Effect of Cattle Diet on Escherichia coli O157:H7 Acid Resistance

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1999, p. 3233–3235
Vol. 65, No. 7
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
CAROLYN J. HOVDE,1* PAULA R. AUSTIN,1 KAREN A. CLOUD,1 CHRISTOPHER J. WILLIAMS,2
AND CARL W. HUNT3
Department of Microbiology, Molecular Biology, and Biochemistry,1 Division of Statistics,2 and Department of
Animal Veterinary Science,3 University of Idaho, Moscow, Idaho 83844
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 signi?cantly impacts public
longer than grain-fed cattle (Fig. 1). All animals were culture
health. Cattle transiently harbor Escherichia coli O157:H7 in
positive for E. coli O157:H7 24 h after inoculation. All animals
their gastrointestinal tracts, and many human infections result
remained healthy, and the concentration of fecal E. coli
from ingestion of contaminated bovine food products (8). E.
O157:H7 gradually decreased, until each animal became cul-
coli O157:H7 contamination, which has caused product recalls
ture negative for the bacteria. Regardless of their diet, animals
and plant closures (3), has an enormous economic impact.
shed similar titers of fecal E. coli O157:H7 that ranged from
Human infections with E. coli O157:H7 result in hemorrhagic
7.1 ? 106 CFU/g of feces to levels detectable only by selective
colitis that can progress to hemolytic-uremic syndrome, a life-
enrichment culture (?102 CFU/g of feces). The average dura-
threatening sequela that is the most common cause of acute
tion grain-fed cattle were culture positive was 4 days. In con-
renal failure in children (8). Diez-Gonzalez et al. recently
trast, cattle fed either alfalfa or grass hay diets shed fecal E.
reported that cattle fed grain diets have large numbers of
coli O157:H7 for longer times, averaging 39 and 42 days, re-
acid-resistant total generic E. coli organisms in their feces,
spectively. The difference between the durations that grain-fed
while cattle fed hay diets do not (4). They suggest that feeding
and hay-fed (grass or alfalfa) cattle were culture positive was
cattle hay diets would reduce the risk of food-borne E. coli
highly signi?cant (paired t test; P ? 0.0004).
O157:H7 infections for humans (4). In view of the apparent
Although these cattle shed E. coli O157:H7 longer when
differences between the published results of this laboratory
they were fed hay than when they were fed grain, Diez-Gonza-
(11, 12), and the conclusions of Diez-Gonzales et al. (4) and in
lez et al. (4) predicted that the pathogens from hay-fed animals
view of the consequences this issue has for public health, we
would be killed by an acid shock similar to the conditions of the
compared the durations for which hay-fed and grain-fed cattle
human stomach and would therefore pose less of a disease risk.
were culture positive for E. coli O157:H7 and assessed the acid
Central to their argument is the extrapolation that E. coli
resistance of fecal E. coli O157:H7 from both groups of cattle.
O157:H7 is similar to the generic nonpathogenic E. coli strains
Eight healthy 1- to 2-year-old Holstein steers were fed dif-
they analyzed. To determine if E. coli O157:H7 from the co-
ferent diets in a modi?ed crossover experimental design. The
lonic digesta of grain-fed ruminants is more acid resistant than
animals were fed once per day, had access to water ad libitum,
E. coli O157:H7 from the colonic digesta of hay-fed ruminants,
and were housed separately in concrete stalls with cedar-chip
we compared the acid resistance of E. coli O157:H7 shed by
bedding. The effects of four diets were assessed: typical ?nish-
the cattle on these two diets.
ing diets containing 82 to 90% grain (either 62.1% barley–
The acid resistance of E. coli O157:H7 was unaffected by the
19.3% corn or 90% corn, with the balance being forage), me-
diets (Table 1). Eight Holstein steers were put on a rotation
dium-quality 100% alfalfa hay, and low-quality 100% timothy
between diets of either grain or hay. When they had adapted to
grass hay. As expected, the grain diets were higher in energy
a diet, animals were inoculated with E. coli O157:H7. Fresh
and lower in ?ber than the hay diets, as determined by stan-
fecal samples were collected by rectal palpation on three sep-
dard techniques (data not shown) (1, 9, 10, 14). After 3 weeks
arate days during the ?rst week postinoculation, and the acid
of adaptation to a particular diet, cattle were inoculated with
resistance of E. coli O157:H7 was assessed. To prevent bacte-
E. coli O157:H7 which had been grown in Luria-Bertani broth
rial growth outside the colonic conditions, only fecal samples
at 37°C with aeration to a cell density of 109 CFU/ml. Each
containing E. coli O157:H7 in concentrations high enough to
animal received 1010 CFU of E. coli O157:H7 via a gastric tube
detect without selective enrichment culture (?102 CFU/g of
placed directly into the rumen. Fresh fecal samples were ob-
feces) were tested. E. coli O157:H7 and total coliform titers in
tained by rectal palpation every 3 to 4 days and were cultured
each sample were compared before and after acid shock and
by a highly sensitive technique to monitor shedding of E. coli
were expressed as percent survival. To mimic exposure to
O157, as previously described (11, 12).
stomach acid, conditions similar to those employed by Diez-
Hay-fed (grass or alfalfa) cattle shed fecal E. coli O157:H7
Gonzalez et al. (4) were used, except that we modi?ed a non-
enrichment culture procedure (12). Fecal samples were sus-
pended in Trypticase soy broth, pH 2.0, incubated 1 h at 37°C,
* Corresponding author. Mailing address: Department of Microbi-
ology, Molecular Biology, and Biochemistry, University of Idaho, Mos-
and neutralized by dilution. Bacteria were enumerated by plate
cow, ID 83844. Phone: (208) 885-5906. Fax: (208) 885-6518. E-mail:
count. Although there was variation in E. coli O157:H7 percent
cbohach@uidaho.edu.
survival among the isolates obtained from the animals, there
3233

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3234
HOVDE ET AL.
APPL. ENVIRON. MICROBIOL.
mals 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.
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 signi?cance
of acid-resistant E. coli O157:H7 in bovine feces is question-
able. For example, humans rarely, if ever, ingest E. coli
O157:H7 directly from the colonic digesta of cattle. The bac-
teria 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 hu-
mans (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
FIG. 1. Effect of diet on the duration for which E. coli O157:H7 is shed from
E. coli O157:H7 ingested by humans is likely to have replicated
cattle. Shown is the ?rst day that each of the cattle tested culture negative for E.
outside the bovine colon and is likely to be on the surface of
coli O157:H7 while being fed grain or hay (alfalfa or grass). Each dot represents
food when ingested, we compared the acid resistance of E. coli
an individual animal. The inoculum was E. coli O157:H7 strain ATCC 43894
O157:H7 from the hay-fed cattle with that from the grain-fed
(American Type Culture Collection, Manassas, Va.). Animals were considered
culture negative for the bacteria after three consecutive culture-negative analy-
cattle under similar conditions (15).
ses, spanning 10 days.
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
was consistently less than 1 log of E. coli O157:H7 death, and
the laboratory (at the original isolation from feces and once
the difference between these two groups was insigni?cant (P ?
before inoculation onto the ground beef) before testing. Iso-
0.77) (Table 1). In fact, E. coli O157:H7 isolates from two
lates were placed on the surface of the ground beef, the inoc-
hay-fed animals (animals 2 and 3) were completely resistant to
ulated ground beef was suspended in Luria-Bertani broth ad-
acid shock under the conditions used. The same analyses on
justed to pH 2.0 for 1 h at 37°C, and the percents survival were
two subsequent days were similar and did not show signi?cant
calculated following the acid shock procedure in reference 15,
differences in E. coli O157:H7 acid resistance (P ? 0.17 and
except that the pH used in this study was 2.0. Percentages
0.69; data not shown). Like Diez-Gonzalez et al. (4), we de-
greater than 100 resulted from the recovery of higher E. coli
termined that nonpathogenic coliforms from hay-fed cattle
O157:H7 titers after pH 2.0 treatment than after pH 7.0 treat-
were more sensitive to acid shock than the coliforms from
ment. The percents survival for E. coli O157:H7 strain ATCC
grain-fed animals (P ? 0.0024). The mean percents coliform
43894 from grain- and hay-fed cattle were 84 and 110, respec-
survival from three sampling days were 50% for hay-fed ani-
tively. 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 ?
TABLE 1. Acid resistance of E. coli O157:H7 from grain-fed and
10%). Interestingly, even E. coli O157:H7 ATCC 43894, which
hay-fed cattle
had previously been reported to be acid sensitive (2), was acid
resistant under these conditions.
% E. coli survival in cattle feda:
Animal no.
Proper preharvest cattle management has the potential to
Grain
Hay
signi?cantly reduce the risk of E. coli O157:H7 human disease
(14). This laboratory has previously shown that hay diets and
1
75
29
abrupt dietary changes extend the duration for which culture-
2
34
100
3
12
100
positive sheep shed E. coli O157:H7 (11, 12). Similarly, the
4
10
15
results of this study con?rm that cattle shed E. coli O157:H7
5
82
23
longer when they are fed hay than when they are fed grain.
6
22
29
Although this diet effect is clear in experimentally inoculated
7
34
19
animals, there has been no epidemiological report showing a
8
23
20
correlation between the incidence of E. coli O157:H7 culture-
positive cattle and their diet (6, 7). Perhaps grain-fed animals
Mean
37
42
are in more heavily contaminated environments or in environ-
a The grain diet contained 90% corn–10% alfalfa silage. The hay diet con-
ments that promote transmission of infection so that differ-
tained 100% timothy. The average pH of the colonic digesta from animals fed
ences in incidence are masked by more frequent reinfection.
grain was 5.5, and the average pH of the colonic digesta from animals fed hay was
Also, the breed, age, or number of animals analyzed could
7.2; both are consistent with the digesta pH values reported in reference 4.
Percents survival were calculated from cultures on differential sorbitol MacCon-
in?uence any inoculation-type study. Since we show here that
key agar supplemented with 4-methylumbelliferyl-?-D-glucuronide (100 mg/liter)
the acid resistance of E. coli O157:H7 is not affected by the diet
before and after acid shock at pH 2.0 for 1 h at 37°C. Presumptive E. coli
of cattle, we caution against preharvest management that in-
O157:H7 isolates were con?rmed serologically. The inoculum was E. coli
cludes an abrupt dietary change from grain to hay. Rather than
O157:H7 strain ATCC 43895 (American Type Culture Collection, Manassas,
Va.). All animals were tested on three separate days; data from one typical
reduce the risk, this change may increase the likelihood that E.
sampling day are shown.
coli O157:H7 culture-positive cattle will enter our food chain.

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VOL. 65, 1999
HAY FEEDING AND ACID RESISTANCE OF E. COLI O157
3235
More extensive scienti?c information about the relationship
sale as ground beef. Int. J. Food Microbiol. 18:321–332.
between the diet of cattle and E. coli O157:H7 is needed before
6. Hancock, D. D., D. H. Rice, D. E. Herriott, T. E. Besser, and E. Ebel. 1997.
management changes are advocated.
Effects of farm manure handling practices on Escherichia coli O157 preva-
lence in cattle. J. Food Prot. 60:363–366.
7. Hancock, D. D., D. H. Rice, L. A. Thomas, D. A. Dargatz, and T. E. Besser.
This work was supported in part by the Idaho Agriculture Experi-
1997. Epidemiology of Escherichia coli O157 in feedlot cattle. J. Food Prot.
ment Station, U.S. Department of Agriculture NRICGP grant 95-
60:462–465.
37201-1979, Public Health Service grant AI33981 from the National
8. Kaper, J. B., and A. D. O’Brien (ed.). 1998. Escherichia coli O157:H7 and
Institutes of Health, and a grant from the Idaho Beef Council.
other Shiga toxin-producing E. coli strains, ASM Press, p. 1–81. Washington,
We thank Jody McKenna for technical assistance and David Breed-
D.C.
ing for assistance in animal handling. We acknowledge Dennis Falk
9. Komarek, A. R. 1993. A ?lter bag procedure for improved ef?ciency of ?ber
and Steven Maki for animal facilities.
analysis. J. Dairy Sci. 76:250.
10. Komarek, A. R. 1993. Improved ef?ciency of ADF analysis using a ?lter bag
procedure. J. Anim. Sci. 71:284.
REFERENCES
11. Kudva, I. T., P. G. Hat?eld, and C. J. Hovde. 1995. Effect of diet on the
1. Association of Of?cial Analytical Chemists. 1990. Of?cial methods of anal-
shedding of Escherichia coli O157:H7 in a sheep model. Appl. Environ.
ysis of the Association of Of?cial Analytical Chemists, 15th ed., p. 82. As-
Microbiol. 61:1363–1370.
sociation of Of?cial Analytical Chemists, Washington, D.C.
12. Kudva, I. T., C. W. Hunt, C. J. Williams, U. M. Nance, and C. J. Hovde. 1997.
2. Benjamin, M. M., and A. R. Datta. 1995. Acid tolerance of enterohemor-
Evaluation of dietary in?uences on Escherichia coli O157:H7 shedding by
rhagic Escherichia coli. Appl. Environ. Microbiol. 61:1669–1672.
sheep. Appl. Environ. Microbiol. 63:3878–3886.
3. Centers for Disease Control and Prevention. 1997. Escherichia coli O157:H7
13. Smulders, F. J. (ed.). 1987. Elimination of pathogenic organisms from meat
infections associated with eating a nationally distributed brand of frozen
and poultry, p. 221–233. Elsevier Science Publishing, Inc., New York, N.Y.
ground beef patties and burgers—Colorado. Morbid. Mortal. Weekly Rep.
14. Van Soest, P. J., J. B. Robertson, and B. A. Lews. 1991. Methods of dietary
43:777.
?ber, neutral detergent ?ber and nonstarch polysaccharides in relation to
4. Diez-Gonzalez, F., T. R. Callaway, M. J. Kizoulis, and J. B. Russell. 1998.
animal nutrition. J. Dairy Sci. 74:3583.
Grain feeding and the dissemination of acid-resistant Escherichia coli from
15. Waterman, S. R., and P. L. C. Small. 1998. Acid-sensitive enteric pathogens
cattle. Science 281:1666–1668.
are protected from killing under extremely acidic conditions of pH 2.5 when
5. Gill, C. O., and C. McGinnis. 1993. Changes in the micro?ora on commercial
they are inoculated onto certain solid food sources. Appl. Environ. Micro-
beef trimmings during their collection, distribution and preparation for retail
biol. 64:3882–3886.

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