safety and quality of meat and poultry are discussed. Irradiation control of the principle microbial pathogens including viruses, the differences among at-risk sub-populations, factors affecting the diminished rate of improvement in food safety and published D values for irradiating raw meat and poultry are presented. Currently permitted levels of irradiation are probably not sufficient to control pathogenic viruses. Typical gram-negative spoilage organisms are very sensitive to irradiation. Their destruction leads to a significant increase in the acceptable shelf life. In addition, the destruction of these normal spoilage organisms did not provide a competitive growth advantage for irradiation injured food pathogens. Another of the main focuses of this review is a detailed compilation of the effects of most of the food additives that have been proposed to minimize the negative quality effect of irradiation. Most of the antimicrobials and antioxidants used singly or in combination produced an increased lethality of irradiation and a decrease in oxidation by-products. Combinations of dosage, temperature, dietary and direct additives, storage temperature and packaging atmosphere can produce meats that the average consumer will find indistinguishable from non-irradiated meats. A discussion of the production of unique radiological by-products is also included.     

Trisodium Phosptiate (TSP) Treatment of Beef Surfaces to Reduce Escherichia coli O157:H7 and Salmonella typhimurium

Trisodium phosphate (TSP) was evaluated for removing attached E. coli O157:H7 and S. typhimurium from beef surfaces using microbiological plating and scanning electron microscopy (SEM). Both fat and fascia surfaces were exposed to 10⁹ CFU/mL of each inoculum for 15 min and rinsed with 10% TSP solution (10°C) for 15 sec. Compared to controls, the level of E. coli O157:H7 was 1.35 and 0.92-logs lower on TSP-treated fat and fascia surfaces, respectively by plating. .S. typhimurium was 0.91- and 0.51-logs lower, respectively. By SEM, TSP-treated fabcia surfaces showed 1.39-log and 0.86-log reductions in E. coli O157:H7 and S. typhimurium, respectively. Overall, TSP was more effective on removing E. coli O157:H7 than S. typhimutium and more efficient in removing both bacteria from fat surfaces than from fascia.     

Efficacy of Ozone Against Escherichia coli O157:H7 on Apples

Apples were inoculated with Escherichia coli O157:H7 and treated with ozone. Sanitization treatments were more effective when ozone was bubbled during apple washing than by dipping apples in pre‐ozonated water. The corresponding decreases in counts of E. coli O157:H7 during 3‐min treatments were 3.7 and 2.6 log₁₀ CFU on apple surface, respectively, compared to < 1 log₁₀ CFU decrease in the stem‐calyx region in both delivery methods. Optimum conditions for decontamination of whole apples with ozone included a pretreatment with a wetting agent, followed by bubbling ozone for 3 min in the wash water, which decreased the count of E. coli O157:H7 by 3.3 log₁₀CFU/g.     

Growth Inhibitory Effect of Chicken Egg Yolk Antibody (IgY) on Escherichia coli O157:H7

Escherichia coli O157:H7‐specific antibodies (immunoglobulin Y [IgY]) were isolated by the water‐dilution method from the egg yolk of chickens that were immunized with E. coli O157:H7 whole cells. The specific‐binding activity of IgY against E. coli O157:H7 as determined by the enzyme immuno assay showed high levels of activity against bacterial whole cells. IgY binding activity was further demonstrated to have an inhibitory effect on E. coli O157:H7 growth in a liquid medium. The antibacterial function of IgY appeared to result from the interaction of IgY with surface components of E. coli O157:H7, as proven from observation of immunofluorescence and immunoelectron microscopy.     

Models of the behavior of Escherichia coli O157:H7 in raw sterile ground beef stored at 5 to 46 degrees C

Escherichia coli O157:H7 can contaminate raw ground beef and cause serious human foodborne illness. Previous reports describe the behavior of E. coli O157:H7 in ground beef under different storage conditions; however, models are lacking for the pathogen's behavior in raw ground beef stored over a broad range of temperature. Using sterile irradiated raw ground beef, the behavioral kinetics of 10 individual E. coli O157:H7 strains and/or a 5- or 10-strain cocktail were measured at storage temperatures from 5° to 46 °C. Growth occurred from 6 to 45 °C. Although lag phase duration (LPD) decreased from 10.5 to 45 °C, no lag phase was observed at 6, 8, or 10 °C. The specific growth rate (SGR) increased from 6 to 42 °C then declined up to 45 °C. In contrast to these profiles, the maximum population density (MPD) declined with increasing temperature, from approximately 9.7 to 8.2 log cfu/g. Bias (B_f) and accuracy (A_f) factors for an E. coli O157:H7 broth-based aerobic growth model (10 to 42 °C) applied to the observations in ground beef were 1.05, 2.70, 1.00 and 1.29, 2.87, 1.03, for SGR, LPD and MPD, respectively. New secondary models increased the accuracy of predictions (5 to 45 °C), with B_f and A_f for SGR, LPD, and MPD of 1.00, 1.06, and 1.00 and 1.14, 1.33, and 1.02, respectively. These new models offer improved tools for designing and implementing food safety systems and assessing the impact of E. coli O157:H7 disease.     

Liquid Smoke Effects on Escherichia Coli O157:H7, and its Antioxidant Properties in Beef Products

Experiments were performed to evaluate the antibacterial properties of liquid smoke (LS), against E. coli O157:H7, in model (agar) and meat systems. The effects of 8% LS on growth of E. coli O157:H7 attached to ground beef, and 1.5% LS on warmed-over flavor (WOF) in precooked beef patties were also studied. E. coli O157:H7 growth was reduced (p<0.05) 2.3 log₁₀ CFU/g in ground beef patties after 3d refrigerated storage. TBA numbers, aroma scores and pH values were lower (p<0.05) in LS treated beef patties. LS reduced undesirable flavor development and may help assure the safety of beef products.     

Influence of autoinducer-2 (AI-2) and beef sample extracts on E. coli O157:H7 survival and gene expression of virulence genes yadK and hhA

ABSTRACT:  Bacterial cell-to-cell communication is mediated by autoinducer (AI) molecules such as AI-2 and has been reported to regulate gene expression in Escherichia coli O157:H7. We have previously shown that ground beef contains compounds that can inhibit sensing of AI-2 like activity. The hypothesis of this study was that AI-2 activity observed in conditioned medium (CM) will enhance E. coli O157:H7 survival and expression of virulence genes, whereas compounds inhibitory (such as those present in ground beef extracts) to AI-2 activity will negate these effects. E. coli O157:H7 luxS mutant strain VS 94 (incapable of synthesizing AI-2) was employed in these studies. The survival of this enteric bacterial pathogen as a function of AI-2 activity and the presence of AI-2 inhibitory compounds was studied at 4 °C. The number of survivors in the presence of AI-2 was significantly higher compared to the absence of AI-2, and the addition of ground beef extracts to conditioned medium negated the influence of AI-2 activity. Autoinducer AI-2 upregulated selected genes virulence genes ( yadK , and hha ), whereas the ground beef extract reversed the effect of AI-2 on the expression of the selected genes.     

Heat Resistance of Salmonella spp., Listeria monocytogenes, Escherichia coli 0157:H7, and Listeria innocua M1, a Potential Surrogate for Listeria monocytogenes, in Meat and Poultry: A Review

ABSTRACT: The heat-resistance data in meat and poultry for various strains of Salmonella, Listeria monocytogenes , and Escherichia coli O157:H7 as well as Listeria innocua M1 are summarized. Heat resistance of these organisms is affected by many factors. Different strains of the same organism have different responses to heat. Heat resistance can also be influenced by the age of the culture, growth conditions, pH, and numerous other factors. Data from this review may prove useful to processors in validating their times and temperatures for thermal processing of meat and poultry. The obvious gaps in the data will provide researchers opportunities to fill those gaps. In addition, it will encourage the development of surrogates, whether biological or otherwise, that will be able to be used in an actual processing environment.