Charles W. Kaspar

Escherichia coli O157:H7 acid tolerance and survival in apple cider

The survival of two Escherichia coli O157:H7 (ATCC 43889 and 43895) and a control strain E. coli was compared in apple cider and in Trypticase soy broth (TSB) adjusted to low and high pH. The O157:H7 strains were detectable in apple cider after 14 to 21 days at 4°C, whereas the control strain could not be detected (> 4-log reduction) after 5 to 7 days. During the first 14 days of storage at 4°C, the levels of strain 43889 decreased by ~3 logs, whereas levels of strain 43895 were unchanged. Survival of O157:H7 strains and the control strain were unaffected by the presence of potassium sorbate or sodium benzoate, except in one instance. Sodium benzoate caused a decrease of 57% in strain 43895 after 21 days, but ~104 CFU/ml still remained. In TSB adjusted to pH 2, 3, 4, 11 or 12, strain 43895 was again the more resistant of the O157:H7 strains, both of which were more durable than the control strain. The O157:H7 strains (especially strain 43895) withstood pH 2 with a minimal drop in CPU after 24 h, whereas no viable organisms were detectable after this time at pH 12. At these extremes of pH, survival was generally greater at 4°C than at 25°C. Despite differences between strains, these results show that E. coli O157:H7 is exceptionally tolerant of acid pH.

Contribution of dps to Acid Stress Tolerance and Oxidative Stress Tolerance in Escherichia coli O157:H7

ABSTRACT An Escherichia coli O157:H7dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log10-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log10-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log10CFU/ml while the parent strain decreased by approximately 2 log10 CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7.

ABSTRACT 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 ςS and its effect on shedding of E. coliO157: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 ofE. 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, ςS 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.

Validation of pepperoni processes for control of Escherichia coli O157:H7

The outbreak of Escherichia coli O157:H7 linked with dry-cured salami in late 1994 prompted regulatory action that required manufacturers of fermented products to demonstrate a 5-log unit reduction in counts of this pathogen during processing. Therefore, pepperoni batter (75% pork:25% beef with a fat content of ca. 32%) was inoculated with a pediococcal starter culture and a five-strain mixture of E. coli O157:H7 (≥2 × 107 CFU/g) and stuffed into 55-mm diameter fibrous casings 47 cm in length. The viability of the pathogen was monitored before stuffing, after fermentation, after thermal processing, and/or after drying. Chubs were fermented at 96°F (36°C) and 85% relative humidity (RH) to pH ≤ 5.0 and then dried at 55°F (13°C) and 65% RH to a moisture/protein ratio of ≤1.6:1 (modified method 6 process). Counts of the pathogen decreased about 1.2 log units after fermentation and drying. In subsequent experiments, heating chubs after fermentation to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min resulted in a ≥5-log unit decrease in numbers of strain O157:H7 without visibly affecting the texture or appearance of the product. These data revealed that a traditional nonthermal, process for pepperoni was only sufficient to eliminate relatively low levels (ca. 2 log CFU/g) of E. coli O157:H7, whereas heating to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min delivered a 5 to 6 log unit reduction in counts of the pathogen in pepperoni.

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.

Viability of Escherichia coli O157:H7 in Salami Following Conditioning of Batter, Fermentation and Drying of Sticks, and Storage of Slices

The fate of Escherichia coli O157:H7 was monitored in salami during conditioning of batter, fermentation and drying of sticks, and storage of slices. The raw batter (75% pork: 25% beef, wt/wt, fat content about 20%) was inoculated with a pediococcal starter culture (about 10(8) CFU/g) and a five-strain cocktail of E. coli O157:H7 ( > or = 2 x 10(7) CFU/g) and stuffed into 104-mm diameter fibrous casings. After being refrigerated at 4 degrees C or being tempered at 13 degrees C, frozen at -20 degrees C, and thawed at 4 degrees C, or being frozen at -20 degrees C, and thawed at 4 degrees C, the inoculated batter was fermented at 24 degrees C and 90% relative humidity (RH) to pH < or = 4.8, dried at 13 degrees C and 65% RH to a moisture/protein ratio of < or = 1.9:1, and then stored at 4 or 21 degrees C under air or vacuum. For salami sticks sampled immediately after drying, appreciable differences were evident among the various batter-conditioning treatments; pathogen numbers were reduced from original levels by 2.1, 1.6, or 1.1 log10 units when batter was tempered, frozen, and thawed, frozen and thawed, or refrigerated, respectively. Similarly, regardless of storage temperature or atmosphere, within 7 days salami slices cut from sticks prepared from batter that was tempered, frozen, and thawed (2.7- to 4.9-log10-unit reduction) or frozen and thawed (2.3- to 4.8-log10-unit reduction) displayed a greater impact on pathogen numbers than slices cut from sticks prepared from batter that was refrigerated (1.6- to 3.1-log10-unit reduction). The effects of batter conditioning notwithstanding, a greater reduction in levels of E. coli O157:H7 was observed when slices were stored at 21 degrees C compared to otherwise similar slices stored at 4 degrees C. After storage for 60 days the pathogen was only detected by enrichment in slices stored at 21 degrees C, whereas pathogen levels ranged from 1.4 to 4.5 log10 CFU/g in slices stored at 4 degrees C. Differences related to storage atmosphere were first observed after slices were stored for 21 days. Such differences were more readily demonstrable after 60 and 90 days, with pathogen numbers for treatments that were statistically different ranging from 0.6- to 1.5-log10 units higher on slices stored under vacuum than in air. These data emphasize the need to implement multiple barriers to appreciably reduce numbers of E. coli O157:H7 in salami.

Decontamination of Plastic and Wooden Cutting Boards for Kitchen Use

Decontamination of Plastic and wooden cutting boards was studied, with a view to preventing cross-contamination of foods in home kitchens. New and used Plastic (four polymers plus hard rubber) and wood (nine hardwoods) boards were cut into 5-cm square blocks (25 cm2 area) for these experiments. Bacterial contaminants-- Escherichia coli (two nonpathogenic strains plus serotype O157:H7), Listeria innocua , L. monocytogenes , or Salmonella typhimurium --applied to the block surface in nutrient broth or chicken juice, were recovered by soaking the surface in nutrient broth or pressing the block onto nutrient agar, within minutes or ≥12 h later. Persistence and overnight multiplication of bacteria on plastic surfaces depended on maintenance of humidity so as to prevent drying of the contaminant. New plastic cutting surfaces were relatively easy to clean and were microbiologically neutral, but plastic boards with extensive knife scars were difficult to clean manually, especially if they had deposits of chicken fat on them. Fewer bacteria were generally recovered from wooden blocks than from plastic blocks. Clean wood blocks rapidly absorbed all of the inoculum, after which the bacteria could not be recovered within 3 to 10 min. If the board surface was coated with chicken fat, some bacteria might be recovered even after 12 h at room temperature and high humidity. Cleaning with hot water and detergent generally removed these bacteria, regardless of bacterial species, wood species, and whether the wood was new or used.

Acid stress damage of DNA is prevented by Dps binding in Escherichia coli O157:H7

BackgroundAcid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7.ResultsThe results from this study showed that acid stress lead to damage of chromosomal DNA, which was accentuated in dps and recA mutants. The use of Bal31, which cleaves DNA at nicks and single-stranded regions, to analyze chromosomal DNA extracted from cells challenged at pH 2.0 provided in vivo evidence of acid damage to DNA. The DNA damage in a recA mutant further corroborated the hypothesis that acid stress leads to DNA strand breaks. Under in vitro assay conditions, Dps was shown to bind plasmid DNA directly and protect it from acid-induced strand breaks. Furthermore, the extraction of DNA from Dps-DNA complexes required a denaturing agent at low pH (2.2 and 3.6) but not at higher pH (>pH4.6). Low pH also restored the DNA-binding activity of heat-denatured Dps. Circular dichroism spectra revealed that at pH 3.6 and pH 2.2 Dps maintains or forms α-helices that are important for Dps-DNA complex formation.ConclusionResults from the present work showed that acid stress results in DNA damage that is more pronounced in dps and recA mutants. The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present. Dps protected DNA from acid damage by binding to DNA. Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed α-helices at low pH. Further investigation into the precise interplay between DNA protection and damage repair pathways during acid stress are underway to gain additional insight.

Survival of Escherichia coli O157:H7 in ground-beef patties during storage at 2, -2, 15 and then -2°C, and -20°C

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (-20 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.

Survival of Escherichia coli O157:H7 in Full- and Reduced-Fat Pepperoni after Manufacture of Sticks, Storage of Slices at 4°C or 21°C under Air and Vacuum, and Baking of Slices on Frozen Pizza at 135, 191 and 246°C

Pepperoni batter was prepared with fat contents of about 15, 20, and 32% (wt/wt) and inoculated with a pediococcal starter culture and ≥2.0 X 10 7 CFU/g of a five-strain inoculum of Escherichia coli O157:H7. The batter was fermented at 96°F (ca. 36°C) and 85% relative humidity (RH) to pH 4.8 and then dried at 55°F (ca. 13°c) and 65% RH to a moisture/protein ratio of ≤1.6:1. For storage, slices were packaged under air or vacuum and stored at 39°F (ca. 4°C) and 70°F (ca. 21°C). For baking, frozen slices were placed on retail frozen cheese pizzas that were subsequently baked at 275°F (ca. 135°C), 375°F (ca. 191°C), or 475°F (ca. 246°C) for 0 to 20 min. Appreciable differences related to fat levels were observed after drying; pathogen numbers decreased by 1.04, 1.31 and 1.62 log 10 units in sticks prepared from batter at initial fat levels of 15, 20, and 32%, respectively. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers, with similar viability observed among the three fat levels tested. At 70°F (ca. 21°C), compared to original levels, pathogen numbers decreased by ≥5.56 and ≥4.53 log,0 units within 14 days in slices stored under air and vacuum, respectively, whereas at 39°F (ca. 4°C) numbers decreased by ≤2.43 log 10 CFU/g after 60 days of storage under either atmosphere. Baking, as expected, resulted in greater reductions in pathogen numbers as the temperature and/or time of baking increased. However, it was still possible to recover the pathogen by enrichment after baking frozen slices on frozen pizza at 475°F (ca. 246°C) for 10 min or at 375°F (ca. 191°C) for 15 min. The calculated D values for all three temperatures tested increased as the fat content of the batter increased from 15 to 20 to 32%. The present study confirmed that fermentation and drying were sufficient to reduce levels of E. coli O157:H7 in pepperoni sticks by 5.5-log 10 -unit total reduction of the pathogen. Baking slices on frozen pizza for at least 15 min at 475°F (ca. 246°C) or 20 min at 375°F (ca. 191°C) was necessary to reduce pathogen numbers to below detection by both direct plating and enrichment.