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...

Enrichment and direct (nonenrichment) rectoanal mucosal swab (RAMS) culture techniques were developed and compared to traditional fecal culture for the detection of Escherichia coli O157:H7 in experimentally infected and naturally infected cattle. Holstein steers (n ‫؍‬ 16) orally dosed with E. coli O157:H7 were sampled after bacterial colonization starting 15 days postinoculation. Enrichment RAMS cultures (70.31% positive) were more sensitive than enrichment fecal cultures with 10 g of feces (46.88% positive) at detecting E. coli O157:H7 (P < 0.01). Holstein bull calves (n ‫؍‬ 15) were experimentally exposed to E. coli O157:H7 by penning them with E. coli O157:H7-positive calves. Prior to bacterial colonization (1 to 14 days postexposure), enriched fecal cultures were more sensitive at detecting E. coli O157:H7 than enriched RAMS cultures (P < 0.01). However, after colonization (40 or more days postexposure), the opposite was true and RAMS culture was more sensitive than fecal culture (P < 0.05). Among naturally infected heifers, enriched RAMS or fecal cultures were equally sensitive (P ‫؍‬ 0.5), but direct RAMS cultures were more sensitive than either direct or enriched fecal cultures at detecting E. coli O157:H7 (P < 0.01), with 25 of 144, 4 of 144, and 10 of 108 samples, respectively, being culture positive. For both experimentally and naturally infected cattle, RAMS culture predicted the duration of infection. Cattle transiently shedding E. coli O157:H7 for <1 week were positive by fecal culture only and not by RAMS culture, whereas colonized animals (which were culture positive for an average of 26 days) were positive early on by RAMS culture. RAMS culture more directly measured the relationship between cattle and E. coli O157:H7 infection than fecal culture.Since Escherichia coli O157:H7 was first identified as a human pathogen (27,33), investigations have demonstrated that human disease outbreaks are often linked to a bovine food source or bovine waste-contaminated water (2,7,30,39). Although most of the known outbreaks of E. coli O157:H7-associated disease in humans are food borne or waterborne, several recent studies indicate that a significant number of human infections are acquired from direct contact with cattle, the environment, or unknown sources (1,12,30,34). An accepted premise is that the reduction of the number of cattle infected with E. coli O157:H7 or the elimination of E. coli O157:H7 from cattle will effect a reduction in the rate of disease in humans. To this end, a great deal of research has focused on describing the ecology and epidemiology of E. coli O157:H7 in cattle, with the hope of identifying interventions to reduce its prevalence in cattle (11,16,17,19,20,23,24,28,29). Within this body of research, numerous methods for detecting E. coli O157:H7 in bovine fecal samples have been developed and used (8,9,22,36,37,40). The reported sensitivities of detection by these various methods vary greatly, and problems occur when data from studies that have used methods with disparate sensitiv...

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...

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening sequela hemolytic-uremic syndrome in humans (6). Healthy cattle are the major reservoir for E. coli O157:H7, and contaminated ground beef, direct animal contact, and bovine manure contamination of other foods or water account for the majority of human infections (2, 3). The predominant colonization site of this microorganism in the bovine gastrointestinal tract (GIT) has been identified as the rectoanal junction (RAJ) mucosa (7). Localization to this site is in contrast with other E. coli serotypes that are present in consistent numbers throughout the large intestine without an increase at the RAJ mucosa. This tissue area comprises a clear junction between the stratified squamous epithelium of the anal canal and the columnar epithelial mucosa of the rectum and is a lymphoid follicle-dense mucosa. Although the mechanisms underlying the tropism of this serotype for this lymphoid follicle-rich site are not known, many studies of experimentally and naturally colonized cattle conclude that the RAJ mucosa plays an important role in E. coli O157:H7 bovine carriage of this human pathogen (5,7,8,10,11). Also, application of E. coli O157:H7 at the RAJ mucosa results in carriage of the bacteria similar to that by naturally infected animals (10).Three distinct patterns of E. coli O157:H7 carriage in cattle have been described previously (1, 9, 12). First, animals can be transiently culture positive for short durations of a few days and are considered passive shedders and are likely not colonized at the RAJ mucosa; second, cattle can be colonized and shed the bacteria for an average of 1 month and typically not longer than 2 months; and third, a few rare animals are colonized for a long duration and shed the bacteria from 3 to 12 months or longer. This unique situation in which a few animals develop long-duration colonization (Ͼ2 months) with E. coli O157 is likely due to bacterial association at the RAJ mucosa; however, it may be due to unique colonization by the bacteria at a site(s) in addition to the RAJ mucosa. For example, the gall bladder can harbor Salmonella spp. in carrier animals and a recent investigation reports E. coli O157:H7 in the gall bladders of some cattle at slaughter; however, this report does not provide information about the E. coli O157:H7 culture history of the animals tested, so the duration for which each animal was culture positive is unknown (4). For the first time, the current study investigated the colonization site of E. coli O157:H7 along the GIT of animals for which a documented history of long-duration carriage of E. coli O157:H7 was available. To identify animals t...

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 ...

Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the ⌬per mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the ⌬per mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the ⌬per mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the ⌬per mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ⌬pO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ⌬pO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ⌬pO157 mutant. The ⌬pO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ⌬pO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ⌬pO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ϳ50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.