Fate of pGFP-bearing Escherichia coli O157:H7 in ground beef at 2 and 10 degrees C and effects of lactate, diacetate, and citrate

Although beef has been implicated in the largest outbreaks of Escherichia coli O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing E. coli O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10 degrees C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2 degrees C, the initial numbers of E. coli O157:H7 (3 and 5 log(10) CFU/g) decreased by approximately 1 log(10) CFU/g when spoilage was manifest (>7 log(10) CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10 degrees C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.     

Elimination of Escherichia coli O157:H7 from Fermented Dry Sausages at an Organoleptically Acceptable Level of Microencapsulated Allyl Isothiocyanate

Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log₁₀ CFU/g Pediococcus pentosaceus and 6 log₁₀ CFU/g Staphylococcus carnosus) and a five-strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log₁₀ CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at ≤26°C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13°C for at least 25 days. The water activity (a_w), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a_w from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log₁₀ CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log₁₀ CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists.     

Evaluation of Culture Methods To Identify Bovine Feces with High Concentrations of Escherichia coli O157

Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log₁₀ CFU/g. The preenrichment streak detected >3.0 log₁₀ CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log₁₀ CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.     

Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7

A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10¹⁰, 10⁹, and 10⁸ PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10⁹ PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.     

Salmonella and Escherichia coli O157:H7 Contamination on Hides and Carcasses of Cull Cattle Presented for Slaughter in the United States: an Evaluation of Prevalence and Bacterial Loads by Immunomagnetic Separation and Direct Plating Methods

The hide and carcass hygiene of cull cattle at slaughter in four geographically distant regions of the United States was examined from July 2005 to April 2006 by measuring the aerobic plate counts (APC) and the prevalences and loads of Salmonella and Escherichia coli O157:H7. The geometric mean log₁₀ APC CFU/100 cm² levels on hides and preevisceration and postintervention carcasses ranged from 6.17 to 8.19, 4.24 to 6.47, and 1.46 to 1.96, respectively, and were highest in the summer (P < 0.0001). The average prevalences of Salmonella on hides and preevisceration and postintervention carcasses were 89.6% (95% confidence interval [CI], 85.1 to 94.0), 50.2% (95% CI, 40.9 to 59.5), and 0.8% (95% CI, 0.18 to 1.42), respectively. The prevalences of E. coli O157:H7 were 46.9% (95% CI, 37.3 to 56.6) and 16.7% (95% CI, 9.8 to 23.6) on hides and preevisceration carcasses, respectively. Examination of the concomitant incidence of Salmonella and E. coli O157:H7 showed that, on average, 33.3% (95% CI, 15.9 to 69.8) of cattle hide and 4.1% (95% CI, 0.98 to 17.3) of preevisceration carcass samples were contaminated with both pathogens. The pathogen prevalence on hides and carcasses was not significantly affected by the season; however, significant differences were observed between plants with respect to the incoming pathogen load and the ability to mitigate hide-to-carcass transfer. In spite of these differences, postintervention carcass contamination was significantly reduced (P < 0.001), likely as a result of the use of one or more of the processing interventions employed at each of the four processing plants examined.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Inactivation of Escherichia coli O157:H7 in Simulated Human Gastric Fluid

Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37°C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were −1.344 ± 0.564 and −0.997 ± 0.388 log₁₀ CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was −0.081 ± 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was −8.784 and −17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.     

Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r² = 0.95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r² = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Effects of Acid Adaptation of Escherichia coli O157:H7 on Efficacy of Acetic Acid Spray Washes To Decontaminate Beef Carcass Tissue

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4°C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.     

Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10⁻⁶ CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10⁻⁴ CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10⁻⁶ CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.     

Combinations of Intervention Treatments Resulting in 5-Log10-Unit Reductions in Numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 Organisms in Apple Cider

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log₁₀ units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log₁₀ units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log₁₀-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log₁₀-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log₁₀-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.     

Reduction of Escherichia coli O157:H7 Populations in Cattle by Addition of Colicin E7-Producing E. coli to Feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10⁷ CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log₁₀ CFU/g was observed, with a maximum decrease of 1.8 log₁₀ CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10⁸ CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

Longitudinal Study of Escherichia coli O157:H7 in a Beef Cattle Feedlot and Role of High-Level Shedders in Hide Contamination

The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>10⁴ CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (≥40 CFU/100 cm²) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (≥200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.It is now well established that at the time of harvest, hides are the major source of Escherichia coli O157:H7 contamination on beef carcasses (1, 4, 22). Thus, reducing the levels of food-borne pathogens on cattle hides has been the focus of many pre- and postharvest research efforts. For postharvest applications, hide interventions (i.e., washing of hide-on carcasses with various antimicrobial agents) are direct approaches and have been shown to be efficacious for reducing hide and carcass contamination rates (2, 4, 5, 22).In the area of preharvest research, several approaches have been taken to reduce the prevalence of E. coli O157:H7 in feces of cattle presented for slaughter. These approaches include, among others, feeding cattle probiotics (dietary administration of beneficial bacteria to compete with E. coli O157:H7), vaccination, and bacteriophage treatment (8, 24, 30). These intervention approaches are indirect. By reducing the fecal pathogen load, the pathogen prevalence and the level on hides are reduced through lower cross-contamination at the feedlot, and subsequently, carcass contamination rates decrease. While the effectiveness of preharvest interventions varies, no preharvest intervention is 100% effective in reducing the fecal prevalence of E. coli O157:H7. It is not known what level of pathogen reduction in feces would be necessary to significantly reduce hide and carcass contamination during processing. Key pieces of information needed to address this question are the number of shedding cattle in a pen needed to contaminate the hides of most of the cattle in the same pen and at what level the shedding cattle are contaminated.Aside from the number of cattle shedding a pathogen, the concentration of the pathogen in feces plays a pivotal role in spreading the pathogen between animals. Recently, cattle shedding E. coli O157:H7 at levels of >10⁴ CFU/g (“supershedders”) have been associated with high rates of transmission of the pathogen between cohort animals (18, 23). Matthews et al. reported that 20% of the E. coli O157:H7 infections in cattle on Scottish farms were responsible for 80% of the transmission of the organism between animals (18). Another study reported similar findings; 9% of the animals shedding E. coli O157:H7 produced over 96% of the total E. coli O157:H7 fecal load for the group (23). While a number of studies have indicated the importance of supershedders in fecal transmission dynamics, there is a general lack of information concerning the effects of high shedding rates on hide prevalence and load. Accordingly, the objectives of this study were (i) to investigate the dynamics of E. coli O157:H7 prevalence and levels in feces and on hides of feedlot cattle over time and (ii) to determine how pathogen prevalence and levels on hides in a pen are affected by individuals shedding E. coli O157:H7 at high levels.In the analysis presented here, fecal shedding was analyzed using the following three categories based on the level of E. coli O157:H7 being shed: shedding positive (presumed concentration, ≥1 CFU/g), high-density shedder (≥200 CFU/g), and supershedder (≥10⁴ CFU/g). Several definitions of E. coli O157:H7 supershedders have been offered previously. One-time shedding levels of >10³ or >10⁴ CFU/g have been used in multiple studies (17, 23, 24), while other groups have required persistent colonization of the rectoanal junction, as well as high cell counts, for an animal to qualify as a supershedder (10). Recently, Chase-Topping et al. (9) reviewed the requirements for supershedder status and provided a working definition: an animal that excretes >10⁴ CFU/g. In doing this, Chase-Topping et al. noted the high stringency of this definition and acknowledged that with such a definition some supershedders will be missed if they are sampled at times other than peak shedding times (9). In the current study, this was a concern. In an attempt to investigate the link between high-shedding-level animals and hide contamination, greater leeway was needed in the classification. When it is sampled on a monthly basis, an animal shedding at high levels can have a large impact on the hide status of pen cohorts between sampling intervals but not be shedding at peak levels on the day of sample collection. Hence, the categories described above were selected to analyze the relationship between fecal shedding and hide contamination.     

Efficacy of Electrolyzed Oxidizing Water for Inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes

The efficacy of electrolyzed oxidizing water for inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes was evaluated. A five-strain mixture of E. coli O157:H7, S. enteritidis, or L. monocytogenes of approximately 10⁸ CFU/ml was inoculated in 9 ml of electrolyzed oxidizing water (treatment) or 9 ml of sterile, deionized water (control) and incubated at 4 or 23°C for 0, 5, 10, and 15 min; at 35°C for 0, 2, 4, and 6 min; or at 45°C for 0, 1, 3, and 5 min. The surviving population of each pathogen at each sampling time was determined on tryptic soy agar. At 4 or 23°C, an exposure time of 5 min reduced the populations of all three pathogens in the treatment samples by approximately 7 log CFU/ml, with complete inactivation by 10 min of exposure. A reduction of ≥7 log CFU/ml in the levels of the three pathogens occurred in the treatment samples incubated for 1 min at 45°C or for 2 min at 35°C. The bacterial counts of all three pathogens in control samples remained the same throughout the incubation at all four temperatures. Results indicate that electrolyzed oxidizing water may be a useful disinfectant, but appropriate applications need to be validated.     

Survival or Growth of Escherichia coli O157:H7 in a Model System of Fresh Meat Decontamination Runoff Waste Fluids and Its Resistance to Subsequent Lactic Acid Stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 ± 0.1) or in water (pH 7.2 ± 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25°C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4°C and lowest at 25°C. The pathogen survived without growth in water washings at 4 and 10°C, while it grew by 0.8 to 2.7 log cycles at 15 and 25°C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10°C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25°C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15°C > 10°C > 4°C, while at 25°C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15°C may maintain a higher acid resistance than when acid habituated at 4°C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Role of Glucose in Enhancing the Temperature-Dependent Growth Inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp.

Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25°C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25°C and grew better in pure culture than in mixed cultures. Pseudomonas sp. inhibited E. coli O157:H7 in cocultures with glucose at 10°C, while at 15°C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15°C and, irrespective of glucose, at 25°C. These results indicate that glucose enhances the growth inhibition of E. coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (≤15°C) temperatures.     

Role of rpoS in Acid Resistance and Fecal Shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator ς^S and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10¹ to 10⁴ CFU, we found the wild-type strain in feces of mice given lower doses (10² versus 10³ CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10⁴ CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P ≤ 0.05). Thus, ς^S appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.     

Comparison of the BAX for Screening/E. coli O157:H7 Method with Conventional Methods for Detection of Extremely Low Levels of Escherichia coli O157:H7 in Ground Beef

Escherichia coli O157:H7 is an important food-borne pathogen. Often E. coli O157:H7 is difficult to detect, because it is present sporadically at very low levels together with very high levels of competitor organisms which can be difficult to distinguish phenotypically. Cultural methods are time-consuming and give variable results in the detection of E. coli O157:H7. This study examined the performance of BAX for Screening/E. coli O157:H7, a new rapid method for the detection of E. coli O157:H7, against traditional and improved cultural methods and an immunodiffusion assay. All cultural methods demonstrated inadequacy in detecting the presence of E. coli O157:H7 in inoculated samples. The limitations of these cultural methods further complicate evaluation of screening methodologies. The BAX for Screening/E. coli O157:H7 assay outperformed the other methods, with a detection rate of 96.5%, compared to 39% for the best cultural method and 71.5% for the immunodiffusion method. The BAX for Screening/E. coli O157:H7 assay proved to be a rapid, highly sensitive test for the detection of low levels of E. coli O157:H7 in ground beef.     

Rectal Carriage of Enterohemorrhagic Escherichia coli O157 in Slaughtered Cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 × 10³ CFU g⁻¹ or <1 × 10³ CFU ml⁻¹ and animals with E. coli O157 levels of ≥1 × 10³ CFU g⁻¹ or ≥1 × 10³ CFU ml⁻¹ in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (≥1 × 10³ CFU g of feces⁻¹) of enterohemorrhagic E. coli O157 results from colonization of this site.