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Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 (O157) is one of the most notorious human pathogens, causing severe disease in humans worldwide. O157 specifically colonizes the large intestine of mammals after passing through the small intestine, and this process is influenced by differential signals between the two regions. Small regulatory RNAs (sRNAs) are able to sense and respond to environmental changes and regulate diverse physiological processes in pathogenic bacteria. Although some sRNAs of O157 have been extensively investigated, whether these molecules can sense differences between the small and large intestine and influence the preferential colonization in the large intestine by O157 remains unknown. In this study, we identified a new sRNA, Esr055 , in O157 which senses the low DNA concentration in the large intestine and contributes to the preferential colonization of the bacteria in this region. The number of O157 wild-type that adhered to the colon is 30.18 times higher than the number that adhered to the ileum of mice, while the number of the Δ Esr055 mutant that adhered to the colon decreased to 13.27 times higher than the number adhered to the ileum. Furthermore, we found that the expression of Esr055 is directly activated by the regulator, DeoR, and its expression is positively affected by DNA, which is significantly more abundant in the ileum than in the colon of mice. Additionally, combining the results of informatics predictions and transcriptomic analysis, we found that several virulence genes are up-regulated in the Δ Esr055 mutant and five candidate genes ( z0568, z0974, z1356, z1926 , and z5187 ) may be its direct targets.

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli. Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study, we screened different strains of serotype O157:H7 for the emergence of strong Congo-red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two major mechanisms which conferred stronger biofilm phenotypes were identified: mutations (insertion, deletion, single nucleotide change) in rcsB region and stx-prophage excision from the mlrA site. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties to all variants. Whereas RcsB mutants showed weaker CR affinity and biofilm properties, it provided more possibilities for phenotypic presentations through heterogenic sequence mutations., (Published by Oxford University Press on behalf of FEMS 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Escherichia coli O157:H7 (O157) is a Gram negative and highly virulent bacteria found in food and water sources, and is a leading cause of chronic diseases worldwide. Diagnosis and prevention from the infection require simple and rapid analysis methods for the detection of pathogens, including O157. Endogenous membrane peroxidase, an enzyme present on the surface of O157, was used for the colorimetric detection of bacteria by catalytic oxidation of the peroxidase substrate. In this study, we have analyzed the impact of the synthesized bare gold nanorods (AuNRs) and silica-coated AuNRs on the growth of E. coli O157. Along with the membrane peroxidase activity of O157, other bacteria strains were analyzed. Different concentrations of nanorods were used to analyze the growth responses, enzymatic changes, and morphological alterations of bacteria by measuring optical density, 3,3',5,5'-tetramethylbenzidine assay, flow cytometry analysis, and microscopy studies. The results revealed that O157 showed higher and continuous membrane peroxidase activity than other bacteria. Furthermore, O157 treated with bare AuNRs showed a decreased growth rate in comparison with the bacteria with surface modified AuNRs. Interestingly, silica-coated AuNRs favored the growth of bacteria and also increased membrane peroxidase activity. This result can be particularly important for the enzymatic analysis of surface treated AuNRs in various microbiological applicants.

Background and Objectives: A diverse group of Escherichia coli are known as enterohemorrhagic Escherichia coli (EHEC) including O157:H7 and non-O157 EHEC. Enterohemorrhagic strains are related to sever clinical conditions in humans including hemorrhagic colitis and hemolytic uremic syndrome, and most of the recorded outbreaks occurred due to O157: H7 E. coli. The aim of the present study was to investigate the presence of O157:H7 E. coli among healthy cattle in Golestan province., Materials and Methods: Fecal samples were collected from 180 clinically healthy cattle in Golestan province. After primary enrichment, samples were streaked on sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC). Non-sorbitol fermenting (NSF) Escherichia coli isolates were subjected to serotyping using commercial O157 antisera and rfb O157 gene PCR. Isolates were additionally tested for major virulence factors of EHEC including stx1, stx2, eae and ehly by multiplex-PCR., Results: Eighteen NSF isolates were recovered from CT-SMAC confirmed as E. coli in biochemical tests. None of the obtained isolates belonged to O157 serogroup. Overall, two isolates harbored the tested virulence genes; one isolate possessed stx2 and ehly, and the other one carried stx2, eae and ehly., Conclusion: The results of this study indicated that cattle in Golestan province could be the reservoir for non-O157 EHEC.

Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC. The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE. This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection.

Appropriate and safe antibacterial agents able to decontaminate meat surfaces have long been big concern of meat industry. In an attempt to manage beef carcass contamination, spray wash treatments utilizing three concentrations (1, 1.5 and 2%) of acetic, lactic, propionic and formic acids were performed to evaluate their efficacy in reducing numbers of Escherichia coli O157:H7 and Staphylococcus aureus on meat tissues. The procured beef pieces of freshly slaughtered animals were decontaminated with hot water and then inoculated with E. coli O157:H7 and S. aureus individually which then were spray washed with organic acids separately. The total plate count of the treated samples showed that the populations of bacteria decreased after being exposed to organic acids. Spray wash of formic acid resulted in the highest reduction of both bacterial species on meat surface. Significantly, higher log reductions were obtained for S. aureus than E. coli O157:H7. It was concluded that organic acids are highly effective in decontaminating meat surfaces and organic acids are shown to be safe, simple, efficient, and cheap modality of meat decontamination which can be highly recommended for industrial scales.

A swine fecal isolate, identified as Escherichia coli ECL12, was found to produce an antimicrobial substance designated as colicin ECL12. Colicin ECL12 was inhibitory against 20 strains of E. coli O157:H7 previously isolated from both human and bovine feces. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Colicin ECL12 was sensitive to several proteolytic enzymes. Adsorption of colicin ECL12 to sensitive cells of E. coli O157:H7 was bactericidal, resulting in a 2 log reduction in viable cell counts. Colicin ECL12 was purified from strain ECL12 by cell extraction and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of colicin ECL12 resolved a single protein with a molecular weight of approximately 65,000.

Escherichia coli O157:H7 and non-O157:H7 survival due to induced acid tolerance or shock responses when exposed to lactic acid over time was studied. Induced acid tolerance or shock responses could allow pathogens, like E. coli O157:H7, to survive acidic conditions in foods during storage. Escherichia coli O157:H7 isolates 932 and E009 and a non-Ol57:H7 strain, 23716, were grown to stationary phase at 32°C and exposed to one of two treatments: acid shock or acid adaption. Acid-shocked cells were exposed to lactic acid at pH 3.5 or 4.0. Acid-adapted cells were exposed to pH 5.5 for an adaptation period and then exposed to an acid challenge of pH 3.5 or 4.0. Samples were incubated at either 25 or 32°C and survival of the isolates at 0, 3, 24, 48, 72, and 168 h (7 days), 336 h (14 days), and 504 h (21 days) was determined. All three isolates survived longer with larger populations at pH 4.0 and 25°C compared to the other treatments. In cases where a difference was observed in the two responses, acid-shocked cells had a higher survival rate (typically less than 2 logs) than acid-adapted cells in most cases. Isolate differences were observed at the two pH and temperature levels. Isolate 932 was the most resistant to the acidic conditions during the incubation period, E009 intermediate, and strain 23716 was the most sensitive.

The combined effect of starter culture (10 7 CPU of lactic acid bacteria per ml), dextrose (0.8%), sodium chloride (2%), nitrite (200 ppm), and temperature (37 and 22°C) on survival of bioluminescent Escherichia coli O157:H7 (l0 0 to 10 5 CPU/ml) in brain heart infusion (BHI) broth was determined. Two model systems representing fermented sausage production were evaluated. The first was the "American style" employing high-temperature (37°C) and short-time (1-day) fermentation. The second was the "European style" in which 22°C was used for 3 days. After fermentation, the samples were stored at 10°C and the survival of E. coli O157:H7 was monitored over an extended period of time. In the "American style" model, E. coli O157:H7 survived a more than 34-day period in BHI containing starter culture and dextrose, while it survived for a more than 51-day period in BHI containing starter culture, dextrose, sodium chloride, and nitrite. In the "European style" model, the survival time of E. coli O157:H7 during fermentation was longer in BHI containing starter culture and dextrose than in BHI containing starter culture, dextrose, sodium chloride, and nitrite. E. coli O157:H7 did not survive past 9 days in BHI containing starter culture and dextrose. Similarly, in BHI containing starter culture, dextrose, sodium chloride, and nitrite the lower E. coli O157:H7 inoculations (l0 0 to 10 4 CFU/ml) did not survive past 9 days; however, at higher inoculum levels (≥10 5 CFU/ml) the organism survived more than 30 days. In conclusion, the lower temperature and longer fermentation time ("European style") is better for elimination of E. coli O157:H7 from a model system representing a fermented sausage production than fermentation at high temperature and short time ("American style").

Three different acid-resistant strains of Escherichia coli O157:H7 were inoculated individually and as a cocktail into sterile apple cider (pH 3.2) at a level of approximately 10 5 cells per ml and incubated at 2°C. Samples were plated on Trypticase soy agar (TSA), violet red bile agar (VRBA), sorbitol MacConkey agar (SMA), and Petrifilm E. coli count plates (Petrifilm) at 24-h intervals. Repair of acid-injured cells was assessed by surface plating cider samples on TSA and allowing a 2-h room-temperature incubation period followed by overlaying with double-strength VRBA or SMA. Since SMA is a surface plate medium, the repair procedure was modified by overlaying SMA with Trypticase soy broth after 2 h of room-temperature incubation. Populations of all three strains and the cocktail of strains decreased rapidly in apple cider and approached undetectable levels within 72 h. At 24 and 48 h, 98.4% and >99% of the E. coli populations were injured, respectively. Repair procedures significantly (α = 0.05) increased detection of E. coli O157:H7. After 72 h E. coli O157:H7 was not detected by using SMA and Petrifilm; however, it was detected using repair procedures. Although detection levels were increased with resuscitation procedures, the levels detected were still lower than those obtained using nonselective TSA. This research confirms the need for special recovery steps when analyzing acidic food products suspected of containing E. coli O157:H7.

Escherichia coli O157:H7 remains a public health problem in the United States despite a dramatic increase in the awareness of, and concern about, foodborne infections since the 1993 multistate E. coli O157:H7 epidemic. Although surveillance data can be difficult to interpret, the incidence of endemic disease caused by this organism is probably not increasing, and might be decreasing, at least in selected populations. With increased recognition of E. coli O157:H7 infection has come the investigation of increasing number of outbreaks, leading to the recognition of many "new" vehicles, including some foods not traditionally associated with enteric infections, such as dry-cured salami and lettuce. Molecular fingerprinting techniques are being used to track the transmission of E. coli O157:H7 through human populations. Analysis of DNA encoding virulence factors and surface antigens suggests that diarrheagenic E. coli have evolved by acquiring large DNA fragments, with subsequent chromosomal recombination. Some Shiga toxin-producing E. coli other than E. coli O157:H7 are no doubt pathogens, but the majority of these toxigenic strains found in food are probably not virulent. More research is needed to define the characteristics that render selected Shiga toxin-producing organisms harmful to humans.

The firefly ( Photinus pyralis ) luciferase ( luc ) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein ( gfp ) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc -bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc -bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.

The inactivation of E. coli O157:H7 in ground beef patties cooked in a skillet was investigated. Ground beef patties inoculated with a mixture of five strains of E. coli O157:H7 were cooked in a Farberware skillet set at a temperature of 275°F (137°C). Eight type K thermocouples connected to a data logger were used to record the temperatures at eight points within the patty. The cooking times studied ranged from 2.25 min to 4 min. Tryptic soy agar plates overlaid with sorbitol MacConkey agar were used for recovery of E. coli O157:H7. Heating of ground beef patties to an internal temperature endpoint of 155°F (68.3°C) resulted in 4-log cycle reductions of the organism. The results of this investigation conducted under conditions simulating those that occur in the retail food industry provide a basis for ensuring safety against E. coli O157:H7 in ground beef patties.

The population of inoculated Escherichia coli O157:H7 was monitored during the manufacture and storage of a semidry beef summer sausage processed by fermentation and cooking at a low temperature by heating to an internal temperature of 130°F (54°C). The all-beef batter (11% fat and nonmeat ingredients) was inoculated with the commercial starter culture Pediococcus acidilactici HP (≥8.6 log CFU/g of batter) and a five-strain mixture of E. coli O157:H7 (≥7 log CFU/g) and then hand stuffed into 2.5-inch (64-mm) diameter fibrous casings. The sausages were fermented at an initial temperature of 85°F (29°C) to a final temperature of 105°F (41°C) over ca. 13 h at 80% relative humidity (RH) to pH 4.6 or pH 5.0. After fermentation to pH 4.6, the internal temperature of the chubs was raised to 130°F (54°C) instantaneous over 3.6 h at 60% RH. After fermentation to pH 5.0, the internal temperature of the chubs was raised to 130°F (54°C) over 3.6 h at 60% RH and the chubs were maintained under these conditions for 0, 30, or 60 min. he chubs were cold water showered for 15 min and then chilled at 39°F (4°C) for 6 h before being vacuum packaged and stored at 39°F (4°C) or 77°F (25°C) for 7 days. Regardless of the target pH, fermentation alone resulted in only a 1.39-log CFU/g decrease in pathogen numbers. However, fermentation to pH 4.6 and heating to an internal temperature of 130°F (54°C) instantaneous reduced counts of E. coli O157:H7 by ≥7.0 log units to below detection levels (<10 CFU/g). Pathogen numbers remained below levels detectable by direct plating, but viable E. coli O157:H7 cells were recovered by enrichment of samples during sausage storage at either refrigeration or abuse temperatures. In contrast, fermentation to pH 5.0 and heating to an internal temperature of 130°F (54°C) instantaneous resulted in a 3.2-log-unit decrease in counts of E. coli O157:H7. No appreciable reductions in pathogen numbers were observed thereafter following storage at either 39°F (4°C) or 77°F (25°C) for 7 days. Fermentation to pH 5.0 and heating to an internal temperature of 130°F (54°C) instantaneous followed by holding for 30 or 60 min resulted in about a 5- or 7-log reduction, respectively, in pathogen numbers. For chubs held for 30 min at 130°F (54°C), pathogen numbers decreased to 2.02 and <1.0 log CFU/g at 39°F (4°C) and 77°F (25°C), respectively, after 7 days; viable cells were only observed by enrichment after storage at 77°F (25°C). For chubs held for 60 min at 130°F (54°C), pathogen numbers remained below levels detectable by direct plating, but viable cells were recoverable by enrichment after 7 days at both storage temperatures. These data will be useful guidelines to manufacturers for developing processing conditions to further ensure the safety of this category of fermented sausages relative to food-borne pathogens such as serotype O157:H7 strains of E. coli .

We studied the contamination of radish sprouts after exposure to Escherichia coli O157:H7-inoculated water in the laboratory. The edible parts, the cotyledons and hypocotyl, became heavily contaminated with E. coli O157:H7 when they were grown from seeds soaked in E. coli O157:H7-inoculated water. These same parts became contaminated with E. coli O157:H7 when their roots were dipped into E. coli O157:H7-inoculated water. These findings suggest that E. coli O157:H7 contamination in the edible parts of radish sprouts could pose a serious hazard if the seeds or hydroponic water are contaminated with the bacterium.

This study examined the effects of a heat shock at 45°C for 30 min on the subsequent heat resistance of Escherichia coli O157:H7 ATCC 43894 in Trypticase soy broth (TSB) and ground beef slurry (GBS). Cultures were grown to stationary phase, stored for 24 h at 4 to 6°C, and then heat shocked to simulate consumer mishandling of meat during the summer. Control or heat-shocked ATCC 43894 cells were then transferred to prewarmed TSB (54, 58, and 62°C) or GBS (58°C) and refrigerated TSB and GBS that were subsequently heated to and held at 58°C (TSB and GBS) and 62°C (TSB only). Heat shock increased D values by 37, 68, and 50% in 54, 58, and 62°C prewarmed TSB, respectively, but had no significant effect on the D value in 58°C GBS. Immediate plating of heated samples yielded greater cell recovery than if samples were held on ice prior to plating. Heat shock did not lead to significant increases in D values when cells were transferred to 4°C TSB and GBS that were heated to the test temperature. This study showed that for E. coli O157:H7 ATCC 43894 the heat-shock effect was lost upon subsequent chilling and rewarming and overshadowed by the protective effects of ground beef constituents. The results do not support the hypothesis that short-term temperature abuse will significantly increase the heat resistance of E. coli O157:H7 in ground beef.

Two 24-hour presumptive enumeration methods for Escherichia coli O157:H7 organisms based on the hydrophobic grid membrane filter (ISO-GRID) and using SD-39 agar, a new selective and differential culture medium, were developed and compared to a 3-tube MPN (most probable number) method using modified tryptone soy broth enrichment. The comparative study comprised 22 combinations of storage conditions and food products, including a variety of raw and cooked meats and several dairy products. The ISO-GRID direct filtration method produced counts which were equivalent to or significantly higher than the 3-tube MPN method for all food-storage combinations except for frozen pasteurized whole egg. The ISO-GRID resuscitation method produced counts equivalent to the 3-tube MPN method for the frozen egg.

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 have been commonly associated with products derived from ground beef, but recently the organism has been implicated as the causative agent in outbreaks involving yogurt and cheese. This finding has raised concern about the potential for its growth and survival in fermented dairy products. A bioluminescent strain of E. coli O157:H7 was used to determine postprocessing survival in yogurt with live cultures at pH 4.17, 4.39, and 4.47 stored at 4 and 10°C. In addition, survival of E. coli O157:H7 was monitored during the manufacture of Cottage, Colby, Romano, and Feta cheeses. Results indicated survival for 8 and 5 days at 4 and 10°C respectively in yogurt at pH 4.17, 17 and 15 days at 4 and 10°C respectively in yogurt at pH 4.39, and 17days at both 4 and 10°C in yogurt at pH 4.47. E. coli O157:H7 did not survive cooking procedures at 56°C in Cottage cheese. However, the pathogen survived for 27, 30, and 27 days in Colby, Romano, and Feta cheeses respectively. A high correlation of r > 0.89 was obtained between counts of bioluminescenct colonies and standard plate count for all yogurt and cheese varieties, indicating that bioluminescence was a sensitive and rapid indicator of cellular viability for E. coli O157:H7. Survival of the pathogen, as indicated by this method, is possible in highly acidic environments even at refrigeration temperatures. This poses a potential hazard should postprocessing contamination occur.2 > 0.89 was obtained between counts of bioluminescenct colonies and standard plate count for all yogurt and cheese varieties, indicating that bioluminescence was a sensitive and rapid indicator of cellular viability for E. coli O157:H7. Survival of the pathogen, as indicated by this method, is possible in highly acidic environments even at refrigeration temperatures. This poses a potential hazard should postprocessing contamination occur.

The purpose of this research was to determine the survival of Escherichia coli O157:H7 when heated in ground turkey containing various additives and fat levels. D values and z values were determined for low (3%)- and high (11 %)- fat ground turkey with or without one of three additives: 8% NaCl, 4% sodium lactate, or a mixture of 8% NaCl, 4% sodium lactate, and 0.5% polyphosphate. Products inoculated with E. coli O157:H7 strain 204P were mixed, aseptically placed into thermal-death-time (TDT) tubes which were sealed and heated at 52, 55, 57 and 60°C. Survival was determined by enumeration on phenol red sorbitol agar, and D values were calculated by two methods. Mean D 52 values ranged from 46.8 to 104.8 min; mean D 55 values ranged from 7.7 to 27.2 min; mean D 57 values ranged from 2.7 to 13.0 min; and mean D 60 values were obtained in turkey meat containing the additive mixture versus other turkey additive formulations. The additives evaluated enhanced survival of E. coli O157:H7 in cooked turkey meat as compared to turkey meat with no additives. In contrast to earlier reports, added fat did not enhance survival ( P < 0.001) D values, occurred in turkey containing the mixture of additives. The z values ranged from 6.09 to 4.08°C, and higher z values were obtained in turkey meat containing the additive mixture versus other turkey additive formulations. The additives evaluated enhanced survival of E. coli O157:H7 in cooked turkey meat as compared to turkey meat with no additives. In contrast to earlier reports, added fat did not enhance survival ( P > 0.05). Product formulation should be a critical consideration when safe cooking processes are developed for ready-to-eat turkey products.

This study examined the effects of adaptation to acid and storage temperature on the survival of a nonpathogenic Escherichia coli strain, three strains of E. coli O157:H7, and three strains of Salmonella spp. in ketchup, mustard, and sweet pickle relish. Stationary-phase cells were adapted to acidic conditions in pH 5.0 Trypticase soy broth for 4 h at 37°C. Samples inoculated with individual strains (10 5 CFU/g for E. coli and 10 6 CFU/g for Salmonella spp.) were stored at 5 and 23°C. Numbers of surviving cells were determined by plating on Trypticase soy agar. Acid adaptation enhanced the survival of all three Salmonella strains (ATCC 6962, 13311, and 25957) and all three E. coli O157:H7 strains (ATCC 43889, 43894, and 43895), with the magnitude of the effect depending on the strain and storage temperature. Acid adaptation enhanced survival of the nonpathogenic E. coli strain at 5°C, but not at 23°C. Cells of all tested strains survived longer at 5°C than at 23°C. In general, the E. coli O157:H7 strains survived longer than the Salmonella and nonpathogenic E. coli strains. Nonadapted E. coli O157:H7 cells were recovered from ketchup stored for I to 7 days at 5°C, whereas only low numbers of nonadapted Salmonella strain ATCC 25957 were recovered after 1 day at 5°C and no nonadapted Salmonella strains survived 2 days at 5°C. All of the E. coli and Salmonella strains died within 1 h in mustard and sweet pickle relish stored at 5 and 23°C. This study showed that adaptation to acid and low temperature enhanced the survival of E. coli O157:H7 and Salmonella strains in ketchup but not in mustard or sweet pickle relish.

This study investigated the influence of low temperature and background flora on growth and verotoxin production by strains of enterohemorrhagic Escherichia coli in milk and ground beef. In the presence of no or low background flora, there was growth of the strains at 8°C. High background flora in ground beef inhibited growth at this temperature. In the foods held at low temperatures, only small amounts of verotoxin were detected; however, even at the optimum 37°C, there was still relatively little verotoxin formed compared to that in broth cultures. Even under nongrowth conditions (high background flora or 5°C holding temperature), the strains remained viable. These data suggest any food contaminated by these bacteria and held at the recommended temperature of 5°C will remain hazardous, and under certain conditions, holding at temperatures :≥8°Cwould increase the hazard.

A total of 750 beef carcasses were assayed to detect E. coli O157:H7 by surface swabbing. Each swab represented a 200-cm 2 composite surface area. Escherichia coli O157:H7 was not detected in any of the carcass samples. Assays were conducted from carcass swab suspensions by direct plating on Petrifilm ™ E. coli Count plates followed by an E. coli O157:H7 assay using the Petrifilm ™ Test Kit-HEC direct blot enzyme immunoassay without sample pre-enrichment, and by enrichment using a modified U.S. Department of Agriculture (USDA) Petrifilm ™ E. coli O157:H7 procedure. Additionally, beef slabs were inoculated with E. coli O157 :H7 to verify that recovery by the direct swab technique was at least as efficient as enrichment of excised tissue samples, which is the usual USDA method. Escherichia coli O157:H7 inoculation of 4,32, and 180 CFU/25 cm 2 were designated low, medium, and high surface contamination levels, respectively. The percentage of recovery of E. coli O157:H7 using the direct swab technique without sample pre-enrichment was 60, 80, and 100% for low, medium, and high surface inoculation levels, respectively. In comparison, recovery from 25-g enrichments of ground excised samples from beef slabs containing 1.4,9.6, and 50 CFU of E. coli O157:H7 was 0, 20, and 73%, respectively. Similarly, the percent recovery of this organism from nonground excised samples containing 56 CFU/25 g enrichment was 68%, versus 100% by direct swab technique without sample pre-enrichment from beef containing 190 CFU/25 cm 2 Direct surface swab tests with the 3M Petrifilm ™ Test Kit-HEC without sample pre-enrichment proved to be a sensitive, nondestructive, alternate means of evaluating beef carcasses for the presence of E. coli O157:H7.

Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic uremic syndrome, has been responsible for several outbreaks associated with consumption of unpasteurized and improperly processed pasteurized milk, and yogurt. Studies were conducted to determine the survival and growth characteristics of this pathogen in unpasteurized milk and pasteurized milk (3.5% fat, 2% fat, skim) at 5,8, 15, and 22°C for up to 28 days. Two levels of inocula (10 3 and 10 5 CFU/ml) of a mixture of five nalidixic acid-resistant E. coli O157:H7 strains were used. E. coli O157:H7 did not grow at 5°C and decreased by 1.6 to 2.0 log CFU/ml in 28 days. Growth occurred at 8°C, with an approximately 1- to 2-log CFU/ml increase within the first 4 days. About a 3- to 5-log CFU/ml increase in E. coli O157:H7 populations was observed at 15°C within the first 3 days. In 3 pasteurized milk samples, E. coli O157:H7 continued to grow to populations of greater than 1.0 × 10 8 CFU/ml at day 7 and remained constant during the remainder of the incubation period. At 22°C, the pH decreased rapidly to less than 4.0 within 4 days and E. coli O157:H7 decreased to undetectable populations within 14 days. E. coli O157:H7 grew more slowly ( P < 0.01) in unpasteurized milk, which had a higher initial microbial population, than in pasteurized milks at 8, 15, or 22°C, likely because of antagonistic activity from preexisting bacteria. No significant differences ( P > 0.05) in survival or growth of E. coli O157:H7 were observed among the pasteurized milk samples, regardless of fat concentration, at all temperatures throughout the study. The data indicate that temperature abuse during shipping and handling can result in significant growth of E. coli O157:H7. Holding milk at ≤5°C is recommended to prevent growth of this pathogen.

A series of progressive experiments was conducted with a model carcass washer using tap water and 2% acetic acid sprays to determine if tissue type, inoculation menstruum, bacterial level, or spray temperature affect removal of bacteria from beef carcass tissue during spray washing. For the first experiment, prerigor (15 min postexsanguination), postrigor (24 h postexsanguination), or postrigor frozen (-20°C, 7 days), thawed, lean beef carcass tissue (BCT) was inoculated with bovine feces and subjected to spray washing (15 s, 56°C) with water or acetic acid. Spray washing with either compound resulted in bacterial populations that were similar for prerigor and postrigor BCT; however, remaining bacterial populations from spray-treated postrigor, frozen BCT were significantly ( P ≤ 0.05) less than for the other two tissue types. For the second experiment, prerigor, lean BCT was inoculated with Escherichia coli O157:H7 suspended in bovine feces or physiological saline and spray washed (15 s, 56°C) with water or acetic acid. Bacterial populations were reduced to similar levels with acid sprays, regardless of menstruum. For the third experiment, E. coli O157:H7 in feces was used to contaminate prerigor lean BCT to obtain different initial bacterial levels (7, 5,3, and 1 log CFU/cm 2 ). Spray washes (15 s, 56°C) with acetic acid reduced the level of the pathogen to 2.51 and 0.30 log CFU/cm 2 when initial bacterial levels were 7 and 5 log CFU/cm 2 , and to undetectable levels when initial bacterial levels were 3 and 1 log CFU/cm 2 . In a fourth experiment, water or acetic acid (15 s), ranging from 30 to 70°C was applied to beef tissue contaminated with E. coli O157:H7 in feces. Remaining bacterial populations were not different between the water treatments or between the acid treatments at any temperature. While variables such as bacterial level and inoculation menstruum may affect the efficacy of spray washing with organic acids, these results indicate that tissue type or spray temperature do not.

A rapid and simple enzyme immunoassay system was developed for detection of the food-borne pathogen Escherichia coli O157. This system is based on the use of anti- E. coli O157 antibody-coated Polymacron™, an inexpensive macroporous polyester fabric, as a high-surface-area immunoadsorbent for the rapid capture and subsequent immunoenzymatic detection of E. coli O157 antigens extracted from test samples by heating at 100°C for 10 min in the presence of sodium cholate. A dot blot format was used which facilitated the assay of multiple samples. The method was specific for all strains tested bearing the O157 and related antigens (e.g., group N Salmonella ), giving positive reactions in the assay of pure cultures of 29 E. coli O157:H7 strains, one E. coli O157:H12, one E. coli O157:NM (nonmotile) and one Salmonella urbana strain, but not in the assay of a variety of other gram-negative and gram-positive bacteria. The method permitted the detection of ca. 400 E. coli O157:H7 CFU in a 5-μl spot applied to Polymacron™, and of as few as 0.4 CFU of E. coli O157:147 cells per g inoculated into ground beef samples, which were assayed after overnight enrichment.

Three strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB) or in a sterile cattle manure extract at 35°C for 18 ± 2 h. Aliquots from both inocula containing 10 6 CFU/ml were used to inoculate 1-cm 3 cubes of beef muscle or adipose tissue by immersion for 20 min at 21°C. After removal from the inoculum, one-half of the samples were analyzed for bacterial cell numbers and pH, and the other half were stored at 4°C for 2 or 3 h before analysis. Samples were analyzed by enumerating bacteria present in liquid droplets deposited on the tissue and bacteria loosely or strongly attached to the tissue in order to determine attachment strength. Total numbers of cells on beef muscle tissue (bacteria in liquid droplets, as well as those loosely and strongly attached) were 5.65 ± 0.14 and 5.76 ± 0.26 log CFU/cm 2 for E. coli O157:H7 inocula grown in TSB and manure extract, respectively. The differences in attachment strength between inocula from the two media were not significant ( P > 0.05). A 2-h storage period after exposure of muscle tissue to an E. coli O157:H7 inoculum did not influence attachment strength. Numbers of bacteria attached to adipose tissue and muscle (5.31 ± 0.08 and 5.48 ± 0.09 log CFU/cm 2 , respectively) were not significantly different ( P > 0.05). After 3 h at 4°C, the attachment strength of E. coli O157:H7 cells on muscle or adipose tissue had not changed. Overall, the culture medium and type of beef tissue did not affect the numbers of E. coli O157:H7 cells attached, nor the strength of their attachment, to muscle or adipose tissue.

The growth of Escherichia coli O157:H7 in raw ground beef was investigated at 12°C, 20°C, and 35°C at pH 5.7 (unadjusted) and adjusted to pH 6.3 to 6.4. These growth data were fitted to the Gompertz equation and the resulting growth kinetics were compared with predictions from the U.S. Department of Agriculture Pathogen Modeling Program. Close agreement with the model was obtained at pH 5.7, but at pH 6.4, growth was more rapid than predicted. The U.S. Department of Agriculture Food Safety and Inspection Service has used this predictive model for developing proposed regulations on time-temperature requirements for carcass cooling. As there may be considerable differences in the microenvironment of raw ground beef and a beef carcass, the validity of using predictive models for estimating growth rates on a carcass should be determined by performing growth studies on carcass surfaces.

Survival of Escherichia coli O157:H7 in fermenting and nonfermenting fresh apple cider was determined. Populations of E. coli O157:H7 were reduced from 6.4 log CFU/ml to undetectable levels (detection limit of 0.5 log CFU/ml) in fermenting cider after 3 days at 20°C and from 6.5 log CFU/ml to 2.9 log CFU/m1 after 10 days at 20°C in nonfermenting cider. After 1 day of incubation, recovery of E. coli O157:H7 from fermenting and nonfermenting cider was statistically ( P < 0.01) lower on sorbitol MacConkey agar than on tryptone soya agar supplemented with cycloheximide. These results suggest that substantial portions of the surviving E. coli O157:H7 populations were sublethally injured by cider components (i.e., acid and ethanol). The pH of fermenting cider was not significantly different ( P > 0.05) from that of nonfermenting cider throughout the 10-day test period. Final ethanol concentrations in fermenting cider reached 6.01% (vol/vol) after 10 days at 20°C. Inactivation of E. coli O157:H7 in fermenting cider is attributed to the combined effects of pH and ethanol. Results of this study indicate that E. coli O157:H7 is capable of survival in fresh apple cider at 20°C, while alcoholic fermentation of fresh cider is an effective means of destroying this pathogen.

The outbreak of Escherichia coli O157:H7 linked with dry-cured salami in late 1994 prompted regulatory action that required manufacturers of fermented products to demonstrate a 5-log unit reduction in counts of this pathogen during processing. Therefore, pepperoni batter (75% pork:25% beef with a fat content of ca. 32%) was inoculated with a pediococcal starter culture and a five-strain mixture of E. coli O157:H7 (≥2 × 10 7 CFU/g) and stuffed into 55-mm diameter fibrous casings 47 cm in length. The viability of the pathogen was monitored before stuffing, after fermentation, after thermal processing, and/or after drying. Chubs were fermented at 96°F (36°C) and 85% relative humidity (RH) to pH ≤ 5.0 and then dried at 55°F (13°C) and 65% RH to a moisture/protein ratio of ≤1.6:1 (modified method 6 process). Counts of the pathogen decreased about 1.2 log units after fermentation and drying. In subsequent experiments, heating chubs after fermentation to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min resulted in a ≥5-log unit decrease in numbers of strain O157:H7 without visibly affecting the texture or appearance of the product. These data revealed that a traditional nonthermal, process for pepperoni was only sufficient to eliminate relatively low levels (ca. 2 log CFU/g) of E. coli O157:H7, whereas heating to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min delivered a 5 to 6 log unit reduction in counts of the pathogen in pepperoni.

Rapid screening of beef for the presence of Escherichia coli O157:H7 was shown to be feasible using a 10-h enrichment in modified buffered peptone water and the antibody-direct epifluorescent filter technique (Ab-DEFT). The Ab-DEFT involved membrane filtration, fluorescent antibody staining and epifluorescence microscopy and was accomplished in less than 1 h. The procedure allowed detection of the pathogen artificially inoculated into beef patties at 0.1 CFU/g. The 10-h nonselective enrichment broth supported rapid growth, which provided sufficient numbers of cells for a positive determination by the Ab-DEFT after fewer than 10 microscope fields were scanned using a 40× objective lens. Immunomagnetic separation using anti- E. coli O157 Dynabeads ® was used to confirm presumptively positive cultures within 24 h. The ease and rapidity of the Ab-DEFT may provide a substantial time and cost savings to the beef industry for screening beef for the presence of E. coli O157:H7.

For rapid and specific identification of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food samples, experimental conditions for a multiplex polymerase chain reaction (PCR) were optimized and a multiple digoxigenin (DIG)-labeled oligonucleotide probe hybridization (DLOPH) assay was developed. A suspect colony from MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-β-d-glucuronide (MSA-BCIG) was used for the multiplex PCR. Three different DNA sequences of E. coli O157:H7 were amplified simultaneously in the PCR: a specific fragment of an attaching and effacing gene ( eae gene), conserved sequences of Shiga-like toxins (SLT) I and II, and a fragment of the 60-MDa plasmid. The identities of PCR products were confirmed by hybridization using DIG-labeled internal oligonucleotide probes and colorimetric detection with anti-DIG Fab fragments conjugated to alkaline phosphatase. This method yielded positive results with all reference strains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, and O157:H - , and negative results were obtained with all strains of nontoxigenic E. coli serogroup O157, other serotypes of E. coli , and other bacterial species. The detection limit of the method was 65 colony-forming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemical and serological tests were positive in the multiplex PCR. EHEC O157:H7 was identified from all of 70 experimentally inoculated ground beef and milk samples which had initial inocula of 4 to 9 CFU/g (ml) and were subjected to a 6-h enrichment culturing. The multiplex PCR procedure could be very useful for routine examinations of food samples for the presence of EHEC O157.

The ability of enterohemorrhagic Escherichia coli O157:H7 to survive a standard Cheddar cheese manufacturing process and subsequent curing was determined. Two treatments with added E. coli O157:H7 were designed with target levels 1 × 10 3 CFU/ml and 1 CFU/ml of cheese milk. Cheese samples were analyzed for E. coli O157:H7 during manufacture at 14, 28, 42, 60, and 74 days, and at 28-day intervals thereafter until the organism could no longer be detected using direct plating or enrichment in two successive samples. Typical colonies on 3M Petrifilm ® E. coli Count Plates were counted as presumptive E. coli O157:H7 and were confirmed with the 3M Petrifilm ® Test Kit-HEC for hemorrhagic E. coli O157:H7. When no E. coli O157:H7 were detected in the cheese with the Petrifilm ® plates, a 25-g sample of cheese was enriched in modified EC broth with novobiocin to detect viable E. coli O157:H7. Cheese made with 10 3 CFU/ml of milk showed a 2-log-unit reduction after 60 days of ripening, with viable E. coli O157:H7 still being detected in 25 g of cheese after 158 days. Cheese made with 1 CFU/ml of milk showed a reduction in E. coli Ol57:H7 to 1 or <1 CFU/g in 60 days, with no E. coli being detected in 25 g of cheese at 158 days. However, both treatments resulted in the survival of E. coli O157:H7 during manufacture and for more than 60 days of curing at 2.75 to 3.76% salt in the moisture phase.

Temperature abuse of foods is often transitory and little information is available describing the response of the foodborne pathogen, Escherichia coli O157:H7, to nonisothermal and/or fluctuating temperature storage. Growth responses were determined for a mixture of three E. coli O157:H7 strains in brain heart infusion (BHI) broth as a function of temperature (static and fluctuating), initial pH (5, 6, and 7), and NaCl content (0.5, 1, 2, and 3%). Five 6-h "square-wave" fluctuating temperature regimes were used: 4 to 12, 4 to 19, 4 to 28, 8 to 19, and 12 to 28°C and compared with growth at 8, 10, 12, 19, and 28°C. The growth curves obtained from fitting the Gompertz equation for the fluctuating temperatures were compared to those obtained for the static temperatures. Increased NaCl concentration decreased growth temperature both for the fluctuating temperature growth curves and the static growth data. The cells grew or remained viable for up to 21 days under all conditions and fluctuating temperatures. Growth kinetics at fluctuating temperatures more closely approximated the higher temperature than the midpoint temperature of each cyclic range. The results indicate that transitory abuse could lead to more rapid growth than expected of E. coli O157:H7 in foods and that given sufficient time E. coli O157:H7 can grow at as low as 8°C.

An enterohemorrhaghic Escherichia coli O157:H7 (EHEC) outbreak in 1993 was epidemioiogically linked to commercial real mayonnaise. This study evaluated EHEC contamination risk during commercial mayonnaise and mayonnaise dressing production, and EHEC behavior in low-pH dressings. Two potential contamination sources, pasteurized liquid eggs and wet environmental areas, were surveyed for 4 months in three processing plants. One hundred eighty-eight egg lots and 114 environmental swabs were collected and analyzed for EHEC by enrichment and direct plating methods. All plant samples were EHEC negative. Commercial mayonnaise plants which use pasteurized eggs and employ effective good manufacturing practices (GMP) sanitation programs are unlikely EHEC harborage and contamination sources. Five commercial real-mayonnaise-based and reduced-calorie and/or fat mayonnaise dressings were inoculated with ≥6 log 10 colony-forming units (CFU)/g EHEC contamination levels and stored at 25°C. The products contained a wide range of acetic acid, NaCl, and preservative levels, while pH varied from 3.21 to 3.94. Products below pH 3.6 rapidly inactivated EHEC, producing ≥7 log 10 CFU/g decreases in ≤1 to ≤3 days. High EHEC lethality was also observed in the pH 3.94, egg white-mayonnaise dressing. Intact packages of commercial mayonnaise and mayonnaise dressings pose negligible EHEC contamination and health hazard risks. As with any food, consumers and food-service workers must use stringent hygienic practices to prevent microbial pathogen contamination during preparation, handling, and storage of mayonnaise-ingredient recipes such as chilled perishable salads and salad bar dressings.

Escherichia coli O157:H7 has been responsible for several recent food-borne outbreaks in the United States. To protect the public health, it is essential that rapid and sensitive methods be developed for detection of this pathogen in foods. Methods were compared for preparation of template DNA for the polymerase chain reaction (PCR) from enrichments of food homogenates seeded with E. coli O157:H7. Samples were enriched for 6 h at 37°C in modified tryptic soy broth supplemented with vancomycin, cefsulodin, and cefixime. Aliquots of the enrichments (10 ml or 1 ml) were analyzed by either washing twice with physiological saline or incubating with antibodies to O157 coupled to immunomagnetic beads (Dynal ® ) followed by resuspending and boiling the samples. A portion of the preparation was used in a multiplex PCR to amplify a 274-bp fragment from the sltI gene and a 364-bp fragment from the sltII gene. PCR amplification of 1-ml portions of enrichment broth was successful at inoculation levels of about 10 cells per g of food. Increasing the test sample volume to 10 ml and/or using an immunomagnetic separation step improved the PCR detection sensitivity to about 1 cell per g; the entire analysis can be completed within 12 h.

The current Food Safety and Inspection Service method for detection and recovery of Escherichia coli O157:H7, (including modified EC broth with novobiocin (mEC+n) and a direct blot ELISA). was used to analyze beef and environmental samples during an investigation of a food-borne disease outbreak attributed to consumption of undercooked hamburger patties. Double-modified trypticase soy broth (dmTSB) and a commercially available dipstick immunoassay were also used to improve detection/recovery of E. coli O157:H7. A total of 1,115 beef and environmental samples was screened with the direct blot ELISA and the dipstick immunoassay; 178 presumptive-positive samples (by either or both of the screening methods) were subjected to recovery/isolation procedures. Toxigenic E. coli O157:H7 was recovered from 45 samples: 40 hamburger-patty samples produced on the epidemiologically identified date, 3 hamburger-patty samples produced on another date, and 2 beef briskets. The organism was not recovered from environmental samples. Limited quantitative analyses indicated that contaminated hamburger patties contained fewer than 4.3 CFU of E. coli O157:H7 per g. Atypical, toxigenic ornithine decarboxylase-negative E. coli O157:H7 and nontoxigenic sorbitol-positive E. coli O157:H29 were also recovered. Both enrichment broths gave strong positive reactions with the two immunoassay screening methods, but E. coli O157:H7 was recovered more often from mEC+n broth than from dmTSB. Both screening methods gave positive results for 44 of the 45 beef samples found to contain E. coli O157:H7. False-positive results were frequently observed with both screening methods.

Efficacies of plating media for recovering heated Escherichia coli O157:H7 were determined and compared. To compare populations of recovered cells, suspensions of cells (three isolates, four replications/isolate) were heated at 50, 55, or 60°C, and then inoculated onto eight media: PCA-PA (plate count agar with 1% pyruvic acid [PA]), MSA (MacConkey sorbitol agar), MSA-Mg (MSA with 0.025% MgSO 4 ), MSA-PA (MSA with 1% PA), MSA-MUG (MSA with 0.005% 4-methylumbelliferyl-β-d-glucuronide (MUG), PRSA-MUG (phenol red sorbitol agar [PSRA] with 0.005% MUG), PRSA-PA (PRSA with 1% PA), and TSA-PA (tryptic soy agar with 1% PA). Recovery was consistently higher ( P < 0.05) with PRSA-MUG and PRSA-PA. At 50, 55, and 60°C, mean numbers (log 10 CFU/ml) of recovered cells on PRSA-MUG were 4.42, 4.62, and 3.32, respectively, as compared to 2.78, 2.08, and 1.63, respectively, on MSA. PCA-PA and TSA-PA were less effective than PRSA media, but better than MSA media. Thus, PRSA with MUG or PA was an effective medium for recovering heated cells of E. coli O157:H7; whereas MSA failed to detect sublethally injured cells. Furthermore, addition of Mg, PA, or MUG to MSA further compromised this medium.

The ability of Escherichia coli O157:H7 to survive and grow on cubes of cantaloupe and watermelon and on the external rind surface of these fruits was investigated. Populations of the pathogen increased on cubes stored at 25°C but remained constant at 5°C over a 34-h storage period. Growth was observed on the rind of melons stored under high relative humidity at 25°C for 14 to 22 days. The pathogen rapidly died on the rind surface of melons stored at 5°C.

Commercial mayonnaise and refrigerated ranch salad dressing were inoculated at two levels with two strains of Escherichia coli O157:H7, a non-pathogenic E. coli , and the non-fecal coliform Enterobacter aerogenes . Results showed that at the high inoculation level (>10 6 colony forming units [CFU]/g) in mayonnaise stored at room temperature (ca. 22°C) both strains of O157:H7 were undetected at 96 h. At the high inoculation level, all strains of coliform bacteria tested survived longer in salad dressing stored at 4°C than in mayonnaise stored at 22°C. The O157:H7 strains were still present at low levels after 17 days. The survival time in the low-level inoculum (10 4 CFU/g) study decreased, but the survival pattern in the two products was similar to that observed in the high-level inoculum study. Slight differences in survival among strains were observed. The greater antimicrobial effect of mayonnaise may be attributable to differences in pH, water activity (a w ), nutrients, storage temperature, and the presence of lysozyme in the whole eggs used in the production of commercial mayonnaise. Coliform bacteria survived longer in refrigerated salad dressing than in mayonnaise particularly at the high-level inoculum. Both mayonnaise (pH 3.91) and salad dressing (pH 4.51) did not support the growth of any of the microorganisms even though survival was observed.

A newly revised enrichment and agar-plating system was tested for selectivity and sensitivity in recovery of unstressed and cold-stressed Escherichia coli O157:H7 from foods. Various foods inoculated with known levels of enterohemorrhagic E. coli O157:H7 (EHEC) were tested by enrichment for 6 h at 37°C in modified tryptic soy broth (mTSB) base supplemented with vancomycin, cefsulodin and cefixime, referred to as EHEC enrichment broth (EEB). Subsequently, portions were spread-plated on sorbitol-MacConkey agar supplemented with tellurite and cefixime (TCSMAC). Further selective enrichment was also examined using immunomagnetic separation (IMS) from the EEB prior to spread-plating on TCSMAC agar. These methods were compared to a procedure of enrichment in mTSB (supplemented with novobiocin) at 37°C for 24 h followed by spread-plating of decimal dilutions on hemorrhagic colitis 4-methylumbelliferyl-B-D-glucuronide (HC-MUG) agar. The new enrichment isolation technique was found to be sensitive at a level of one EHEC organism per 10 g of food in four food types. This represents an approximate l00-fold to 1,000-fold enhancement in sensitivity over the comparative method for foods with high levels of competitive microflora. These enrichment-isolation protocols also were compared in analysis of naturally contaminated raw or undercooked ground beef samples implicated in foodborne illness. EEB-TCSMAC with and without IMS were combined with rapid biochemical tests, and with O157 latex agglutination and confirmation of toxin genes by polymerase chain reaction (PCR) to provide a completed test within 30 h of initiating testing. The new system was successful in 15 of 17 samples, where only 6 of 17 were found positive by the comparative technique.

Enumeration of Escherichia coli O157:H7 from Petrifilm ™ E. coli (PEC) Count plates was studied incorporating the Petrifilm ™ -HEC kit without sample pre-enrichment. Samples containing various E. coli O157:H7 inoculum levels were plated according to PEC package insert instructions. Plates were incubated at 32°C for dairy products and at 35°C for other samples. Total coliform, E. coli and E. coli O157:H7 levels were quantified in both control and inoculated samples. The average recovery from various food matrices was 74.8%. Food samples included retail soft cheese, clams, ground beef, chicken, salad mix, apple juice, cantaloupe and raw milk. The overall recovery was also determined on surface samples including cutting boards and preparation tables, at 116.3%. Results suggest that enumeration of E. coli O157:H7 from PEC Count plates is possible for products containing background coliforms as high as 40,000 colony forming units (CFU)/g, and with minimal background coliforms at <10 CFU/g. This method is a fast and efficient means of quantifying levels of presumptive positive E. coli O157:H7 from PEC Count plates.

The fate of enterohemorrhagic Escherichia coli O157:H7 was determined in three different lots of commercial mayonnaise, including four different samples from a lot implicated in an outbreak of E. coli O157:H7 infection. The initial pH of the products ranged from 3.6 to 3.9. Products were inoculated with 6.5 × 10 3 E. coli O157:H7/g and incubated at 5 or 20°C. Escherichia coli O157:H7 did not grow at either temperature but survived for 34 to 55 days at 5°C and for 8 to 21 days at 20°C, depending on the lot. Survival was greatest in real mayonnaise purchased at retail among six mayonnaise samples which included a reduced calorie mayonnaise. Escherichia coli O157:H7 populations decreased between 2- and 100-fold by 3 weeks at 5°C, and between 10- and 1,000-fold by 7 days at 20°C. There was little or no change in pH (<0.1 unit), aerobic plate count, mold and yeast count or Lactobacillus count (< 1 log 10 CFU/g) for the duration of the study. Commercial mayonnaise manufactured under good manufacturing practices is not a public health concern. Abusive handling of mayonnaise resulting in cross-contamination with E. coli O157:H7-contaminated food or contamination by an infected foodhandler is the principal basis for concern.

Escherichia coli O157:H7 is an important and common human pathogen which causes diarrhea, bloody diarrhea (hemorrhagic colitis) and the life threatening post-diarrheal disorder, hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 produces one or two potent cytotoxins, designated Shiga-like toxins (or verocytotoxins) I and II. While additional serotypes of cytotoxin-producing E. coli may cause human disease, E. coli O157:H7 is the most important such enteric pathogen in the United States. Epidemiologic data suggest that the incidence of hemolytic uremic syndrome is probably increasing. Until data emerge from controlled studies, conservative management of infected patients remains the mainstay of therapy, rather than specific antibacterial or antitoxin therapy. The serious nature of illness caused by E. coli O157:H7 should make prevention of human infection with this pathogen a high priority for the food industry.

Use of antisense oligonucleotide probes (OP) for detection and genotyping of Shiga-like toxin producing Escherichia coli (SLTEC) was evaluated. Based on published deoxyribonucleic acid (DNA) sequences of the A subunits of Shiga-like toxin (SLT) I and II genes, three synthetic antisense OP were constructed (OP-1, -2 and -3). Their use for detection and genotyping of SLTEC was evaluated and the results were compared to those obtained using cloned toxin-gene probe fragments. Both the OP-1 and OP-2 hybridized with all 69 SLT-I and II producing strains. Furthermore, the OP-1 hybridized only with all 18 SLT-I-only producing strains, and the OP-2 hybridized only with all 48 SLT-II-only producing strains. OP-3, a pool of four OP, detected all SLTEC strains regardless of toxin genotype. None of the OP hybridized to any of the 91 SLT-negative strains. These results demonstrate the three OP to be unique reagents for SLTEC epidemiological studies.