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Applied and Environmental Microbiology, July 1999, p. 3233-3235, Vol. 65, No. 7
Department of Microbiology,
Received 10 February 1999/Accepted 6 April 1999
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.
Preharvest cattle management
significantly impacts public health. Cattle transiently harbor
Escherichia coli O157:H7 in their gastrointestinal tracts,
and many human infections result from ingestion of contaminated bovine
food products (8). E. coli O157:H7 contamination,
which has caused product recalls and plant closures (3), has
an enormous economic impact. Human infections with E. coli
O157:H7 result in hemorrhagic colitis that can progress to
hemolytic-uremic syndrome, a life-threatening sequela that is the most
common cause of acute renal failure in children (8). Diez-Gonzalez et al. recently reported that cattle fed grain diets have
large numbers of acid-resistant total generic E. coli
organisms in their feces, while cattle fed hay diets do not
(4). They suggest that feeding cattle hay diets would reduce
the risk of food-borne E. coli O157:H7 infections for humans
(4). In view of the apparent differences between the
published results of this laboratory (11, 12), and the
conclusions of Diez-Gonzales et al. (4) and in view of the
consequences this issue has for public health, we compared the
durations for which hay-fed and grain-fed cattle were culture positive
for E. coli O157:H7 and assessed the acid resistance of
fecal E. coli O157:H7 from both groups of cattle.
Eight healthy 1- to 2-year-old Holstein steers were fed different diets
in a modified crossover experimental design. The animals were fed once
per day, had access to water ad libitum, and were housed separately in
concrete stalls with cedar-chip bedding. The effects of four diets were
assessed: typical finishing diets containing 82 to 90% grain (either
62.1% barley-19.3% corn or 90% corn, with the balance being
forage), medium-quality 100% alfalfa hay, and low-quality 100%
timothy grass hay. As expected, the grain diets were higher in energy
and lower in fiber than the hay diets, as determined by standard
techniques (data not shown) (1, 9, 10, 14). After 3 weeks of
adaptation to a particular diet, cattle were inoculated with E. coli O157:H7 which had been grown in Luria-Bertani broth at 37°C
with aeration to a cell density of 109 CFU/ml. Each animal
received 1010 CFU of E. coli O157:H7 via a
gastric tube placed directly into the rumen. Fresh fecal samples were
obtained by rectal palpation every 3 to 4 days and were cultured by a
highly sensitive technique to monitor shedding of E. coli
O157, as previously described (11, 12).
Hay-fed (grass or alfalfa) cattle shed fecal E. coli O157:H7
longer than grain-fed cattle (Fig. 1).
All animals were culture positive for E. coli O157:H7
24 h after inoculation. All animals remained healthy, and the
concentration of fecal E. coli O157:H7 gradually decreased,
until each animal became culture negative for the bacteria. Regardless
of their diet, animals shed similar titers of fecal E. coli
O157:H7 that ranged from 7.1 × 106 CFU/g of feces to
levels detectable only by selective enrichment culture
(<102 CFU/g of feces). The average duration grain-fed
cattle were culture positive was 4 days. In contrast, cattle fed either
alfalfa or grass hay diets shed fecal E. coli O157:H7 for
longer times, averaging 39 and 42 days, respectively. The difference
between the durations that grain-fed and hay-fed (grass or alfalfa)
cattle were culture positive was highly significant (paired
t test; P = 0.0004).
Although these cattle shed E. coli O157:H7 longer when they
were fed hay than when they were fed grain, Diez-Gonzalez et al. (4) predicted that the pathogens from hay-fed animals would be killed by an acid shock similar to the conditions of the human stomach and would therefore pose less of a disease risk. Central to
their argument is the extrapolation that E. coli O157:H7 is similar to the generic nonpathogenic E. coli strains they
analyzed. To determine if E. coli O157:H7 from the colonic
digesta of grain-fed ruminants is more acid resistant than E. coli O157:H7 from the colonic digesta of hay-fed ruminants, we
compared the acid resistance of E. coli O157:H7 shed by the
cattle on these two diets.
The acid resistance of E. coli O157:H7 was unaffected by the
diets (Table 1). Eight Holstein steers
were put on a rotation between diets of either grain or hay. When they
had adapted to a diet, animals were inoculated with E. coli
O157:H7. Fresh fecal samples were collected by rectal palpation on
three separate days during the first week postinoculation, and the acid
resistance of E. coli O157:H7 was assessed. To prevent
bacterial growth outside the colonic conditions, only fecal samples
containing E. coli O157:H7 in concentrations high enough to
detect without selective enrichment culture (>102 CFU/g of
feces) were tested. E. coli O157:H7 and total coliform titers in each sample were compared before and after acid shock and
were expressed as percent survival. To mimic exposure to stomach acid,
conditions similar to those employed by Diez-Gonzalez et al.
(4) were used, except that we modified a nonenrichment culture procedure (12). Fecal samples were suspended in
Trypticase soy broth, pH 2.0, incubated 1 h at 37°C, and
neutralized by dilution. Bacteria were enumerated by plate count.
Although there was variation in E. coli O157:H7 percent
survival among the isolates obtained from the animals, there was
consistently less than 1 log of E. coli O157:H7 death, and
the difference between these two groups was insignificant (P = 0.77) (Table 1). In fact, E. coli O157:H7 isolates
from two hay-fed animals (animals 2 and 3) were completely resistant to
acid shock under the conditions used. The same analyses on two
subsequent days were similar and did not show significant differences
in E. coli O157:H7 acid resistance (P = 0.17
and 0.69; data not shown). Like Diez-Gonzalez et al. (4), we
determined that nonpathogenic coliforms from hay-fed cattle were more
sensitive to acid shock than the coliforms from grain-fed animals
(P = 0.0024). The mean percents coliform survival from
three sampling days were 50% for hay-fed animals and 86% for
grain-fed animals. However, this difference was small compared to the
>3-log10-fold difference reported by Diez-Gonzalez et al.
(4); it may be due to the differences in the methods of acid
exposure or bacterial enumeration.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effect of Cattle Diet on Escherichia
coli O157:H7 Acid Resistance
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FIG. 1.
Effect of diet on the duration for which E. coli O157:H7 is shed from cattle. Shown is the first day that each
of the cattle tested culture negative for E. coli O157:H7
while being fed grain or hay (alfalfa or grass). Each dot represents an
individual animal. The inoculum was E. coli O157:H7 strain
ATCC 43894 (American Type Culture Collection, Manassas, Va.). Animals
were considered culture negative for the bacteria after three
consecutive culture-negative analyses, spanning 10 days.
TABLE 1.
Acid resistance of E. coli O157:H7 from
grain-fed and hay-fed cattle
Despite the fact that acidic contents of the human stomach provide some defense against pathogens and that acid-resistant E. coli O157:H7 isolates are more likely to survive passage through the acidic conditions of the stomach, the significance of acid-resistant E. coli O157:H7 in bovine feces is questionable. For example, humans rarely, if ever, ingest E. coli O157:H7 directly from the colonic digesta of cattle. The bacteria very likely replicate outside the bovine colon on the hides of the animals, in manure, in low-nutrient environments, or in contaminated food or water before they are ingested by humans (5, 13). In addition, Waterman and Small (15) recently showed that E. coli O157:H7 and other enteric pathogens may be protected from killing under extreme acidic conditions when they are on solid food surfaces. Bacteria inoculated onto the surface of ground beef survived acid shock conditions that a pure culture of the same organism did not (15). Because the E. coli O157:H7 ingested by humans is likely to have replicated outside the bovine colon and is likely to be on the surface of food when ingested, we compared the acid resistance of E. coli O157:H7 from the hay-fed cattle with that from the grain-fed cattle under similar conditions (15).
E. coli O157:H7 isolates from grain- or hay-fed cattle were acid resistant when they were placed on the surface of ground beef. Isolates from cattle feces were minimally subcultured in the laboratory (at the original isolation from feces and once before inoculation onto the ground beef) before testing. Isolates were placed on the surface of the ground beef, the inoculated ground beef was suspended in Luria-Bertani broth adjusted to pH 2.0 for 1 h at 37°C, and the percents survival were calculated following the acid shock procedure in reference 15, except that the pH used in this study was 2.0. Percentages greater than 100 resulted from the recovery of higher E. coli O157:H7 titers after pH 2.0 treatment than after pH 7.0 treatment. The percents survival for E. coli O157:H7 strain ATCC 43894 from grain- and hay-fed cattle were 84 and 110, respectively. For E. coli ATCC 43895 from grain- and hay-fed cattle, the percentages were 133 and 100, respectively. Data are the averages of three separate experiments (standard error = 10%). Interestingly, even E. coli O157:H7 ATCC 43894, which had previously been reported to be acid sensitive (2), was acid resistant under these conditions.
Proper preharvest cattle management has the potential to significantly reduce the risk of E. coli O157:H7 human disease (14). This laboratory has previously shown that hay diets and abrupt dietary changes extend the duration for which culture-positive sheep shed E. coli O157:H7 (11, 12). Similarly, the results of this study confirm that cattle shed E. coli O157:H7 longer when they are fed hay than when they are fed grain. Although this diet effect is clear in experimentally inoculated animals, there has been no epidemiological report showing a correlation between the incidence of E. coli O157:H7 culture-positive cattle and their diet (6, 7). Perhaps grain-fed animals are in more heavily contaminated environments or in environments that promote transmission of infection so that differences in incidence are masked by more frequent reinfection. Also, the breed, age, or number of animals analyzed could influence any inoculation-type study. Since we show here that the acid resistance of E. coli O157:H7 is not affected by the diet of cattle, we caution against preharvest management that includes an abrupt dietary change from grain to hay. Rather than reduce the risk, this change may increase the likelihood that E. coli O157:H7 culture-positive cattle will enter our food chain. More extensive scientific information about the relationship between the diet of cattle and E. coli O157:H7 is needed before management changes are advocated.
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ACKNOWLEDGMENTS |
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This work was supported in part by the Idaho Agriculture Experiment Station, U.S. Department of Agriculture NRICGP grant 95-37201-1979, Public Health Service grant AI33981 from the National Institutes of Health, and a grant from the Idaho Beef Council.
We thank Jody McKenna for technical assistance and David Breeding for assistance in animal handling. We acknowledge Dennis Falk and Steven Maki for animal facilities.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844. Phone: (208) 885-5906. Fax: (208) 885-6518. E-mail: cbohach{at}uidaho.edu.
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REFERENCES |
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