Effects of acid adaptation, product pH, and heating on survival of Escherichia coli O157:H7 in pepperoni

The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log(10) units.     

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.     

Antimicrobial effects of mustard flour and acetic acid against Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella enterica serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22 degrees C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10(6) to 10(7) CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22 degrees C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5 degrees C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22 degrees C than at 5 degrees C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

Effects of sodium chlorate on toxin production by Escherichia coli O157:H7

Chlorate kills E. coli O157:H7 and has been proposed as a feed additive to be included in cattle rations immediately prior to slaughter to reduce E. coli O157: H7 populations in the gut. Antibiotic usage is not recommended in cases of E. coil O157:H7-induced hemorrhagic colitis because some antibiotics stimulate increased toxin production. This study was undertaken to determine if chlorate treatment affected toxin production. Pure cultures of E. coil O157:H7 were treated with 1/4 MIC of antibiotics (ampicillin, tetracycline, ceftiofur, gentamicin, monensin, tylosin, penicillin, ciprofloxacin, and novobiocin); toxin production was significantly increased by some antibiotics, but not by chlorate. Studies with mixed fecal bacteria demonstrated that chlorate killed E. coli O157:H7, but again did not stimulate toxin production. Chlorate appears to be an effective method to reduce shiga toxin-producing E. coil (STEC) populations in food animals, but additional studies are warranted before it is used to control infections.     

Osmotic stress on dilution of acid injured Escherichia coli O157:H7

To determine surviving numbers of Escherichia coli from cultures or food systems, dilution with 0.1% peptone is regularly used. Higher numbers of survivors could be obtained from an acid-treated culture if 0.5 mol l-1 sucrose was added to the 0.1% peptone. Sorbitol, glucose or sodium chloride, but not glycerol, could be used in place of sucrose. Using electron microscopy distinct differences could be seen between acid-treated and untreated cells. The osmolarity of the diluents ranged from 5 to 500 mosmol kg-1 H2O for the 0.5 mol l-1 sugar or glycerol solutions, to about 1000 mosmol kg-1 H2O for the salt solution. Maximum recovery diluent has an osmolarity of about 300 mosmol kg-1 H2O and resulted in recovery of similar numbers of injured cells as a 0.5 mol l-1 solution of sugar in 0.1% peptone. Taking into account the observed damage to acid-treated cells and the differences in osmolarity of the diluents, it is likely that dilution in 0.1% peptone imposed additional stress on the acid-injured cells which caused further cell damage. Dilution in a more osmotically stable solution alleviated this osmotic stress.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

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.     

Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.     

Starvation- and stationary-phase-induced acid tolerance in Escherichia coli O157:H7.

Stationary phase and the starvation of log-phase cells increased the acid tolerance of Escherichia coli O157:H7 strains. Although the degree of acid tolerance varied, the survival of most O157:H7 strains exceeded that of other, related, pathogens in a synthetic gastric fluid.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Effect of cattle diet on Escherichia coli O157:H7 acid resistance.

The duration of shedding of Escherichia coli O157 isolates by hay-fed and grain-fed steers experimentally inoculated with E. coli O157:H7 was compared, as well as the acid resistance of the bacteria. The hay-fed animals shed E. coli O157 longer than the grain-fed animals, and irrespective of diet, these bacteria were equally acid resistant. Feeding cattle hay may increase human infections with E. coli O157:H7.     

Distinct acid resistance and survival fitness displayed by Curli variants of enterohemorrhagic Escherichia coli O157:H7

Curli are adhesive fimbriae of Enterobacteriaceae and are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagic Escherichia coli O157:H7. The relative proportions of curli-producing variants (C(+)) and curli-deficient variants (C(-)) in an E. coli O157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C(+) and C(-) subpopulations occurred mostly in response to starvation and was unidirectional from C(-) to C(+); in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C(+) variants grew to higher concentrations in nutrient-limited conditions than C(-) variants, whereas C(-) variants were significantly more acid resistant than C(+) variants. This difference in acid resistance does not appear to be linked to the curli fimbriae per se, since a csgA deletion mutant in either a C(+) or a C(-) variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants of E. coli O157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in an E. coli O157:H7 population may provide a survival strategy in which C(+) variants are selected in a nutrient-limited environment, whereas C(-) variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.     

The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7

The survival characteristics of a non-toxigenic, antibiotic-resistant strain of Escherichia coli O157:H7 in bovine faeces were investigated. Faecal samples were inoculated with 10(8-9) cfu g-1 of the organism and (i) stored in closed plastic containers at 10 degrees C, (ii) stored in closed plastic containers placed outside or (iii) decanted onto the surface of grazing land. Recovery and enumeration on Sorbitol MacConkey Agar (SMAC) and Tryptic Soya Agar (TSA) revealed that the E. coli O157:H7 numbers in both enclosed samples (i and ii) had decreased by 4.5-5.5 log10 cfu g-1 within 99 d. Numbers in samples decanted onto grassland (iii) decreased by 4.0-5.0 log10 cfu g-1 within 50 d but the organism was still detectable in the surrounding soil for up to 99 d. Persistence of E. coli O157:H7 in bovine faeces and contaminated pastures may therefore be an important factor in the initial infection and re-infection of cattle.     

Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice

Outbreaks of diarrhoea and haemolytic uraemic syndrome have been associated with the consumption of apple cider and apple juice. The organism implicated in these outbreaks has been Escherichia coli O157:H7, indicating the resistance of the serotype to acidic pH. On comparing the growth of this serotype with a control strain of E. coli, it was found that strain O157:H7 grew well in trypticase soy broth at pH levels ranging from 2.0 to 9.0, while control strains failed to grow at pH levels below 4.0 and above 9.0. The growth of both strains were inhibited by adding 0.05% of either benzoic acid or sorbic acid. Similarly, O157:H7 grew well in both natural (unpasteurized) as well as in pasteurized apple juice and the growth was inhibited by adding 0.1% of either benzoic acid or sorbic acid. Control strains of E. coli failed to grow in either types of apple juice. The possible sources of contamination of natural apple juice with O157:H7 serotype are discussed.     

Survival of Escherichia coli O157:H7 on cattle hides

The objective of this study was to determine the time period that Escherichia coli O157:H7 survives on the hides of cattle. Extensive research has been conducted and is ongoing to identify and develop novel preharvest intervention strategies to reduce the presence of E. coli O157:H7 on live cattle and subsequent transfer to processed carcasses. If a reduction of E. coli O157:H7 levels in feces can be achieved through preharvest intervention, it is not known how long it would take for such reductions to be seen on the hide. In the study presented herein, three trials were conducted to follow E. coli O157:H7 hide prevalence over time. For each trial, 36 animals were housed in individual stanchions to minimize or prevent hide contamination events. Through prevalence determination and isolate genotyping with pulsed-field gel electrophoresis, survival of E. coli O157:H7 on the hides of live cattle was determined to be short lived, with an approximate duration of 9 days or less. The results of this study suggest that any preharvest interventions that are to be administered at the end of the finishing period will achieve maximum effect in reducing E. coli O157:H7 levels on cattle hides if given 9 days before the cattle are presented for processing. However, it should be noted that interventions reducing pathogen shedding would also contribute to decreasing hide contamination through lowering the contamination load of the processing plant lairage environment, regardless of the time of application.