INTRODUCTION

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During the past decade, Escherichia coli O157:H7, an important human pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, has been reported as a cause of human illness with increased frequency (1, 10, 16). Cattle, especially young animals, have been implicated as a principal reservoir of E. coli O157:H7 (4, 7, 19, 21, 22, 24, 26), with undercooked ground beef being a major vehicle of food-borne outbreaks.

A recent national survey performed by the National Animal Health Monitoring System of the U.S. Department of Agriculture revealed that 1.6% of feedlot cattle shed E. coli O157:H7 in their feces and that 0.4% shed E. coli O157:nonmotile (O157:NM) in their feces (6). A major study of calves on dairy farms revealed that 1.5 to 2.9% of animals between 24 h of age and weaning and 4.9 to 5.3% of animals between the age of weaning and 4 months shed E. coli O157:H7 in their feces (26). Experimental infection of calves and adult cattle with E. coli O157:H7 infection revealed that shedding of E. coli O157:H7 varies widely among animals of the same age group (14 to >20 weeks) but persists longer in calves than in adults, and previous infection does not prevent reinfection with the same strain of E. coli O157:H7 (5).

Many concerns have been raised regarding E. coli O157:H7 contamination of foods. Such concerns have been heightened by the tolerance of E. coli O157:H7 to acidic conditions. Proper cooking is an effective method of killing E. coli O157:H7 in foods. Insanitary practices in preparing foods often result in food-borne illness; hence, methods for reducing or eliminating the carriage of E. coli O157:H7 in cattle are needed to reduce the level of exposure to the pathogen in food and the environment (23). The purpose of this study was to isolate potential probiotic bacteria and to evaluate their efficacy at reducing the level of carriage of E. coli O157:H7 by cattle. Probiotic bacteria are those that beneficially affect the host by improving its microbial balance, including eliminating or reducing microorganisms that are carried by the host and that are harmful to humans.

	MATERIALS AND METHODS

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Source and isolation of potential probiotic bacteria. Bacteria to be screened for activity bactericidal for or inhibitory to E. coli O157:H7 were isolated from cattle feces or cattle gastrointestinal tissue (intestine and colon). Fecal samples were collected from cattle that were confirmed to be negative for E. coli O157:H7 by fecal testing (26). Fifty-five fecal samples were serially diluted (1:10) in 0.1 M phosphate buffer (phosphate-buffered saline [PBS]; pH 7.2), 0.1 ml of each dilution was plated onto sorbitol MacConkey agar (SMA), and the plates were incubated for 16 h at 37°C. Up to 10 colonies were randomly selected, and each one was transferred to a test tube containing 10 ml of Trypticase soy broth (TSB; BBL, Becton Dickinson Co., Cockeysville, Md.). Cultures were incubated for 16 h at 37°C. Sixty-eight tissue samples (1 g each in 9 ml of PBS) were homogenized individually (Ultra-Turrax T25 homogenizer; Janke & Kunkel IKA-Labortechnik, Staufen, Germany) at 9,500 rpm for 1 min, and 0.1-ml portions were plated onto the surfaces of SMA plates. The plates were incubated for 16 h at 37°C. Up to 10 colonies were each transferred to test tubes containing 10 ml of TSB, and the tubes were incubated for 16 h at 37°C.

Screening of cultures for anti-E. coli O157:H7 properties. A five-strain mixture of E. coli O157:H7 from our culture collection, including strains 932 (human isolate), C7927 (human isolate), E009 (meat isolate), E0018 (cattle isolate), and E0122 (cattle isolate), was used to screen culture supernatants for anti-E. coli O157:H7 activity. Approximately 10⁷ cells of E. coli O157:H7 of approximately equal populations of each strain in 0.1 ml were plated onto the surfaces of duplicate SMA and Trypticase soy agar (TSA) plates. Cultures were sedimented by centrifugation (4,000 × g for 20 min), and the supernatant from each culture was filter sterilized (0.2-µm-pore-size cellulose acetate membrane; Nalgene Co., Rochester, N.Y.) for determination of anti-E. coli O157:H7 properties. A disc (diameter, 12 mm; Dispens-O-Disc; Difco Laboratories, Detroit, Mich.) was placed on the surface of each SMA and TSA plate, and 0.1 ml of filter-sterilized supernatant from a single culture was applied to the surface of the disc. In addition, a disc with Vibax LA-5 (Triclosan) (1:2,000 in PBS; Benchmark Co., Salt Lake City, Utah) and another disc with 70% ethanol, which were used as positive controls, and a disc with PBS, which was used as a negative control, were applied to each plate. The cultures were incubated for 18 h at 37°C and were observed for zones of growth inhibition. Probiotic bacteria were selected as those which produced a clear zone of 1 mm or greater surrounding each disc.

Preparation of E. coli O157:H7 cultures for inoculation into calves. The same five-strain mixture of E. coli O157:H7 described above was used to inoculate calves. To facilitate enumeration of these bacterial isolates, the strains were selected for resistance to nalidixic acid (50 µg/ml) by exposure to serially increased (1:2; i.e., 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25, and 50 µg/ml) concentrations of nalidixic acid in MacConkey agar every 24 h. Each strain of nalidixic acid-resistant E. coli O157:H7 was transferred into 10 ml of TSB containing nalidixic acid (50 µg/ml) and was incubated for 16 to 18 h at 37°C with agitation (150 rpm). A 2-ml suspension of each isolate was transferred to 300 ml of TSB. After incubating at 37°C for 16 to 18 h with agitation (150 rpm), the bacteria were sedimented by centrifugation (4,000 × g for 20 min) and were washed three times in PBS. PBS was added to sedimented bacteria in an amount needed to obtain an optical density (OD) at 630 nm of 0.5 (ca. 10⁸ CFU/ml). The five isolates (2 × 10⁹ CFU of each strain) of E. coli O157:H7 were mixed in 250 ml of 2% sterilized skim milk just prior to oral inoculation of the calves. Enumeration of the bacteria was confirmed by plate counts on TSA and SMA plates containing nalidixic acid at 50 µg/ml (SMA-NA plates).

Preparation of probiotic bacteria for inoculation into calves. All 18 probiotic bacterial isolates were selected for resistance to nalidixic acid (50 µg/ml) by the procedure described above for ease of enumeration of the organisms in feces. The bacteria were grown individually in 10 ml of TSB containing nalidixic acid (50 µg/ml). A 1-ml portion of each isolate was transferred to 100 ml of TSB. After incubation at 37°C for 16 to 18 h, the bacteria were sedimented by centrifugation (4,000 × g for 20 min), washed, and adjusted to an OD at 630 nm of 0.5 by the method described above. The 18 strains of probiotic bacteria (10¹⁰ CFU), with approximately equal populations of each strain, were mixed into 250 ml of 2% sterilized skim milk immediately before oral inoculation of the calves. The bacterial population was confirmed by enumeration of serial dilutions on TSA and SMA-NA plates, in duplicate.

Preparation of calves before receipt of bacterial inoculation. Fifteen single-source male dairy calves were reared on milk replacer and were weaned at 6 weeks of age prior to transfer to the University of Georgia. The calves were housed individually in climate-controlled biolevel-2 concrete rooms. Each room had an individual floor drain and was cleaned once daily. Calves were fed a mixture of alfalfa pellets and sweet feed twice daily and had free access to water. During a 2-week conditioning period, feces from all calves were sampled by fluorescent-antibody staining for bovine diarrhea virus, coronavirus, rotavirus, E. coli pilus antigens, and cryptosporidia, and all samples tested negative. Fecal flotation for intestinal parasites and bacterial culture for Salmonella and E. coli O157:H7 were also negative prior to inoculation. After a 2-week preconditioning period, calves were surgically fitted with rumen cannulas (flexible rumen cannulas).

Rumen cannulation and surgical aftercare. Feed was withheld from the calves for 12 h. The left paralumbar fossa was clipped and scrubbed for standard surgical preparation. The fossa was anesthetized with lidocaine by using a paravertebral nerve block. A circular incision slightly smaller than the diameter of the cannula was made, and the circular piece of skin and underlying cuticular and external abdominal oblique muscles were removed. Vessels were ligated as necessary, and internal abdominal oblique muscles, transverse muscles, and the peritoneum were bluntly separated and retracted to create an opening to expose the rumen wall. The rumen wall was grasped with two large towel clamps for traction to exteriorize the rumen. The rumen wall was then sutured to the skin with no. 3 catgut, incorporating the muscle layers with a continuous suture pattern. The rumen wall was incised, and a circular piece of rumen was removed and the cannula was inserted.

Inoculation of calves with probiotic and challenge bacteria. Following a 12-h fast, six calves were inoculated via a rumen cannula with 250 ml of skim milk containing probiotic bacteria. After 48 h, the five-strain mixture of E. coli O157:H7 was inoculated via the same route. Nine control calves were challenged with the five-strain mixture of E. coli O157:H7 only. Following challenge, the calves were examined daily for clinical signs including depression, pyrexia, diarrhea, and anorexia. Rectal feces or rumen samples collected from the fistula were assayed for pH (rumen) and counts of E. coli O157:H7 and probiotic bacteria (rumen contents and feces). Six of the nine control calves (calves 2, 3, 4, 7, 8, and 9) were twice fasted for 48 h each at days 15 and 16 or days 16 and 17 and at days 22 and 23 or days 23 and 24 to provide conditions that can exhance the fecal excretion of E. coli O157:H7 (3). Similarly, four of six probiotic bacteria-treated calves (calves 1, 3, 4, and 6) were twice fasted at days 15 and 16 or days 16 and 17 and at days 22 and 23 or days 23 and 24.

Recovery of bacteria inoculated into calves. A sample of 10 g of feces or rumen content was collected by retrieval from the rectum or cannulation of the rumen daily up to the end of the study following inoculation with E. coli O157:H7. Samples were placed in a tube containing 15 ml of Cary-Blair transport medium, kept at 5°C, and transported to the Center for Food Safety and Quality Enhancement for analysis. A volume containing 1 g of feces or rumen fluid was serially (1:10) diluted in 0.85% NaCl to 10⁶, and 0.1 ml of each dilution was plated in duplicate onto SMA-NA plates. Tissue samples from sites throughout the entire gastrointestinal tract collected at necropsy (see below) were held at 5°C until analysis. Luminal contents from each segment were separated and weighed, and the tissue was rinsed with 100 ml of PBS. The rinsed tissue was added to 9 ml of PBS and was homogenized for 1 min at 9,500 rpm with an Ultra-Turrax tissue homogenizer. A 0.1-ml sample of tissue or tissue content suspension was inoculated onto SMA-NA plates in quadruplicate, and the plates were incubated at 37°C for 24 h for enumeration of E. coli O157:H7 or probiotic bacteria. If these bacteria were not detected by the direct plating method, a selective enrichment method (17) (modified TSB containing 50 µg of nalidixic acid/ml) was performed. The samples were each placed in 100 ml of selective enrichment medium and were incubated at 37°C for 24 h with agitation at 150 rpm. Dilutions of cultures were plated onto SMA-NA plates, and isolates were selected and further tested. Colonies typical of E. coli O157:H7 (sorbitol negative) were replated onto SMA-NA plates and were confirmed to be E. coli by biochemical methods and as O157 (26) and H7 (8) by serological methods. Colonies typical of probiotic bacteria (sorbitol positive) were randomly selected from plates with the highest dilution having colonies and were confirmed to be the inoculated probiotic bacteria by genomic DNA fingerprinting by a pulsed-field gel electrophoresis (PFGE) procedure (14).

Genomic fingerprinting of bacterial isolates. PFGE procedures similar to those described previously were used (14). Bacteria were grown in 10 ml of TSB at 37°C for 24 h with agitation at 200 rpm. The bacteria were sedimented by centrifugation (4,000 × g for 20 min), washed three times in 75 mM NaCl containing 25 mM EDTA at pH 7.4 (SE), and resuspended in 0.5 ml of SE. The bacterial suspension was mixed with 0.5 ml of 2% (wt/vol) low-melting-point agarose in buffer consisting of 10 mM Tris, 10 mM MgCl₂, and 0.1 mM EDTA (pH 7.5). This mixture was dispensed into sample molds, and the agarose plugs were digested with proteinase K (2 mg of proteinase K, 50 mM Tris, 50 mM EDTA, 1% N-lauroylsarcosine/ml [pH 8.0]) at 56°C overnight. The samples were washed in 10 mM Tris-5 mM EDTA (pH 7.5) and digested with 50 U of XbaI. After incubation at 37°C overnight, the reaction was stopped by the addition of 20 µl of 0.5 M EDTA. The DNA samples were electrophoresed on a 1.2% agarose gel in 0.5× TBE buffer (10× TBE buffer is 0.89 M Tris, 0.025 M EDTA, and 0.89 M boric acid) with a contour-clamped homogeneous electric field device (CHEF MAPPER; Bio-Rad). After electrophoresis for 24 h at 200 V with pulse times of 5 to 50 s and linear ramping and an electrical field angle of 120° at 14°C, the gels were stained with ethidium bromide and the bands were visualized and photographed with UV transillumination.

Necropsy of calves. The calves were killed with intravenous sodium pentobarbital. The gastrointestinal tract was clamped at the esophagus and rectum and was removed in toto. Lengths (4 to 6 cm) of duodenum, proximal, middle, and distal jejunum, proximal and distal ileum, proximal and distal cecum, proximal loop of the ascending colon, centripetal turn and centrifugal turn of the spiral colon, transverse colon, and descending colon were double tied to allow sampling of all sections for the enumeration of E. coli O157:H7 and probiotic bacteria in both the tissues and their contents with minimal cross-contamination. Sections and contents of the rumen, reticulum, omasum, and abomasum and sections of the kidney, spleen, liver, gall bladder, jejunal lymph node, ileal lymph node, cecal lymph node, and tonsil also were collected for culture and enumeration of E. coli O157:H7 and/or probiotic bacteria. Sections from all of these sites, as well as sections of prescapular lymph node, skeletal muscle, skin, tonsil, thyroid, thymus, esophagus, heart, pancreas, umbilicus, adrenal, urinary bladder, and testes, also were placed in 10% buffered formalin for histologic examination.

Histopathology of tissues. Fixed tissues were embedded in paraffin by standard methods, sectioned at 5 µm, and stained with hematoxylin and eosin by the protocol described by Brown et al. (3). Selected sections were Gram stained (3).

Statistical analysis. Significant differences between treatments were determined by using the Statistical Analysis System's t test procedure (20).

	RESULTS

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In vitro screening of potential probiotic bacteria that secrete a metabolite(s) inhibitory to E. coli O157:H7. A total of 1,200 bacterial colonies were isolated from the gastrointestinal tissues and feces of cattle determined not to excrete E. coli O157:H7 in their feces. These bacteria were screened for their ability to inhibit the growth of or kill E. coli O157:H7 in vitro, and 18 were determined to secrete antimicrobial metabolites. Among them, five colonies were isolated from feces, five were isolated from the small intestine, and eight were isolated from the colon. Seventeen of the 18 colonies were identified as E. coli and the other was identified as Proteus mirabilis (Table 1). All colonies were assayed for Shiga toxin production (26), and none produced Shiga toxin. Genomic DNA fingerprinting by PFGE revealed 13 different profiles among the 18 isolates.

Colonization of calves by probiotic bacteria. One calf was initially fed one strain of probiotic bacteria (E. coli 271 at 10¹⁰ CFU). The calf appeared to be clinically normal, and this E. coli strain was recovered by the enrichment procedure only from the contents of the ileum and the cecum at the termination of the experiment (12 days). Two calves were then fed all 18 strains (at approximately equal concentrations of each strain; 5 × 10⁸ CFU each) of probiotic bacteria (1 × 10¹⁰ CFU per calf) as a mixture. The calves' feces were of normal consistency, and the bacteria were isolated from the gastrointestinal tract (both tissue and the contents of the rumen and colon) for up to 27 days (at the termination of the study, the counts were 50 to 200 CFU/g of feces).

Efficiency of probiotic bacteria in reducing carriage of E. coli O157:H7 in calves. Of the nine control calves administered only E. coli O157:H7, all remained clinically healthy, with no evidence of fever or diarrhea. E. coli O157:H7 was isolated intermittently from the rumen fluid of all animals during the first 3 weeks postinoculation (Fig. 1). Shedding of E. coli O157:H7 in the feces at various levels was generally detected continuously for the duration of the experiment (mean, 28 days) (Fig. 2). At necropsy, E. coli O157:H7 was isolated from all nine calves, as follows: rumen contents of five of nine calves, reticulum contents of three of nine calves, omasum contents of one of nine calves, and the colons of nine of nine calves (Table 2). No pathological changes were observed in any of the tissue samples examined microscopically.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Detection of E. coli O157:H7 in rumen fluid of nine control calves administered only E. coli O157:H7. Bacterial enumeration was performed by surface plating on SMA-NA plates, in duplicate. The arrow indicates that detection of E. coli O157:H7 was by an enrichment procedure in which 9 ml of rumen fluid was positive for E. coli O157:H7.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Detection of E. coli O157:H7 in feces of nine control calves administered only E. coli O157:H7. Bacterial enumeration was performed by surface plating on SMA-NA plates, in duplicate. The arrow indicates that detection of E. coli O157:H7 was by an enrichment procedure in which 9 g of feces was positive for E. coli O157:H7.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Detection of E. coli O157:H7 in rumen fluid of six calves administered probiotic bacteria and administered E. coli O157:H7 2 days later. Bacterial enumeration was performed by surface plating on SMA-NA plates, in duplicate. The arrow indicates that detection of E. coli O157:H7 was by an enrichment procedure in which 9 ml of rumen fluid was positive for E. coli O157:H7.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Detection of E. coli O157:H7 in feces of six calves administered probiotic bacteria and administered E. coli O157:H7 2 days later. Bacterial enumeration was performed by surface plating on SMA-NA plates, in duplicate. The arrow indicates that detection of E. coli O157:H7 was by an enrichment procedure in which 9 g of feces was positive for E. coli O157:H7.

Sites of localization of probiotic bacteria. All calves treated with probiotics were necropsied between 29 and 30 days after the administration of E. coli O157:H7. Of the six calves administered probiotic bacteria 2 days before treatment with E. coli O157:H7, probiotic bacteria were recovered at necropsy from the contents of the rumens, reticulums, omasa, and colons of five of six calves. However, probiotic bacteria were not isolated at necropsy from the one animal from which E. coli O157:H7 was isolated from cecal and colonic contents at populations of 2.5 × 10³ to 5.0 × 10³ CFU/g (Tables 3 and 4).

	DISCUSSION

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Ruminants including cattle (3, 5), deer (12), and sheep (13) have been identified as carriers of E. coli O157:H7. The primary sites of E. coli O157:H7 localization in calves are the rumen and colon (3). The rumen appears to be the most important site for long-term carriage of E. coli O157:H7 because it may serve as the source of the bacteria found in the colon (3). Histologic examination of colonic tissue revealed no evidence of attachment of E. coli O157:H7 to colonic tissue. Hence, the presence of E. coli O157:H7 in the colon may be a transient state whereby the bacteria are passing through rather than colonizing the colon. In addition, changes that affect the conditions of the rumen, such as fasting, may influence the presence of E. coli O157:H7. Studies by Rasmussen et al. (18) revealed that E. coli O157:H7 could grow unrestricted in rumen fluid collected from fasted animals. Factors influencing the conditions in the rumen include nutrition, feeding regimens, and animal handling at the farm (9).

The presence of bacteria that produce metabolites inhibitory to E. coli O157:H7 at sites where O157:H7 strains localize is another factor that may influence the localization of O157:H7 in the gastrointestinal tract. Some strains of E. coli can produce colicins that are inhibitory in vitro to diarrheagenic E. coli strains, including strains of serotype O157:H7 (2, 15). Murinda et al. (15) assayed 24 E. coli colicin-producing strains and determined that all E. coli O157:H7 strains evaluated were sensitive to ColE1 to ColE8, K, and N on mitomycin C-containing agar and to ColG, ColH, and Mcc B17 on Luria agar. Colicins could be one of many metabolites produced by the probiotic bacteria in the rumen and other sections of the gastrointestinal tract. The findings from this study indicate that the probiotic bacteria localize at the same sites of calves as E. coli O157:H7 (Tables 2 and 4); hence, the anti-E. coli O157:H7 metabolites that the probiotic bacteria produce would be in the same proximity as the target bacteria.

The administration of probiotic bacteria, used in this study to treat calves prior to exposure to E. coli O157:H7, decreases the duration of ruminal carriage of E. coli O157:H7. Our studies with probiotic bacteria revealed that E. coli O157:H7 was detectable in rumen fluid an average of 14 days (range, 9 to 17 days) postinoculation in five of six animals given probiotic bacteria, whereas O157:H7 was detected in rumen fluid for an average of 26 days (range, 22 to 32 days) in control calves not receiving probiotic bacteria. At necropsy, E. coli O157:H7 was no longer detected in the rumens of six of six calves receiving probiotic bacteria. In contrast, E. coli O157:H7 was detected at necropsy in the contents of the rumen, reticulum, or omasum of seven of nine control animals.

Cray and Moon (5) reported that experimentally treated calves shed E. coli O157:H7 in their feces at least 7 weeks postinoculation. In our study, fecal shedding of E. coli O157:H7 was reduced from 25 to 32 days, when the calves were necropsied (control group), to 14 to 19 days in five of the six calves treated with probiotic bacteria. At necropsy, E. coli O157:H7 was recovered from the feces of only one of the six probiotic-treated animals, whereas it was recovered from all nine of the control group from which E. coli O157:H7 was recovered at 25 to 32 days postinoculation. The persistence of E. coli O157:H7 in one animal in the group treated with probiotic bacteria may be due to the apparent failure of probiotic bacteria to colonize this animal. It is possible that greater protection or clearance can be conferred by multiple treatments with probiotic bacteria.

Although the mechanism(s) by which our selected probiotic bacteria reduced the level of carriage of E. coli O157:H7 remains to be elucidated, these studies indicate that microbial interactions could be an important factor that contributes to the homeostasis of the bacterial flora in the gastrointestinal tract (11, 25). Hence, treatment of cattle with probiotic bacteria can reduce the level of carriage and fecal shedding of E. coli O157:H7 and may thereby reduce environmental contamination with this pathogen. Reducing E. coli O157:H7 carriage in cattle should decrease the likelihood of meat, vegetable, fruit, and water contamination, thereby decreasing the potential for food-, water-, and environment-associated E. coli O157:H7 illness in humans.