A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios >10 2 terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10 10 PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be >10 2 . In addition, phages were maintained at 10 6 PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.Since its association with disease in 1982 (33), Escherichia coli O157:H7 has become a worldwide threat to public health and is one of today's most troubling food-borne pathogens. Human illness with E. coli O157:H7 ranges from self-limited watery diarrhea and hemorrhagic colitis to life-threatening manifestations such as the hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Cattle and sheep, important domestic ruminants, are the primary reservoirs for this human pathogen and are the most common sources for foodborne, waterborne, and direct-animal-contact infections (9-11, 18, 48, 51). In contrast to humans, the carrier ruminants remain healthy, and E. coli serotype O157:H7 is a transient member of the ruminant gastrointestinal flora. Persistence of E. coli O157:H7 carriage in naturally and experimentally infected animals varies from days to months (6,8,12,13,19,35,38).Successful strategies to control or reduce the carriage and prevalence of E. coli O157:H7 in live ruminants would reduce the risk of human exposure to this pathogen. There is currently no reliable intervention or animal vaccine available to curb carriage of E. coli O157:H7. Probiotics aimed at creating an intestinal microenvironment inhibitory to E. coli O157:H7 have been tested, but without consistent success (14,22,28,29,50). A lytic bacteriophage that would specifically target the E. coli O157 serotype is another appealing approach because phage therapy has been successful in animal trials against a broad range of bacterial pathogen...

The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ϳ92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, ⌬tir, and ⌬eae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.Enterohemorrhagic Escherichia coli (EHEC) strains are a subset of Shiga toxin-producing E. coli (STEC) that can cause human disease and are threats to public health worldwide (46,49). Human illnesses caused by EHEC range from self-limiting watery diarrhea or hemorrhagic colitis to life-threatening sequelae, the hemolytic-uremic syndrome or thrombotic thrombocytopenic purpura. The predominant EHEC serotype associated with the most severe disease in North America, the United Kingdom, and Japan is O157:H7 (23,42,44,46,59).Cattle are considered the primary reservoir for E. coli O157:H7 and the most common source for food-borne and direct animal contact infections (5, 25, 69). Healthy cattle carry E. coli O157:H7 transiently without suffering pathological symptoms (2,4,26). Individual animals can passively shed E. coli O157:H7 in their feces for a short time (a few days) without establishing a colonized state or can pass fecal E. coli O157:H7 for a longer time (a month or more) if the bacteria colonize and persist (22). The conditions that lead to these different host-bacterium interactions are not understood.It is well accepted that reducing the carriage or prevalence of E. coli O157:H7 in cattle would reduce the risk of human exposure to this pathogen (61). Recently, the recto-anal junction (RAJ) mucosa was identified as the primary site of E. coli O157:H7 colonizatio...

Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening complications. Because healthy cattle are reservoirs for the bacterium, ruminant infection models have applications in analyzing the relationship between cattle and this human pathogen and in testing interventions to reduce or prevent bovine colonization with this bacterium. Current approaches often do not reliably mimic natural, long-term bovine colonization with E. coli O157:H7 in older calves and adult animals (ages that enter our food chain). Based on the recent identification of the bovine rectoanal junction mucosa as a site of E. coli O157:H7 colonization, we developed a novel rectal swab administration colonization model. We compared this method with oral dosing and direct contact transmission (Trojan) methods. E. coli O157:H7 carriage status was determined by fecal or rectoanal mucosa swab culture. High (ϳ10 10 CFU) and low (ϳ10 7 CFU) oral doses of E. coli O157:H7 in sheep and cattle resulted in variable infection with the bacterium. Some animals became colonized with the bacteria and remained culture positive for several weeks, and some animals did not become colonized and rapidly cleared the bacteria in a few days. Pen mates of E. coli O157:H7 culture-positive Trojan cattle had a low infection rate and variable colonization status. However, rectal swab administration of E. coli O157:H7 to cattle resulted in consistent long-term colonization in all animals. The surprising ease with which long-term infections resulted from a single application of bacteria to the rectoanal mucosa also strongly supported this location as a site of E. coli O157:H7 colonization in cattle.Escherichia coli O157:H7 is an enteric pathogen of humans that causes a spectrum of illnesses, including hemorrhagic colitis and renal failure, and can be fatal (14,24,29). Domestic cattle are an important reservoir of this pathogen and are the source for most food-borne infections (8). Experimental and field studies of E. coli O157:H7 in beef and dairy cattle have found within-group variation between individual animals with respect to occurrence and duration of fecal culture-positive status (2,3,6,10,15,17,20,21,27,28). This variation suggests that host factors play an important role in E. coli O157:H7 colonization of cattle. A thorough understanding of the hostbacterium interaction could lead to the development of novel interventions, and in order to investigate interactions between the ruminant host and E. coli O157:H7 researchers must recreate infections in an experimental setting. Moreover, to evaluate the efficacy of traditional types of interventions, such as vaccinations (25), probiotics (4, 5), and antimicrobials, it is necessary to be able to reproduce the state of colonization.The earliest publications of ruminant E. coli O157:H7 inoculation studies reported on the use of very young calves, either preweaned or early postweaning, receiving doses of 10 10 CFU administered orally (6, 10) or through rumen cannulae (16,30). An oral dose of 10 8 CFU given to four ...

Among bovine fecal and recto-anal mucosal swab samples cultured in our laboratory for Escherichia coli O157:H7, we frequently isolated E. coli organisms that were phenotypically similar to the O157:H7 serotype as non-sorbitol fermenting and negative for ␤-glucuronidase activity but serotyped O nontypeable:H25 (ONT: H25). This study determined the prevalence and virulence properties of the E. coli ONT:H25 isolates. Among dairy and feedlot cattle (n ‫؍‬ 170) sampled in Washington, Idaho, and Alberta, Canada, the percentage of animals culture positive for E. coli ONT:H25 ranged from 7.5% to 22.5%, compared to the prevalence of E. coli O157:H7 that ranged from 0% to 15%. A longitudinal 8-month study of dairy heifers (n ‫؍‬ 40) showed that 0 to 15% of the heifers were culture positive for E. coli O157:H7, while 15 to 22.5% of the animals were culture positive for E. coli ONT:H25. As determined by a multiplex PCR, the E. coli ONT:H25 isolates carried a combination of virulence genes characteristic of the enterohemorrhagic E. coli, including intimin, translocated intimin receptor, Stx2, and hemolysin (eae-␤, tir, stx 2vh-a , and hly). E. coli ONT:H25 isolates from diverse geographic locations and over time were fingerprinted by separating XbaI-restricted chromosomal DNA by pulsed-field gel electrophoresis (PFGE) separation. Two strains of E. coli ONT:H25 were highly similar by PFGE pattern. Experimental inoculation of cattle showed that E. coli ONT:H25, like E. coli O157:H7, colonized the bovine recto-anal junction mucosa for more than 4 weeks following a single rectal application of bacteria.Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms that cause hemorrhagic colitis and the potentially fatal hemolytic uremic syndrome (HUS) are classed as enterohemorrhagic E. coli (EHEC) (16,28,45). Although the majority of HUS cases in humans have been associated with the EHEC serotype O157:H7 (27, 32), non-O157 serotypes also cause disease and are more common than E. coli O157:H7 in some geographical areas (18,22,36,46). There are over 200 different serotypes of STEC (32), and the number associated with human illness exceeds 100 (6). Domestic ruminants are important reservoirs of STEC and are the most common source for foodborne and animal-contact infections (13). Investigations throughout the world reveal that 10 to 80% of cattle are infected with STEC (5, 6, 49; http://www.who.int/emc-documents /zoonoses/whocsraph988c.html). Non-O157 STEC strains are more prevalent in animals and as contaminants in foods and water than E. coli O157 and, therefore, presumably humans are more frequently exposed to them (9, 10, 27). In the United States, it is estimated that 20% to 50% of STEC human infections are caused by non-O157:H7 serotypes (1, 2).The recto-anal junction (RAJ) mucosa has recently been identified as a site of E. coli O157:H7 colonization in the bovine gastrointestinal tract (33). This finding was supported by earlier observations in mature cattle that E. coli O157:H7 persists in the lower gastrointestinal tract an...