Grain Feeding and the Dissemination of Acid-Resistant Escherichia coli from Cattle

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Science  11 Sep 1998:
Vol. 281, Issue 5383, pp. 1666-1668
DOI: 10.1126/science.281.5383.1666


The gastric stomach of humans is a barrier to food-borne pathogens, but Escherichia coli can survive at pH 2.0 if it is grown under mildly acidic conditions. Cattle are a natural reservoir for pathogenic E. coli, and cattle fed mostly grain had lower colonic pH and more acid-resistant E. coli than cattle fed only hay. On the basis of numbers and survival after acid shock, cattle that were fed grain had 106-fold more acid-resistantE. coli than cattle fed hay, but a brief period of hay feeding decreased the acid-resistant count substantially.

Foods can be cooked or irradiated to kill bacteria, but there are ∼30 million food-borne illnesses each year in the United States (1). A variety of hypotheses have been formulated to explain the increased incidence of food-borne illness (2, 3). Modern societies tend to “dine out” more often and consume more processed food. Modern detection methods for pathogenic bacteria are more sensitive, and this sensitivity has heightened our awareness of the problem (4). Some experts have suspected a more rapid evolution of bacterial virulence factors, but this evolution is poorly understood (5).

Although Escherichia coli is a normal inhabitant of the gastrointestinal tract, some strains (for example, O157:H7) produce toxins and are pathogenic (6). Hamburger has frequently been contaminated with pathogenic E. coli, and vegetables and fruit juices have also been sources of infection (4). Cattle, a natural reservoir for pathogenic strains, have often been implicated in E. coli infection (7). It is virtually impossible to prevent all fecal contamination of meat at slaughter, and vegetables are sometimes fertilized with cattle manure. The ability of E. coli to cause food-borne illness is enhanced by its low infective dose, and as few as 10 cells can cause infection (8).

The ability of bacteria to act as food-borne pathogens depends on their capacity to survive the low pH of the gastric stomach and to colonize the intestinal tract of humans (9), but the role of acid resistance in the dissemination of pathogenic bacteria has often been ignored. Pathogenic and nonpathogenic E. coli cultures develop extreme acid resistance only when they are grown at mildly acidic pH. If E. coli is grown at neutral pH, it is acid sensitive and killed by the low pH of gastric juice (10).

Since the Second World War, fattening beef cattle in the United States have been fed large amounts of grain (starch) and very little hay (11), but the impact of grain feeding on acid-resistantE. coli had not been examined. Many forms of starch pass through the pregastric stomach (rumen) to the intestines (11), and cattle are deficient in the starch-degrading enzyme, amylase (12). Starch can be fermented in the colon, and E. coli ferments maltose, an extracellular degradation product of starch (13). Starch fermentation in the colon produces volatile fatty acids (acetate, butyrate, and propionate) that decrease pH (12).

To determine the potential impact of grain feeding on E. coli in cattle, we removed colonic digesta from the rectums of cattle that were fed hay, grass, and varying amounts of rolled corn. Digesta were diluted 10-fold with sterile anaerobic water and mixed vigorously with a vortex mixer for 1 min. The pH was measured with a combination electrode. Coliforms were enumerated by visually monitoring turbidity after serial dilution in lauryl sulfate broth (14). Escherichia coli was determined by screening the bacteria on the basis of lactose fermentation, gas production, indole production, the methyl red reaction, Voges-Proskauer test, and citrate fermentation (14). Acid shock was performed by diluting digesta samples 100-fold into Luria broth that had been adjusted to pH 2.0 (14). After 1.0 hour at pH 2.0, viable cell numbers were determined by serially diluting into lauryl sulfate broth.

A survey of 61 cattle indicated that grain supplementation could increase total and acid-resistant E. coli numbers (Table 1). Cattle fed either hay or fresh grass (pasture) had a colonic pH greater than 7.0, the total E. coli count was only 20,000 cells per gram, and virtually all of these bacteria were killed by an acid shock that mimicked the pH of gastric juice. Moderate amounts of grain (60% of dry matter) did not cause a statistically significant decrease in pH (P < 0.05), but the total E. coli population was 6.3 × 106 viable cells per gram of digesta. Some of the E. coli were killed by acid shock, but the acid-resistant count was greater than 25,000 viable cells per gram. When animals were fed more than 80% grain, the pH was significantly lower (P < 0.05), and the acid-resistant E. coli count was 250,000 viable cells per gram.

Table 1

The effect of grain feeding on the colonic pH andE. coli counts of cattle fed various amounts of grain.

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To define more precisely the role of grain in promoting the growth of acid-resistant E. coli, we performed highly controlled experiments. Mature, nonlactating Holstein cows were surgically modified so that ruminal contents could be removed directly from the rumen (IACUC protocol 95-1-97). Cattle of similar size (600 kg) were fed every 2 hours with a rotary feeder (10 kg of dry matter per day). The feeds used were medium-quality timothy hay (14% crude protein, 40% neutral detergent fiber) and a grain mixture [89% rolled (cracked) corn and 11% soybean meal]. The diets were 0, 45, and 90% grain with the remainder being hay.

Samples of digesta were obtained from the rumen as well as the colon. Ruminal contents were squeezed through cheesecloth and purged with oxygen-free carbon dioxide. Colonic samples were processed as described above. Samples were centrifuged at 13,000g for 10 min to remove bacteria and feed particles, and fermentation acids were analyzed by high-pressure liquid chromatography. The total count of anaerobic bacteria was determined by serially diluting the digesta in a nonselective medium designed for strictly anaerobic bacteria (15). Samples of colonic digesta were processed as described above. E. coli strains arising from isolated colonies were obtained from MacConkey's plates supplemented with sorbitol as an energy source. E. coli strains isolated from cattle andE. coli O157:H7 were given an even longer acid shock (6 hours), and in this case the recovery medium was Luria broth.

The randomized block design was a 3 × 3 Latin square (three animals × three diets) with 14 days of adaptation and 4 days of sample collection (total of 54 days). Because the animals were mature, and the environment of the barn was carefully controlled, the effect of time was judged to be inconsequential. The data were first analyzed by two-way analysis of variance (diet versus animal), and the Fvalues indicated that P < 0.05 in all cases. Student-Newman-Keuls test (16) was used to estimate differences among means, and the variance estimates were pooled (17).

When cattle were fed increasing amounts of grain, the volatile fatty acid (acetic, propionic, and butyric) concentration of the rumen did not increase significantly (P > 0.05), but the concentration in the colon increased ∼fourfold (P < 0.05) (Fig. 1A). Under these conditions, ruminal pH remained essentially constant (P > 0.05), but the pH of the colon decreased (P < 0.05) when the volatile fatty acids accumulated (Fig. 1B). Lactic and succinic acids were never detected in rumen samples, but small amounts of both acids were observed in colon samples when 90% grain was fed. Grain supplementation had little effect on the numbers of anaerobic bacteria in the rumen, but the colon count increased 1000-fold (Fig. 2A). Hay-fed cattle had less than 105 colonic coliforms, but those fed 90% grain had ∼108 coliforms per gram of digesta (Fig. 2B). Only a small fraction of the ruminal coliforms were E. coli, but virtually all of the colonic coliforms were identified as E. coli (Fig. 2C).

Figure 1

The effect of grain feeding (percentage of diet dry matter) on (A) the volatile fatty acid concentration of the rumen and colon and (B) pH. The error bars indicate standard deviations of the mean (three animals, four sampling days).

Figure 2

The effect of grain feeding (percentage of diet dry matter) on (A) total anaerobes, (B) coliforms, and (C) coliforms that were identified as E. coli. The error bars indicate standard deviations of the mean (three animals, four sampling days).

Hay-fed cattle had a low concentration of volatile fatty acids in their colons (Fig. 1B), and acid shock killed more than 99.99% of theE. coli (Fig. 3A). When diets were supplemented with either 45 or 90% grain, acids accumulated, colonic pH declined (Fig. 1B), and a much larger percentage of theE. coli survived acid shock (Fig. 3B). The idea that grain, by promoting acid production in the colon, was regulating acid resistance in vivo, was corroborated by in vitro experiments. WhenE. coli strains isolated from the cattle were grown in the laboratory with a high concentration of glucose, acetic acid accumulated in the medium, pH declined, and the cell survival after acid shock was high (Fig. 3B). If the glucose concentration of the medium was low, little acid was produced, and cell survival was extremely low. Strains isolated from cattle fed forage or grain, andE. coli O157:H7 (ATCC 43895, CDC EDL 933) behaved similarly, and this result indicated that grain feeding was inducing acid resistance rather than selecting a different population of E. coli.

Figure 3

(A) The survival of E. coli in colonic fluid (percentage of initial count) after acid shock (pH 2.0, Luria broth, 1 hour), and (B) the effect of glucose and final pH on the survival of colonic E. coli isolates (hay versus grain) and E. coli O157:H7 after acid shock (pH 2.0, Luria broth 6 hours). When the cultures were cultivated overnight in broth containing large amounts of glucose (10 mg solids/ml) (filled bars), the final pH was 4.8. Cultures with small amounts of glucose (0.5 mg/ml) (open columns) produced less acid and the final pH was 6.8. The error bars indicate standard deviations of the mean (10 strains, two replicates per strain).

About 5% of our E. coli isolates (n = 155) were sorbitol negative, a diagnostic trait of O157:H7 (14), but none of these strains tested positive for O157:H7 antigens (18). The absence of E. coli O157:H7 in our cattle is not surprising. Previous workers have noted that nonpathogenic E. coli can often outgrow pathogenic strains, and this point is illustrated by at least three observations: (i) The percentage of O157:H7-positive animals in herds directly linked to outbreaks was less than 2% (19); (ii) even cattle experimentally inoculated with E. coli O157:H7 did not shed the bacterium for long periods of time (20); and (iii)E. coli O157:H7 numbers can be reduced by giving animals doses of nonpathogenic E. coli (21).

The finding that grain feeding increased both the number and acid resistance of E. coli in cattle could have significant implications for food safety. Although not all E. coli are pathogenic, there is always the risk that at least some cattle will harbor pathogenic strains. Acid resistance appears to be a factor in the dissemination (transmission) of E. coli from cattle to humans. Therefore, it is reasonable to suggest that the induction of acid resistance could increase the risk of food-borne illness. Our studies indicated that the time needed to decrease E. colinumbers was relatively short (Fig. 4A). Cattle adapted to a 90% grain diet had an acid-resistant E. coli count greater than 106 viable cells per gram. After change to a hay diet, the viable cell number immediately declined, and after 5 days the E. coli population was nearly 106-fold lower (Fig. 4B).

Figure 4

The effect of hay on the total numbers of colonic E. coli in cattle that had been consuming the 90% grain diet. (A) Cattle were switched from 90% grain to hay on day zero. (B) The numbers of E. coli that were able to survival acid shock (pH 2.0, Luria broth, 1 hour). The bars indicate standard deviations of the mean (three animals, one replicate per animal, two independent experiments). The dotted lines show the detection limit of our enumerations.

Grain feeding is a practice that promotes the production and efficiency of cattle production, and it is unlikely that American cattle will ever be fed diets consisting only of hay. However, our studies indicate that cattle could be given hay for a brief period immediately before slaughter to significantly reduce the risk of food-borne E. coli infection.

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