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Experimental Biology and Medicine 228:365-369 (2003)
© 2003 Society for Experimental Biology and Medicine

SUPPLEMENT. FOOD SAFETY CONCERNS OF VEROTOXIN-PRODUCING ESCHERICHIA COLI

Influence of pH Treatments on Survival of Escherichia coli O157:H7 in Continuous Cultures of Rumen Contents

Brandolyn H. Thran*, Hussein S. Hussein*,1, Doug Redelman{dagger} and George C.J. Fernandez{ddagger}

* Department of Animal Biotechnology,
{dagger} Cytometry Center, and
{ddagger} Nevada Agricultural Experiment Station, University of Nevada, Reno, Nevada 89557

Abstract

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 109 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 106 to 104/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.

Key Words: Escherichia coli • cattle • continuous culture • food-borne pathogens

Escherichia coli O157:H7 has been associated with numerous worldwide outbreaks of human food-borne illnesses (1–3). Because studies have directly linked several human illness outbreaks to consumption of undercooked ground beef or other beef products, cattle are considered reservoirs of this pathogen (4–7). At slaughter, contamination of beef with E. coli O157:H7 usually occurs from feces on the hide or from the digesta released by nicking the gastrointestinal tract. Because contamination is confined to the carcass surface (8), efforts have been devoted to develop and implement postharvest control methods to reduce E. coli O157:H7 contamination. These efforts resulted in developing and implementing the Hazard Analysis and Critical Control Point system to assure safety of beef and other meat products (9). The postharvest control methods and their efficacy have been reviewed recently (10). These methods include trimming and washing (11, 12), spraying with sanitizers (13–15), hot water washing (16, 17), using dips (18, 19), using food additives (20), and irradiation (21, 22).

Recently, attention has been given to development and evaluation of preharvest strategies that would minimize or reduce E. coli O157:H7 contamination before sending cattle to slaughter. Implementation of control measures at the farm level combined with effective postharvest measures would improve beef safety and reduce consumers’ concern. Several on-farm factors have been found to interact simultaneously and affect carriage and shedding of E. coli O157:H7 by cattle. These include animal factors (23), manure handling (24), drinking water (25), using feed additives (26), feeding probiotics (26), feeding management (27), and dietary ingredients (28–32).

Diet composition (33) has been shown to significantly alter survival, proliferation, and shedding of E. coli O157:H7 by sheep (28, 34) and cattle (32). However, the modes and sites (rumen, small intestine, or large intestine) of such actions are not identified. Recently, the diet effects on ruminal proliferation and fecal shedding of E. coli O157:H7 in inoculated cattle were investigated (26, 33). Regardless of the various daily rumen pH ranges (i.e., 5.6 to 6.6, 5.9 to 7.0, and 5.9 to 6.8) created by feeding different concentrates (corn, cottonseed/barley, or barley, respectively), E. coli O157:H7 was quickly eliminated from the rumen but persisted in the feces for 67 days (33). Similar responses were observed when high-concentrate or high-forage diets were fed (26). Therefore, it was suggested that rumen pH may have less effect on survival and proliferation of E. coli O157:H7. In these studies (26, 33), a limited number of rumen pH treatments (created by the diets fed) were tested and the response may have been masked by animal variations. Therefore, this study was designed to assess survival and proliferation of E. coli O157:H7 under rumen pH treatments of practical implication. To achieve this, eight pH treatments were selected to cover the wide range of ruminal pH under normal feeding conditions (34), which vary from high concentrate (in the feedlot) to high or all forage (under grazing conditions). The pH treatments were tested by using a dual-flow continuous culture fermenter system that mimics ruminal microbial fermentation while minimizing variations.

Materials and Methods

Animals and Collection of Rumen Fluid.
Two ruminally cannulated mature Angus steers were used as donors of rumen fluid to inoculate the dual-flow continuous culture fermenter system. The steers were gradually (over a 1-month period) adapted to a diet containing 50% forage (grass hay) and 50% concentrate (corn) on a dry matter basis and had ad libitum access to this diet for 2 weeks before collection of rumen fluid. The diet was formulated to meet or exceed nutrient requirements of the steers (35). The rumen fluid was collected (by using a vacuum pump) from each steer approximately 2 hr postfeeding and was strained immediately through four layers of cheesecloth into a prewarmed, insulated thermos.

Continuous Culture System and Operation.
The dual-flow continuous culture fermenter system was developed (36) and modified (37) to simulate differential solid-liquid removal rates occurring in the rumen environment. Evaluation and validation of the efficacy of this system in simulating ruminal fermentation in cattle (37, 38) and sheep (39, 40) were documented. The system at the University of Nevada-Reno consists of eight fermenters of 1020 ml of working volume each. Upon arrival to the laboratory, the rumen fluids from both steers were combined (on an equal volume basis) and used to inoculate the fermenters. Each fermenter was supplied daily with 75 g of dry matter of the ground (2-mm screen) diet (13.3% protein; containing, on dry matter basis, 49.6% grass hay, 35.8% corn, 11.9% soybean meal, 1.59% limestone, 0.67% Na2SO4, and 0.42% mineral/vitamin mix) by an automated feeding mechanism adjusted to deliver the diet in 12 equal portions (every 2 hr) over a 24-hr period to establish steady-state conditions. Each fermenter was continuously infused with a mineral buffer (artificial saliva) solution (41) containing urea (0.5 g/l) at a rate of 1.8 ml/min to obtain a liquid dilution rate of 10% hr-1. Solid (overflow) dilution rate was maintained at 5% hr-1 by removing liquid through a filter at 0.9 ml/min. Anaerobic conditions were achieved by continuous infusion of N2 at a rate of 40 ml/min. Maintaining the fermenters’ temperature at 39°C and mixing of their contents were achieved by using the VirTis Omni-Culture fermenter base units (The VirTis Company, Gardiner, NY). The test pH of each fermenter (i.e., 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, and 7.25 ± 0.05) was maintained by automated infusion of 3 N HCl or 5 N NaOH regulated by a pH controller (Cole-Parmer, Vernon Hills, IL).

Experimental Design.
The experimental design was a randomized complete block design (42) and consisted of three inoculation periods (blocks). The eight pH treatments were allocated at random to the eight fermenters. At the beginning of the experiment, the first 3 days were used for gradual adjustment of the microbial population in the rumen fluid (pH 6.2) inoculum to the test pH. The following 4 days were used for adjustment of the microbial population to the diet at the test pH. On Day 8, each fermenter was inoculated with 1 ml of E. coli O157:H7 (ATCC 43888) suspension (109 cells/ml). On Day 13 (5 days postinoculation), no E. coli O157:H7 was found in the fermenters even after enrichment (43). Therefore, inoculation of the fermenters with E. coli O157:H7 was repeated on Days 17 and 22 to allow for three replications for each pH treatment. No carry over effect was expected because on Day 5 of each inoculation, no E. coli O157:H7 was detected in the fermenter contents before or after enrichment.

E. coli O157:H7 Inoculum.
The E. coli O157:H7 inoculum was prepared by growing the cells in tryptic soy broth at 37°C for 10 hr and then its concentration was estimated spectrophotometrically (OD600) based on previously established growth curves. E. coli O157:H7 cells were concentrated by centrifugation at 3000g for 10 min and resuspended in sterile saline at approximately 109 cells/ml. Actual starting concentrations were confirmed by plating serial dilutions of the inoculum on tryptic soy agar.

Sample Collection.
Samples (1 ml each) were collected from each fermenter 1 hr before and every hour for 12 hr postinoculation. Samples then were collected every 2 hr for an additional 12 hr. Samples were immediately placed on ice and stored at 4°C. At the end of the 24-hr sampling period, all samples were analyzed to detect and quantify E. coli O157:H7 by using a flow cytometric method (43).

Sample Analysis.
From each sample, a 100-µl aliquot was taken and labeled with 100 µg of fluorescein (FITC)-labeled affinity purified antibody to E. coli O157:H7 (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Positive controls were pure cultures of E. coli O157:H7 (ATCC 43888). The measurements were performed with a four-color XL-MCL flow cytometer with System II software (Beckman Coulter, Fullerton, CA). The instrument was operated at the high flow rate that examines samples at approximately 40 µl/min. Because the samples contained large numbers of extraneous particles (i.e., feed or other bacterial species), the instrument was normally thresholded on green fluorescence to collect relatively larger numbers of the specifically labeled E. coli O157:H7 (43). Data files were collected for standard times and were used in combination with the known flow rates to calculate approximate cell (i.e., E. coli O157:H7) concentrations.

Statistical Analysis.
The starting concentrations of E. coli O157:H7 for the first, second, and third inoculations were confirmed by plate counts to be 1.3 x 1010, 2.9 x 1010, and 3.9 x 109 cells/ml of inoculum (i.e., 1.3 x 107, 2.9 x 107, and 3.9 x 106 cells/ml of fermenter contents), respectively. Because of these variations in the starting concentrations, the numbers of E. coli O157:H7 in every fermenter at each sampling time (measured by flow cytometry) were standardized and expressed as percentages of the initial dose. These percentages of E. coli O157:H7 remaining in the fermenters at a given time were analyzed as a repeated measure in a randomized complete block design with a factorial (pH x time) treatment structure by using Proc MIXED of SAS (44). The covariance structures (ar[1], cs, and toeplitz) among the different sampling times were compared by using the type option in SAS Proc MIXED (44). The ar(1) covariance structure was selected to model the correlations among the sampling times based on the smallest AIC value (44). The sampling time (number of hours postinoculation) was treated as quantitative, and the significance (P < 0.05) of the linear, quadratic, linear x pH interaction, and quadratic x pH interaction were tested. The percentage of E. coli O157:H7 remaining at any given time for each pH treatment was estimated from the linear and quadratic coefficients of the quadratic regression model included in the MIXED model. Significant differences (P < 0.05) among linear or quadratic coefficients for the various pH treatments were tested by using t test in the Proc MIXED solution option (44). At 12 hr postinoculation, the percentages of E. coli O157:H7 remaining in the continuous culture system for the pH tested were compared by using the least-squares means option in Proc MIXED (44). At 24 hr postinoculation, the percentage of E. coli O157:H7 remaining were also compared in a similar fashion.

Results

Preinoculation (1 hr before) samples from each of the eight fermenters were negative for E. coli O157:H7 when tested directly or after enrichment. After inoculation, the actual numbers of E. coli O157:H7 (data not shown) decreased immediately for all pH treatments except for pH 7.0. At this pH, E. coli O157:H7 numbers increased for 4 hr before starting to decrease in a fashion similar to that for the remaining pH treatments.

The regression lines depicting the decrease in E. coli O157:H7 numbers as a percentage of initial dose are illustrated in Figure 1Go. Each line represents the fitting of data collected from the three inoculations (i.e., three replications) for each pH treatment. The decrease in E. coli O157:H7 numbers was quadratic (P < 0.05) for all pH treatments. This is confirmed by the coefficients of the quadratic effects presented in Table IGo. Based on the data in Figure 1Go, the numbers of E. coli O157:H7 remaining in the fermenters decreased at a slower rate for the culture at pH 7.0 when compared with those at other pH treatments. This observation is also confirmed by the magnitude of the linear and quadratic coefficients of the quadratic model (Table IGo).



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  		Figure 1. Remaining numbers of E. coli O157:H7 (expressed as a percentage of the initial dose) in continuous cultures of rumen contents as influenced by culture pH.

 

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  		Table I. Comparisons Among the Intercepts (ß0) and Each of the Linear (ß1) and Quadratic (ß2) Sets of Coefficients (at a given time [t]) of the Quadratic Regression Modela Used to Examine the Effect of pH on the Remaining Number of E. coli O157:H7 (as a percentage of initial dose) Over Time in Continuous Cultures of Rumen Contents
 
A preliminary experiment was conducted to assess the effects of the above mentioned pH treatments on the same E. coli O157:H7 (at the same inoculation dose) used in the current study. In this preliminary experiment, samples were collected every hour and analyzed by flow cytometer. Results (data not shown) revealed that E. coli O157:H7 numbers decreased rapidly during the first 12 hr postinoculation and then decreased at a slower rate for the following 12 hr. At 24 hr postinoculation, the number of E. coli O157:H7 remaining in the fermenters fell below the sensitive range (i.e., <104 cells/ml) of E. coli O157:H7 quantification (43). Therefore, it was decided to terminate the sampling at 24 hr postinoculation.

Regardless of the pH treatment, the rates of reduction of E. coli O157:H7 numbers in the fermenters were fast during the first 12 hr postinoculation. Therefore, we attempted to estimate the percentages of E. coli O157:H7 numbers remaining in the fermenters at 12 hr postinoculation for each of the pH treatments by using the quadratic regression model (i.e., Y = ß0 - ß1 t + ß2 t2, where Y is the percentage of remaining E. coli O157:H7 numbers at t incubation time, ß0 is the Y intercept, and both ß1 and ß2 are the linear and quadratic coefficients for a specific pH treatment). The estimated percentages (data not shown) were then compared for each two pH treatments at 12 hr postinoculation. The difference (percentage units) between the estimated values for each set of two pH treatments and the significance of such difference are presented in Table IIGo. At 12 hr postinoculation (Table IIGo), only the effect of pH 7.0 was different (P < 0.05) from those of other pH treatments that were all similar (P > 0.05). This observation suggests that regardless of culture pH (except for pH 7.0), E. coli O157:H7 numbers decreased at a fast rate. Although the decrease in E. coli O157:H7 numbers at pH 7.0 during the first 12 hr postinoculation was slower than that for other pH treatments, the final levels of number reduction at 24 hr postinoculation were not different (P > 0.05; data not shown). This could be explained by the fact that rate of number reduction was faster at pH 7.0 during the second 12 hr postinoculation, whereas the rates of reduction were slower for the other pH treatments during that time.


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  		Table II. Comparisons Among Estimated Percentages of E. coli O157:H7 Remaining in Continuous Cultures of Rumen Contents for the pH Tested at 12-hr Postinoculationa
 
Discussion

The rumen environment is very complex and has evolved over many years to allow for survival and proliferation of a specific anaerobic microbial population that supports survival and production of the host animal through fermentation (45). Therefore, it is difficult for a transient organism to survive in this highly competitive environment. Within the rumen, there are many inhibitory factors (e.g., rumen pH and fermentation end-products) that limit growth of transient bacterial species.

The observation that E. coli O157:H7 could not proliferate and decreased in numbers within 24 hr in the continuous culture system at the wide pH range (i.e., 5.5–7.25) tested (Fig. 1Go) is in agreement with results from studies investigating survival of E. coli O157:H7 in vivo (26, 33). Buchko et al. (33) and Tkalcic et al. (26) reported that E. coli O157:H7 was rapidly removed from cattle with rumens of varying pH (i.e., ranging from 5.6 to 7.0).

Because fermentation of different diets results in various profiles of acid end-products that alter rumen pH, it was suggested (28, 30, 32) that dietary manipulation may affect survival and proliferation of E. coli O157:H7 in the rumen environment. However, our results (Fig. 1Go) suggest that competitive exclusion by the rumen microorganisms most probably decreased the numbers of E. coli O157:H7 within 24 hr from 106 and 107 to 104 and 105 cells/ml of artificial rumen contents, regardless of culture pH. It appears that the rumen pH, as a function of fermentation of dietary components, is less effective in decreasing the numbers of E. coli O157:H7 entering cattle rumens. Therefore, our data and those of others (26, 33) suggest that the diet effects on E. coli O157:H7 entering the gastrointestinal tract of cattle is postruminally through specific ingredients or components. Diet effects on the small or large intestinal environments may be through altering colonization, proliferation, and the subsequent shedding of this food-borne pathogen.

Footnotes

This work was supported by the U.S. Department of Agriculture Integrated Research, Education, and Extension Competitive Grants Program Grant 2001-05062. This research was also supported in part by Nevada Agricultural Experiment Station (publication no. 5302450).

1 To whom request for reprints should be addressed at Animal Biotechnology, University of Nevada, Mail Stop 202, Reno, NV 89557. E-mail: hhussein{at}agnt1.ag.unr.edu Back

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