H7 Populations Research Articles

Effect of Simulated Spray Chilling with Chemical Solutions on Acid-Habituated and Non–Acid-Habituated Escherichia coli O157:H7 Cells Attached to Beef Carcass Tissue

Samples (10 by 20 by 2.5 cm) of beef carcass tissue were inoculated (104 to 105 CFU/cm2) with Escherichia coli O157: H7 that was either non–acid habituated (prepared by incubating at 15°C for 48 h in inoculated filter-sterilized composite [1:1] of hot and cold water meat decontamination runoff fluids, pH 6.05) or acid habituated (prepared in inoculated water fluids mixed with filter-sterilized 2% lactic acid [LA] runoff fluids in a proportion of 1/99 [vol/vol], pH 4.12). The inoculated surfaces were exposed to conditions simulating carcass chilling (−3°C for 10 h followed by 38 h at 1°C). Treatments applied to samples (between 0 and 10 h) during chilling included the following: (i) no spraying (NT) or spraying (for 30 s every 30 min) with (ii) water, (iii) cetylpyridinium chloride (CPC; 0.1 or 0.5%), (iv) ammonium hydroxide (AH; 0.05%), (v) lactic acid (LA; 2%), (vi) acidified sodium chlorite (ASC; 0.12%), (vii) peroxyacetic acid (PAA; 0.02%), (viii) sodium hydroxide (SH; 0.01%), or (ix) sodium hypochlorite (SC; 0.005%) solutions of 4°C. Samples were taken at 0, 10, 24, 36, and 48 h of the chilling process to determine changes in E. coli O157:H7 populations. Phase 1 tested water, SH, PAA, LA, and 0.5% CPC on meat inoculated with non–acid-habituated pathogen populations, whereas phase 2 tested water, SC, AH, ASC, LA, and 0.1% CPC on meat inoculated with acid- and non–acid-habituated populations. Reductions in non–acid-habituated E. coli O157:H7 populations from phase 1 increased in the order NT = water = SH < PAA < LA < CPC. Reductions from phase 2 for acid-habituated cells increased in the order NT = water = SC < ASC = LA = AH < CPC, whereas on non–acid-habituated cells the order observed was NT = water = SC < AH = ASC < LA < CPC. Previous acid habituation of E. coli O157:H7 inocula rendered the cells more resistant to the effects of spray chilling, especially with acid; however, the trend of reduction remained spray chilling with water = non–spray chilling < spray chilling with chemical solutions.

Nonimmunized egg yolk powder can suppress the colonization of Salmonella typhimurium, Escherichia coli O157:H7, and Campylobacter jejuni in laying hens

If colonized, poultry shed enteric pathogens in the feces. Of those that colonize poultry Salmonella spp., Campylobacter jejuni (C. jejuni), and Escherichia coli (E. coli) O157:H7 are the most concern to the industry. The authors previously discovered that the introduction of 5% dried nonimmunized egg yolk powder in the regular feed could eliminate and prevent Salmonella enteritidis intestinal colonization of laying hens. Hence, the efficacy of nonimmunized dried egg yolk powder supplement in controlling the colonization of laying hens with Salmonella typhimurium (S. typhimurium), C. jejuni, and E. coli O157:H7 was investigated in this study. The 10% (wt/ wt) egg yolk powder eliminated S. typhimurium after 2 wk of feeding and the 5 and 7.5% (wt/wt) supplements significantly (P < 0.05) reduced the frequency of colonization. Similarly, 7.5 and 10% egg yolk powder reduced the colonization of C. jejuni and E. coli O157:H7 significantly (P < 0.05) after 1 wk of feeding. In the prevention trial, pathogen-free chickens were fed supplemented feed (10% egg yolk powder) for 4 wk and were then infected. S. typhimurium was prevented from colonizing the intestine throughout the 4-wk test, and E. coli O157:H7 colonization was delayed for 2 wk. C. jejuni and E. coli O157:H7 populations in the intestine were significantly (P < 0.05) suppressed by egg yolk powder supplementation throughout the test. None of the supplement concentrations affected BW or the mean weight and number of the eggs laid per hen. At the end of the study, the organisms were absent or significantly (P < 0.05) reduced in the intestine, ovary, oviduct, and spleen of hens fed the egg yolk powder treatment. This study indicates that nonimmunized egg yolk powder is able to reduce the frequency of colonization of foodborne pathogens and prevent these organisms from colonizing the intestinal tract. Addition of egg yolk powder to the regular feed at a concentration of 7.5 to 10% (wt/wt) may be a simple means of reducing or inhibiting S. typhimurium, C. jejuni, and E. coli O157:H7 colonization in laying hens.

In Vitro Inactivation of Escherichia coli O157:H7 in Bovine Rumen Fluid by Caprylic Acid

The antibacterial effect of caprylic acid (35 and 50 mM) on Escherichia coli O157:H7 and total anaerobic bacteria at 39° C in rumen fluid (pH 5.6 and 6.8) from 12 beef cattle was investigated. The treatments containing caprylic acid at both pHs significantly reduced (P < 0.05) the population of E. coli O157:H7 compared with that in the control samples. At pH 5.6, both levels of caprylic acid killed E. coli O157:H7 rapidly, reducing the pathogen population to undetectable levels at 1 min of incubation (a more than 6.0-log CFU/ml reduction). In buffered rumen fluid at pH 6.8, 50mM caprylic acid reduced the E. coli O157:H7 population to undetectable levels at 1 min of incubation, whereas 35mM caprylic acid reduced the pathogen by approximately 3.0 and 5.0 log CFU/ml at 8 and 24 h of incubation, respectively. At both pHs, caprylic acid had a significantly lesser (P < 0.05) and minimal inhibitory effect on the population of total anaerobic bacteria in rumen compared with that on E. coli O157:H7. At 24 h of incubation, caprylic acid (35 and 50 mM) reduced the population of total anaerobic bacteria by approximately 2.0 log CFU/ml at pH 5.6, whereas at pH 6.8, caprylic acid (35 mM) did not have any significant (P > 0.05) inhibitory effect on total bacterial load. Results of this study revealed that caprylic acid was effective in inactivating E. coli O157:H7 in bovine rumen fluid, thereby justifying its potential as a preslaughter dietary supplement for reducing pathogen carriage in cattle.

Reduction of Escherichia coli O157:H7 and Salmonella enterica Serovar Enteritidis in Chicken Manure by Larvae of the Black Soldier Fly

Green fluorescent protein–labeled Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were inoculated at 107 CFU/g into cow, hog, or chicken manure. Ten- or 11-day-old soldier fly larvae (Hermetia illucens L.) (7 to 10 g) were added to the manure and held at 23, 27, or 32°C for 3 to 6 days. Soldier fly larvae accelerated inactivation of E. coli O157: H7 in chicken manure but had no effect in cow manure and enhanced survival in hog manure. The initial pH values of the hog and chicken manure were 6.0 to 6.2 and 7.4 to 8.2, respectively, and it is surmised that these conditions affected the stability of the larval antimicrobial system. Reductions of E. coli O157:H7 populations in chicken manure by larvae were affected by storage temperature, with greater reductions in samples held for 3 days at 27 or 32°C than at 23°C. Pathogen inactivation in chicken manure by larvae was not affected by the indigenous microflora of chicken manure, because Salmonella Enteritidis populations in larvae-treated samples were approximately 2.5 log lower than control samples without larvae when either autoclaved or nonautoclaved chicken manure was used as the contaminated medium during 3 days of storage. Extending the storage time to 6 days, larvae again accelerated the reduction in Salmonella Enteritidis populations in chicken manure during the first 4 days of storage; however, larvae became contaminated with the pathogen. After 2 days of feeding on contaminated manure, Salmonella Enteritidis populations in larvae averaged 3.3 log CFU/g. Populations decreased to 1.9 log CFU/g after 6 days of exposure to contaminated chicken manure; however, the absence of feeding activity by the maggots in later stages of storage may be responsible for the continued presence of Salmonella Enteritidis in larvae. Transfer of contaminated larvae to fresh chicken manure restored feeding activity but led to cross-contamination of the fresh manure.

Reduction of Microbial Pathogens during Apple Cider Production Using Sodium Hypochlorite, Copper Ion, and Sonication

Sodium hypochlorite (100 ppm), copper ion water (1 ppm), and sonication (22 to 44 kHz and 44 to 48 kHz) were assessed individually and in combination for their ability to reduce populations of Escherichia coli O157:H7 and Listeria monocytogenes on apples and in apple cider. Commercial unpasteurized cider was inoculated to contain approximately 106 CFU/ml of either pathogen and then sonicated at 44 to 48 kHz, with aliquots removed at intervals of 30 to 60 s for up to 5 min and plated to determine numbers of survivors. Subsequently, whole apples were inoculated by dipping to contain approximately 106 CFU/g E. coli O157:H7 or L. monocytogenes, held overnight, and then submerged in 1 ppm copper ion water with or without 100 ppm sodium hypochlorite for 3 min with or without sonication at 22 to 44 kHz and examined for survivors. Treated apples were also juiced, with the resulting cider sonicated for 3 min. Populations of both pathogens decreased 1 to 2 log CFU/ml in inoculated cider following 3 min of sonication. Copper ion water alone did not significantly reduce populations of either pathogen on inoculated apples. However, when used in combination with sodium hypochlorite, pathogen levels decreased approximately 2.3 log CFU/g on apples. Sonication of this copper ion–sodium hypochlorite solution at 22 to 44 kHz did not further improve pathogen reduction on apples. Numbers of either pathogen in the juice fraction were approximately 1.2 log CFU/ml lower after being juiced, with sonication (44 to 48 kHz) of the expressed juice decreasing L. monocytogenes and E. coli O157:H7 populations an additional 2 log. Hence, a 5-log reduction was achievable for both pathogens with the use of copper ion water in combination with sodium hypochlorite followed by juicing and sonication at 44 to 48 kHz.

Acid Tolerance of Acid-Adapted and Nonadapted Escherichia coli O157:H7 following Habituation (10°C) in Fresh Beef Decontamination Runoff Fluids of Different pH Values

This study evaluated survival of Escherichia coli O157:H7 strain ATCC 43895 during exposure to pH 3.5 following its habituation for 2 or 7 days at 10°C in fresh beef decontamination waste runoff fluid mixtures (washings) containing 0, 0.02, or 0.2% of lactic or acetic acids. Meat washings and sterile water (control) were initially inoculated with approximately 5 log CFU/ml of acid- and nonadapted E. coli O157:H7 cells cultured (30°C, 24 h) in broth with and without 1% glucose, respectively. After 2 days, E. coli O157:H7 survivors from acetate washings (pH 3.7 to 4.7) survived at pH 3.5 better than E. coli O157:H7 survivors from lactate washings (pH 3.1 to 4.6), especially when the original inoculum was acid adapted. Also, although E. coli O157:H7 habituated in sterile water for 2 days survived well at pH 3.5, the corresponding survivors from nonacid water meat washings (pH 6.8) were rapidly killed at pH 3.5, irrespective of acid adaptation. After 7 days, E. coli O157:H7 survivors from acetate washings (pH 3.6 to 4.7) continued to resist pH 3.5, whereas those from lactate washings died off. This loss of acid tolerance by E. coli O157:H7 was due to either its low survival in 0.2% lactate washings (pH 3.1) or its acid sensitization in 0.02% lactate washings, in which a Pseudomonas-like natural flora showed extensive growth (>8 log CFU/ml) and the pH increased to 6.5 to 6.6. Acid-adapted E. coli O157:H7 populations habituated in water washings (pH 7.1 to 7.3) for 7 days continued to be acid sensitive, whereas nonadapted populations increased their acid tolerance, a response merely correlated with their slight (<1 log) growth at 10°C. These results indicate that the expression of high acid tolerance by acid-adapted E. coli O157:H7 can be maintained or enhanced in acid-diluted meat decontamination waste runoff fluids of pH levels that could permit long-term survival at 10°C. Previous acid adaptation, however, could reduce the growth potential of E. coli O157:H7 at 10°C in nonacid waste fluids of high pH and enriched in natural flora. These conditions might further induce an acid sensitization to stationary E. coli O157:H7 cells.

Effects of Sodium Chlorate on Antibiotic Resistance inEscherichia coliO157:H7

The spread of antibiotic resistance from farm animals to human pathogens has become a matter of great public health significance, and methods to reduce foodborne pathogenic bacteria without utilizing traditional antibiotics have been sought. Chlorate kills Escherichia coli O157:H7 in vitro and in vivo and has been proposed as a feed additive to be included in food animal rations immediately prior to slaughter to reduce E. coli O157:H7 populations in the animal gastrointestinal tract. This study was designed to examine the effect that the development of chlorate resistance in E. coli O157:H7 has on resistance to 32 medically important antibiotics. Chlorate resistance numerically increased antibiotic resistance in E. coli O157:H7 strain 6058 and strain 933 to six and four antibiotics, respectively, but chlorate resistance affected resistance to only two antibiotics (cefoxotin and sulfadimethoxine) in both strains examined. Based on the fact that chlorate resistant mutants do not appear to compete well in vivo and based on the results of the current study, it appears unlikely that chlorate treatment will result in a dissemination of antibiotic resistance. However, further research is needed to confirm these results.

Reduction of E. coli O157:H7 populations in sheep by supplementation of an experimental sodium chlorate product

Ruminant animals are naturally infected with the pathogen Escherichia coli O157:H7, annually responsible for numerous meat recalls, foodborne illnesses and deaths. E. coli are equipped with the enzyme nitrate reductase, which not only enables this bacteria to respire anaerobically, but also converts chlorate to the toxic metabolite chlorite. This enzyme system is particular to only a few intestinal bacteria, therefore the vast majority are not affected by chlorate. Sodium chlorate has been shown to effectively decrease foodborne pathogens in several livestock species, including ruminants. However, because infection and proliferation of E. coli occurs primarily in the lower intestine, there is interest in “by-passing” the rumen, thereby, delivering chlorate directly to the largest population of pathogens. The objective of the current study was to evaluate the ability of an experimental sodium chlorate product (ECP II), designed to by-pass the rumen, in reducing fecal shedding and gut concentrations of E. coli O157:H7. Twenty crossbred mature ewes were adapted to a high grain ration and experimentally inoculated with E. coli O157:H7. Thirty-six hours following inoculation, sheep received in their feed one of the following ECP treatments: (1) control (CON), no chlorate; (2) 1 X (LOW); (3) 2 X (MED); and (4) 4 X (HIGH) where X=1.1 g chlorate ion equivalents/kg BW (five sheep per treatment). Fecal samples were collected every 12 h following inoculation and 24 h following the feeding of chlorate, all animals were euthanized and tissue samples and their respective contents collected from the rumen, cecum and rectum. The MED and HIGH chlorate treatments significantly reduced fecal shedding of E. coli O157:H7 compared to the CON treatment [1.53, 1.11, and 3.89 CFU/g feces (log 10), respectively]. Ruminal contents were similar among treatments, while chlorate tended to decrease ( P=0.08) and reduced ( P<0.05) E. coli O157:H7 populations in the cecum and rectum, respectively. Populations of generic E. coli in the cecal contents were numerically lower ( P=0.11) in the LOW treatment and tended to decrease ( P=0.06) in the MED and HIGH chlorate treatments, respectively. Fermentation profiles through the gastrointestinal tract were unaffected as indicated by slight, but not significant, changes in volatile fatty acids (VFA) profiles in sheep fed chlorate. Results from this study indicate that this experimental chlorate product, administered in the feed, was effective in reducing E. coli O157:H7 from the lower gut of sheep as evidenced by the lower cecal and rectal but not ruminal concentrations. Feeding chlorate may be an effective method to decrease E. coli O157:H7 populations in ruminant animals prior to slaughter.

Critical Factors Affecting the Destruction of Escherichia coli O157:H7 in Apple Cider Treated with Fumaric Acid and Sodium Benzoate

ABSRACTDestruction of Escherichia coli O157:H7 in apple cider treated with fumaric acid and sodium benzoate (0.15% and 0.05% w/v, respectively) was determined under pH and storage temperatures that commonly occur in apple cider. At 5°C storage, while destruction of E. coli O157:H7 in the presence of preservatives increased with time, there was little decline in E. coli O157:H7 populations in the absence of the preservatives. Increasing storage temperatures to 15°C and 25°C significantly increased the rate of destruction of E. coli O157:H7 in cider with the preservatives (P &lt; 0.05). Increasing the pH of cider (from 3.2 to 4.7) decreased the rate of destruction of E. coli O157:H7.

APPLICATION OF OZONE FOR INACTIVATION OF ESCHERICHIA COLI 0157:H7 ON INOCULATED ALFALFA SPROUTS

Chemical treatments to eliminate pathogens on inoculated seed sprouts have shown little success. This study investigated the effectiveness of aqueous ozone in killing Escherichia coli 0157:H7on alfalfa sprouts. Alfalfa sprouts inoculated with a five strain cocktail ofE. coli 0157:H7were immersed in water containing 21 ppm ozone for 2, 4, 8, 16, 32, 64 min at 4C. To increase availability and accessibility of ozone into sprout crevices, alternative treatments with continuous ozone sparging with and without pressurization were evaluated. Ozone treated sprouts were subjected to 12 psi hydrostatic pressure with pressurized ozone gas for 5 min to aid introduction of aqueous ozone into areas where pathogens may be hidden. Immersion of inoculated alfalfa sprouts (∼10 7 CFU/g) in water containing 21 ppm ozone reduced the population of E. coli 0157:H7 by 0.85 log 10 CFU/g after 64 min. There were no significant differences in log 10 reductions between the ozone treatments and control or between treatment times. With the exception of 2 min treatments, continuous ozone sparging resulted in reductions of 0.83 - 2.20 logo CFU/g, significantly higher (P ≤ 0.05) than reductions obtained by sparging with air (0.83 -1.29 logo CFU/g). Application of low hydrostatic pressure (12 psi) for 5 min subsequent to continuous ozone sparging for 2 to 64 min reduced E. coli 0157:H7 populations by 2 logo 10 CFU/g. Reductions resulting from pressurized ozone treatments did not differ significantly from those resulting from unpressurized ozone treatments, except at 32 min. Ozone treatments did not exhibit a visible detrimental effect on sprout quality. Further investigation is required to develop methods applying ozone for the purpose of decontaminating sprouts and reducing health risks. Ozone exhibits the potential for successful post harvest treatment of sprouts and also to replace other chemical treatments currently being used in the seed sprout industry.

Influence of pH treatments on survival of Escherichia coli O157:H7 in continuous cultures of rumen contents.

The pH (i.e., 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, and 7.25) effect on Escherichia coli O157:H7 in an artificial rumen model was investigated. Eight fermenters were inoculated with bovine rumen fluid and were supplied with a diet (75 g of dry matter daily in 12 equal portions [every 2 hr]) containing similar forage-to-concentrate ratio. After an adaptation period (i.e., 3 days for adjusting the rumen fluid [pH 6.2] microbial population to the test pH and 4 days for adjustment to the diet at the test pH), each fermenter was inoculated with 10(9) cells of E. coli O157:H7. Samples were collected hourly for 12 hr and every 2 hr for an additional 12 hr and were analyzed by flow cytometer. E. coli O157:H7 could not be quantified after 24 hr, and detection was only possible after enrichment. Because the pathogen could not be detected 5 days postinoculation (i.e., Day 13), the fermenters were reinoculated with E. coli O157:H7 on Days 17 and 22. E. coli O157:H7 numbers decreased from 10(6) to 10(4)/ml of fermenter contents in a quadratic (P < 0.05) fashion over the 24-hr sampling period, and the rate of reduction was slower (P < 0.05) for pH 7.0 than for other pH treatments. Results suggested that E. coli O157:H7 population were decreased by competitive exclusion and were not affected by culture pH.

HEAT INACTIVATION OF ESCHERICHIA COLI 0157:H7 IN APPLE CIDER IN THE PRESENCE OF GLYCEROL MONOLAURATE AND A SYNTHETIC ANTIMICROBIAL PEPTIDE, PR-26

The effect of moderate temperatures in combination with glycerol monolaurate and an antimicrobial peptide, PR-26 on E. coli 0157:H7 in apple cider was determined. A five-strain mixture of E. coli 0157:H7 was inoculated into apple cider (pH 3.53) containing 0.01% PR-26 or 0.02% glycerol monolaurate or a combination of 0.01% PR-26 and 0.02% glycerol monolaurate. The apple cider was incubated at 40, 45 or 50C for 30 s, 5 min, 10 min, and 15 min. At each of the specified time intervals, the surviving population of E. coli 0157:H7 was determined. At all the three temperatures, there was no significant difference (P > 0.05) between the E. coli 0157:H7 populations of the control and the treatment containing PR-26. However, the treatments containing glycerol monolaurate alone or combination of glycerol monolaurate and PR-26 resulted in significant reductions (P < 0.05) in E. coli 0157:H7 counts. Results indicate that addition of glycerol monolaurate (0.02%) to apple cider and heating at 50C for 5 min can reduce E. coli 0157:H7 by 5.0 log CFU/mL.

Escherichia coli O157:H7 Populations in Sheep Can Be Reduced by Chlorate Supplementation

Ruminant animals are a natural reservoir of the foodborne pathogen Escherichia coli O157:H7. Some foodborne pathogens (e.g., E. coli) are equipped with a nitrate reductase that cometabolically reduces chlorate. The intracellular reduction of chlorate to chlorite kills nitrate reductase–positive bacteria; however, species that do not reduce nitrate are not affected by chlorate. Therefore, it has been suggested that ruminants be supplemented with chlorate prior to shipment for slaughter in order to reduce foodborne illnesses in human consumers. Sheep (n = 14) were fed a high-grain ration and were experimentally infected with E. coli O157:H7. These sheep were given an experimental product (XCP) containing the equivalent of either 2.5mM NaNO3 and 100mM NaCl (control sheep; n = 7) or 2.5mM NaNO3 and 100mM NaClO3 (chlorate [XCP]–treated sheep; n = 7). Control and XCP-treated sheep were treated for 24 h; XCP treatment reduced the population of inoculated E. coli O157:H7 (P < 0.05) from 102, 105, and 105 CFU/g in the rumen, cecum, and rectum, respectively, to <101 CFU/g in all three sections of the gastrointestinal tract. The number of sheep testing positive for E. coli O157:H7 was significantly reduced by XCP treatment. In a similar fashion, total E. coli and coliforms were also reduced (P < 0.05) in all three compartments of the intestinal tract. Intestinal pH, total volatile fatty acid production, and the acetate/propionate ratio were unaffected by XCP treatment. On the basis of these results, it appears that chlorate treatment can be an effective method for the reduction of E. coli O157:H7 populations in ruminant animals immediately prior to slaughter.

Fate of Escherichia coli O157:H7 during Composting of Bovine Manure in a Laboratory-Scale Bioreactor

Inactivation profiles of Escherichia coli O157:H7 in inoculated bovine manure–based compost ingredients were determined by composting these ingredients in a bioreactor under controlled conditions. A 15-liter bioreactor was constructed to determine the fate of E. coli O157:H7 and changes in pH, moisture content, temperature, and aerobic mesophilic and thermophilic bacterial counts during composting. Fresh cow manure, wheat straw, cottonseed meal, and ammonium sulfate were combined to obtain a moisture content of ca. 60% and a carbon/nitrogen ratio of 29:1. The compost ingredients were held in the bioreactor at a constant external temperature of 21 or 50°C. Self-heating of the ingredients due to microbial activity occurred during composting, with stratified temperatures occurring within the bioreactor. At an external temperature of 21°C, self-heating occurred for 0 to 3 days, depending on the location within the bioreactor. E. coli O157:H7 populations increased by 1 to 2 log10 CFU/g during the initial 24 h of composting and decreased by ca. 3.5 log10 CFU/g near the bottom of the bioreactor and by ca. 2 log10 CFU/g near the middle and at the top during 36 days of composting. At an external temperature of 50°C, E. coli O157:H7 was inactivated rapidly (by ca. 4.9 log10 CFU/g at the top of the bioreactor, by 4.0 log10 CFU/g near the middle, and by 5.9 log10 CFU/g near the bottom) within 24 h of composting. When inoculated at an initial level of ca. 107 CFU/g, E. coli O157:H7 survived for 7 days but not for 14 days at all three sampling locations, as indicated by either direct plating or enrichment culture. At the top of the bioreactor a relatively constant moisture content of 60% was maintained, whereas the moisture content near the bottom decreased steadily to 37 to 45% over 14 days of composting. The pH of the composting mixture decreased to ca. 6 within 1 to 3 days and subsequently increased to 8 to 9. Results obtained in this study indicate that large populations (104 to 107 CFU/g) of E. coli O157:H7 survived for 36 days during composting in a bioreactor at an external temperature of 21°C but were inactivated to undetectable levels after 7 to 14 days when the external temperature of the bioreactor was 50°C. Hence, manure contaminated with large populations (e.g., 107 CFU/g) of E. coli O157:H7 should be composted for more than 1 week, and preferably for 2 weeks, when held at a minimum temperature of 50°C.

Selection of Recently Isolated Colicinogenic Escherichia coli Strains Inhibitory to Escherichia coli O157:H7

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.

Sodium chlorate supplementation reduces E. coli O157:H7 populations in cattle.

Cattle are a natural reservoir of the food-borne pathogen Escherichia coli O157:H7. Therefore, strategies that reduce E. coli O157:H7 prior to slaughter will reduce human exposures to this virulent pathogen. When bacteria that can anaerobically respire on nitrate (e.g., E. coli) are exposed to chlorate, they die because the intracellular enzyme nitrate reductase converts nitrate to nitrite, but also co-metabolically reduces chlorate to cytotoxic chlorite. Because chlorate is bactericidal only against nitrate reductase-positive bacteria, it has been suggested that chlorate supplementation be used as a strategy to reduce E. coli O157:H7 populations in cattle prior to harvest. Cattle (n = 8) were fed a feedlot-style high-grain diet experimentally infected with three strains of E. coli O157:H7. Cattle were given access to drinking water supplemented with 2.5 mM KNO3 and 100 mM NaCl (controls; n = 4) or 2.5 mM KNO3 and 100 mM NaClO3 (chlorate-treated; n = 4). Sodium chlorate treatment for 24 h reduced the population of all E. coli O157:H7 strains approximately two logs (10(4) to 10(2)) in the rumen and three logs (10(6) to 10(3)) in the feces. Chlorate treatment reduced total coliforms and generic E. coli from 106 to 10(4) in the rumen and by two logs throughout the rest of the gastrointestinal tract (ileum, cecum, colon, and rectum). Chlorate treatment reduced E. coli O157:H7 counts throughout the intestinal tract but did not alter total culturable anaerobic bacterial counts or the ruminal fermentation pattern. Therefore, it appears that chlorate supplementation is a viable potential strategy to reduce E. coli O157:H7 populations in cattle prior to harvest.

Fate of Escherichia coli O157:H7 in Coleslaw during Storage

An outbreak of Escherichia coli O157:H7 infection associated with the consumption of coleslaw in several units of a restaurant chain prompted a study to determine the fate of the pathogen in two commercial coleslaw preparations (pH 4.3 and 4.5) held at 4, 11, and 21°C for 3 days. At an initial population of 5.3 log10 CFU/g of coleslaw, E. coli O157:H7 did not grow in either coleslaw stored at the three temperatures. Rather, the population of E. coli O157:H7 decreased by 0.1 to 0.5 log10 CFU/g within 3 days. The greatest reduction (0.4 and 0.5 log10 CFU/g) in population occurred at 21°C, whereas only slight decreases (0.1 to 0.2 log10 CFU/g) occurred at 4 and 11°C. A pH of 4.3 to 4.5 of coleslaw had little effect on reducing E. coli O157:H7 populations. Results suggest that the tolerance of E. coli O157:H7 to acid pH, not temperature abuse, is a major factor influencing the pathogen's fate in restaurant-prepared coleslaw.

Behavior of Escherichia coli O157:H7 during the Manufacture and Ripening of Feta and Telemes Cheeses

Pasteurized whole ewe's and cow's milk was used in the manufacture of Feta end Telemes cheeses, respectively, according to standard procedures. In both cases, the milk had been inoculated with Escherichia coli O157:H7 at a concentration of ca. 5.1 log CFU/ml and with thermophilic or mesophilic starter cultures at a concentration of ca. 5.3 to 5.6 log CFU/ml. In the first 10 h of cheesemaking, the pathogen increased by 1.18 and 0.82 log CFU/g in Feta cheese and by 1.56 and 1.35 log CFU/g in Telemes cheese for the trials with thermophilic and mesophilic starters, respectively. After 24 h of fermentation, a decrease in E. coli O157:H7 was observed for all trials. At that time, the pH was reduced to 4.81 to 5.10 for all trials. Fresh cheeses were salted and held at 16°C for ripening until the pH was reduced to 4.60. Cheeses were then moved into storage at 4°C to complete ripening. During ripening, the E. coli O157:H7 population decreased significantly (P ≤ 0.001) and finally was not detectable in Feta cheese after 44 and 36 days and in Telemes cheese after 40 and 30 days for the trials with thermophilic and mesophilic starters, respectively. The estimated times required for one decimal reduction of the population of E. coli O157:H7 after the first day of processing were 9.71 and 9.26 days for Feta cheese and 9.09 and 7.69 days for Telemes cheese for the trials with thermophilic and mesophilic starters, respectively.

Addition of Fumaric Acid and Sodium Benzoate as an Alternative Method To Achieve a 5-log Reduction of Escherichia coli O157:H7 Populations in Apple Cider

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

The fate of Escherichia coli O157 : H7 in Myzithra, Anthotyros, and Manouri whey cheeses during storage at 2 and 12°C

Myzithra, Anthotyros and Manouri whey cheeses were inoculated the day after production withEscherichia coli O157 : H7 at concentrations of approx. 1·8×106cfu g−1, and stored at 2 and 12°C for 30 and 20 days, respectively. The pH of the whey cheeses decreased from an initial value of approx. 6·20 to 5·83 or 5·60 (Myzithra) 5·75 or 5·20 (Anthotyros) and 5·80 or 5·30 (Manouri) by the end of the corresponding storage periods at 2 and 12°C, respectively. Escherichia coli O157 : H7 populations in the whey cheeses at the end of the 12°C storage period, had grown with an increase of approx. 1·3 log10cfu g−1. E. coli O157 : H7 populations in whey cheeses at the end of the 2°C storage period did not grow and decreased, with an approx. 2·5 log10cfu g−1reduction. Results showed that E. coli O157 : H7 can grow at 12°C and survive at 2°C storage in Myzithra, Anthotyros and Manouri whey cheeses, and therefore post-manufacturing contamination with this pathogen must be avoided by employing hygienic control programmes such as HACCP.