Biotic and abiotic factors influencing in vitro growth of Escherichia coli O157:H7 in ruminant digestive contents

The gastrointestinal tract (GIT) of ruminants is the main reservoir of enterohemorrhagic Escherichia coli, which is responsible for food-borne infections in humans that can lead to severe kidney disease. Characterization of biotic and abiotic factors that influence the carriage of these pathogens by the ruminant would help in the development of ecological strategies to reduce their survival in the GIT and to decrease the risk of contamination of animal products. We found that growth of E. coli O157:H7 in rumen fluid was inhibited by the autochthonous microflora. Growth was also reduced when rumen fluid came from sheep fed a mixed diet composed of 50% wheat and 50% hay, as opposed to a 100% hay diet. In fecal suspensions, E. coli O157:H7 growth was not suppressed by the autochthonous flora. However, a probiotic strain of Lactobacillus acidophilus inhibited E. coli O157:H7 growth in fecal suspensions. The inhibitory effect was dose dependent. These lactic acid bacteria could be a relevant tool for controlling O157:H7 development in the terminal part of the ruminant GIT, which has been shown to be the main site of colonization by these pathogenic bacteria.     

Gastrointestinal Tract Location of Escherichia coli O157:H7 in Ruminants

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of Escherichia coli O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with E. coli O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of E. coli O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum. E. coli O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of E. coli O157:H7 persistence was the lower GIT. E. coli O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for E. coli O157:H7 persistence and proliferation in mature ruminant animals.     

Gastrointestinal tract location of Escherichia coli O157:H7 in ruminants

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of Escherichia coli O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with E. coli O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of E. coli O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum. E. coli O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of E. coli O157:H7 persistence was the lower GIT. E. coli O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for E. coli O157:H7 persistence and proliferation in mature ruminant animals.     

Effect of Forage or Grain Diets with or without Monensin on Ruminal Persistence and Fecal Escherichia coli O157:H7 in Cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10¹⁰ CFU/animal) made resistant to nalidixic acid (Nal^r). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal^r E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal^r E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Escherichia coli O157:H7 in Environments of Culture-Positive Cattle

Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.     

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

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.     

Rectal Administration of Escherichia coli O157:H7: Novel Model for Colonization of Ruminants

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 (~10¹⁰ CFU) and low (~10⁷ 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 Colonization at the Rectoanal Junction of Long-Duration Culture-Positive Cattle

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.     

Capacity of the bovine intestinal mucus and its components to support growth of Escherichia coli O157:H7

Colonization of the gastrointestinal tract of cattle by Shiga toxin-producing Escherichia coli increases the risk of contamination of food products at slaughter. Our study aimed to shed more light on the mechanisms used by E. coli O157:H7 to thrive and compete with other bacteria in the gastrointestinal tract of cattle. We evaluated, in vitro, bovine intestinal mucus and its constituents in terms of their capacity to support growth of E. coli O157:H7 in presence or absence of fecal inoculum, with and without various enzymes. Growth of E. coli O157:H7 and total anaerobic bacteria were proportionate to the amount of mucus added as substrate. Growth of E. coli O157:H7 was similar for small and large intestinal mucus as substrate, and was partially inhibited with addition of fecal inoculum to cultures, presumably due to competition from other organisms. Whole mucus stimulated growth to the greatest degree compared with other compounds evaluated, but the pathogen was capable of utilizing all substrates to some extent. Addition of enzymes to cultures failed to impact growth of E. coli O157:H7 except for neuraminidase, which resulted in greater growth of E. coli O157 when combined with sialic acid as substrate. In conclusion, E. coli O157 has capacity to utilize small or large intestinal mucus, and growth is greatest with whole mucus compared with individual mucus components. There are two possible explanations for these findings (i) multiple substrates are needed to optimize growth, or alternatively, (ii) a component of mucus not evaluated in this experiment is a key ingredient for optimal growth of E. coli O157:H7.     

Effects of Common Forage Phenolic Acids on Escherichia coli O157:H7 Viability in Bovine Feces

Ruminant animals are carriers of Escherichia coli O157:H7, and the transmission of E. coli O157:H7 from cattle to the environment and to humans is a concern. It is unclear if diet can influence the survivability of E. coli O157:H7 in the gastrointestinal system or in feces in the environment. Feces from cattle fed bromegrass hay or corn silage diets were inoculated with E. coli O157:H7, and the survival of this pathogen was analyzed. When animals consumed bromegrass hay for <1 month, viable E. coli O157:H7 was not recovered after 28 days postinoculation, but when animals consumed the diet for >1 month, E. coli O157:H7 cells were recovered for >120 days. Viable E. coli O157:H7 cells in feces from animals fed corn silage were detected until day 45 and differed little with the time on the diet. To determine if forage phenolic acids affected the viability of E. coli O157:H7, feces from animals fed corn silage or cracked corn were amended with common forage phenolic acids. When 0.5% trans-cinnamic acid or 0.5% para-coumaric acid was added to feces from silage-fed animals, the E. coli O157:H7 death rate was increased significantly (17-fold and 23-fold, respectively) compared to that with no addition. In feces from animals fed cracked corn, E. coli O157:H7 death rates were increased significantly with the addition of 0.1% and 0.5% trans-cinnamic acid (7- and 13-fold), 0.1% and 0.5% p-coumaric acid (3- and 8-fold), and 0.5% ferulic acid (3-fold). These data suggest that phenolic acids common to forage plants can decrease viable counts of E. coli O157:H7 shed in feces.     

Rectoanal Junction Colonization of Feedlot Cattle by Escherichia coli O157:H7 and Its Association with Supershedders and Excretion Dynamics

Feedlot cattle were observed for fecal excretion of and rectoanal junction (RAJ) colonization with Escherichia coli O157:H7 to identify potential “supershedders.” RAJ colonization and fecal excretion prevalences were correlated, and E. coli O157:H7 prevalences and counts were significantly greater for RAJ samples. Based on a comparison of RAJ and fecal ratios of E. coli O157:H7/E. coli counts, the RAJ appears to be preferentially colonized by the O157:H7 serotype. Five supershedders were identified based on persistent colonization with high concentrations of E. coli O157:H7. Cattle copenned with supershedders had significantly greater mean pen E. coli O157:H7 RAJ and fecal prevalences than noncopenned cattle. Cumulative fecal E. coli O157:H7 excretion was also significantly higher for pens housing a supershedder. E. coli O157:H7/E. coli count ratios were higher for supershedders than for other cattle, indicating greater proportional colonization. Pulsed-field gel electrophoresis analysis demonstrated that isolates from supershedders and copenned cattle were highly related. Cattle that remained negative for E. coli O157:H7 throughout sampling were five times more likely to have been in a pen that did not house a supershedder. The data from this study support an association between levels of fecal excretion of E. coli O157:H7 and RAJ colonization in pens of feedlot cattle and suggest that the presence of supershedders influences group-level excretion parameters. An improved understanding of individual and population transmission dynamics of E. coli O157:H7 can be used to develop preslaughter- and slaughter-level interventions that reduce contamination of the food chain.     

Effect of Dietary Stress on Fecal Shedding of Escherichia coli O157:H7 in Calves

Two groups of calves were subjected to dietary stress by withholding of food beginning 1 or 14 days after inoculation with 10¹⁰ CFU of Escherichia coli O157:H7. Following treatment, neither group had a significant increase in fecal shedding of E. coli O157:H7. A third group of calves had food withheld for 48 h prior to inoculation with 10⁷ CFU of E. coli O157:H7. These calves were more susceptible to infection and shed significantly more E. coli O157:H7 organisms than calves maintained on a normal diet.     

Experimental and Field Studies of Escherichia coli O157:H7 in White-Tailed Deer

Studies were conducted to evaluate fecal shedding of Escherichia coli O157:H7 in a small group of inoculated deer, determine the prevalence of the bacterium in free-ranging white-tailed deer, and elucidate relationships between E. coli O157:H7 in wild deer and domestic cattle at the same site. Six young, white-tailed deer were orally administered 10⁸ CFU of E. coli O157:H7. Inoculated deer were shedding E. coli O157:H7 by 1 day postinoculation (DPI) and continued to shed decreasing numbers of the bacteria throughout the 26-day trial. Horizontal transmission to an uninoculated deer was demonstrated. Although E. coli O157:H7 bacteria were recovered from the gastrointestinal tracts of deer necropsied from 4 to 26 DPI, attaching and effacing lesions were not apparent in any deer. Results are similar to those of inoculation studies in calves and sheep. In field studies, E. coli O157 was not detected in 310 fresh deer fecal samples collected from the ground. It was detected in feces, but not in meat, from 3 of 469 free-ranging deer in 1997. In 1998, E. coli O157 was not detected in 140 deer at the single positive site found in 1997; however, it was recovered from 13 of 305 dairy and beef cattle at the same location. Isolates of E. coli O157:H7 from deer and cattle at this site differed with respect to pulsed-field gel electrophoresis patterns and genes encoding Shiga toxins. The low overall prevalence of E. coli O157:H7 and the identification of only one site with positive deer suggest that wild deer are not a major reservoir of E. coli O157:H7 in the southeastern United States. However, there may be individual locations where deer sporadically harbor the bacterium, and venison should be handled with the same precautions recommended for beef, pork, and poultry.     

Acyl-Homoserine-Lactone Autoinducer in the Gastrointesinal Tract of Feedlot Cattle and Correlation to Season, <Emphasis Type="Italic">E. Coli</Emphasis> O157:H7 Prevalence,and Diet

Acyl-homoserine-lactone autoinducer (AHL) produced by nonenterohemorrhagic Escherichia coli species in cattle appears to be required for enterohemorrhagic E. coli (EHEC) colonization of the gastrointestinal tract (GIT). The current research aimed to examine the effect of season, diet, EHEC shedding, and location within the GIT on AHL prevalence in the ruminant. Luminal content samples were collected from the rumen and rectum of feedlot cattle at slaughter in the spring, summer, fall, and winter for culture of E. coli O157:H7 and AHL determination. During the spring collection, samples were additionally collected from the cecum and small intestine, but these samples all were AHL negative and therefore not examined again. To assess the influence of diet on AHL prevalence, 14 lambs were fed either 100% forage or 80% concentrate diets and experimentally inoculated with EHEC. At 8 days after infection, all the lambs were killed, and necropsies were taken, with luminal contents collected from the GIT. The collections from the feedlot cattle had AHL in 100% of the rumen content samples from the spring, summer, and fall, but not in any of the winter samples. No other GIT samples from feedlot cattle were AHL positive, and all the samples from the sheep study were AHL negative. The cattle seemed to show a weak correlation between ruminal AHL and EHEC prevalence. This research found AHL only in the rumen and not in the lower GIT of feedlot cattle. However, it is unclear whether this is because the pH of the lower gut destroys the AHL or because a lack of certain bacteria in the lower gut producing AHL. Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

In Vivo and Ex Vivo Evaluations of Bacteriophages e11/2 and e4/1c for Use in the Control of Escherichia coli O157:H7

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10¹⁰ CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10¹¹ PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.Escherichia coli O157:H7 has become a worldwide public health concern since it was first identified as a human pathogen in 1982 (31). This pathogen has a very low infectious dose (approximately 10 cells) in humans, and symptoms of infection range from watery diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and in some cases, death (22, 39). Ruminants are recognized as reservoirs for this pathogen and are the most common sources for food-borne outbreaks (8, 13, 25). It has been reported that the occurrence of E. coli O157:H7 in the feces and, in particular, the hide of cattle is a significant source of the pathogen on the carcass and in derived meat products (11, 12, 25). The control of this pathogen within the animal is difficult, because carriage in ruminants is asymptomatic and shedding can be intermittent and seasonal (12, 19).Research has highlighted the necessity for preharvest intervention strategies to control or reduce E. coli O157:H7 in the food chain (17, 18). Successful strategies to reduce the carriage of E. coli O157:H7 in ruminant animals could potentially reduce the risk of human exposure to this pathogen. There are currently no effective and reliable commercially available intervention strategies to control the carriage of E. coli O157:H7 in ruminants. However, research in this area is increasing, and numerous agents, such as vaccines, probiotics, and bacteriophages (phages), are being evaluated (15, 17, 18). The use of phages for the control of food-borne pathogens in the food chain is desirable, as they are natural, nontoxic viruses that target only specific bacteria (2) and are already being used in human and veterinary medicine, particularly prior to antibiotics (6, 14, 15, 30, 37). Many studies have investigated the use of different phages for the control of E. coli O157:H7 in various animals, including mice, calves, and sheep (4, 5, 35, 37, 41). Although the results between studies vary, some have reported the successful reduction of E. coli O157:H7 levels in animals (4), and one study has resulted in a U.S. patent (41). There are very few commercially available phage products to date, but research indicates promising outcomes for the use of phages for the control of E. coli O157:H7 within the food chain.The E. coli O157:H7-specific phages e11/2 and e4/1c were isolated from bovine slurry in a previous study (26) and have the potential to be used as biocontrol agents for E. coli O157:H7. Both phages have been found to be active against E. coli O157:H7 in a number of relevant test conditions involving different pHs, water activity, and temperatures (B. Coffey, L. Rivas, G. Duffy, A. Coffey, R. P. Ross, and O. McAuliffe, unpublished data). In addition, whole-genome sequencing revealed that neither phage encodes undesirable properties, such as virulence factors, that would hinder its use as a biocontrol agent for E. coli O157:H7 (B. Coffey, G. O''Flynn, A. Coffey, O. O''Sullivan, O. McAuliffe, and R. P. Ross, unpublished data). The objective of the present study was, first, to evaluate the effect of phages e4/1c and e11/2 against inoculated E. coli O157:H7 in an ex vivo model rumen system, and second, to assess the ability of a phage cocktail (e11/2 and e4/1c) to reduce the shedding of E. coli O157:H7 in experimentally inoculated cattle. Findings from ex vivo studies determined our phages to be effective against E. coli O157:H7 in a model rumen system; however, complete eradication of E. coli O157:H7 from cattle was not achieved.     

Survival in acidic and alcoholic medium of Shiga toxin-producing Escherichia coli O157:H7 and non-O157:H7 isolated in Argentina

Background

In spite of Argentina having one of the highest frequencies of haemolytic uraemic syndrome (HUS), the incidence of Escherichia coli O157:H7 is low in comparison to rates registered in the US. Isolation of several non-O157 shiga toxin-producing Escherichia coli (STEC) strains from cattle and foods suggests that E. coli O157:H7 is an uncommon serotype in Argentina. The present study was undertaken to compare the survival rates of selected non-O157 STEC strains under acidic and alcoholic stress conditions, using an E. coli O157:H7 strain as reference.

Results

Growth at 37°C of E. coli O26:H11, O88:H21, O91:H21, O111:H^-, O113:H21, O116:H21, O117:H7, O157:H7, O171:H2 and OX3:H21, was found to occur at pH higher than 4.0. When the strains were challenged to acid tolerance at pH as low as 2.5, viability extended beyond 8 h, but none of the bacteria, except E. coli O91:H21, could survive longer than 24 h, the autochthonous E. coli O91:H21 being the more resistant serotype. No survival was found after 24 h in Luria Bertani broth supplemented with 12% ethanol, but all these serotypes were shown to be very resistant to 6% ethanol. E. coli O91:H21 showed the highest resistance among serotypes tested.

Conclusions

This information is relevant in food industry, which strongly relies on the acid or alcoholic conditions to inactivate pathogens. This study revealed that stress resistance of some STEC serotypes isolated in Argentina is higher than that for E. coli O157:H7.     

Impact of the Direct Application of Therapeutic Agents to the Terminal Recta of Experimentally Colonized Calves on Escherichia coli O157:H7 Shedding

Enterohemorrhagic Escherichia coli O157:H7 is an important intestinal pathogen of humans with a main reservoir of domesticated ruminants, particularly cattle. It is anticipated that the risk of human infection can be reduced by controlling the organism within its reservoir hosts. Several options for the control of E. coli O157:H7 in cattle have been proposed, but none have been demonstrated to be successful in the field. Here we describe a novel experimental method, based on the terminal-rectum-restricted colonization described previously, to eliminate fecal carriage of E. coli O157:H7. In experimentally challenged calves, direct application to the rectal mucosa of either of two therapeutic agents, polymyxin B or chlorhexidine, greatly reduced bacterial shedding levels in the immediate posttreatment period. The most efficacious therapeutic agent, chlorhexidine, was compared in orally and rectally challenged calves. The treatment eliminated high-level shedding and reduced low-level shedding by killing bacteria at the terminal rectum. A rapid-detection system based on the ability to identify E. coli O157:H7 from swabs of the rectal mucosa was also assessed. This test was sufficiently sensitive to identify high-level bacterial carriage. Thus, a combination of the detection method and treatment regimens could be used in the field to eliminate high-level fecal excretion of E. coli O157:H7, so greatly reducing its prevalence within this host and the risk of human infection.     

Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing Escherichia coli Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics

Healthy ruminants are the main reservoir of Shiga toxin-producing Escherichia coli (STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic Saccharomyces cerevisiae CNCM I-1077 or Lactobacillus acidophilus BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition, S. cerevisiae did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that cause human diseases ranging from uncomplicated diarrhea to hemorrhagic colitis (HC), as well as life-threatening complications, such as hemolytic-uremic syndrome (HUS). Most outbreaks and sporadic cases of HC and HUS have been attributed to O157:H7 STEC (http://www.cdc.gov/ecoli/outbreaks.html; http://www.euro.who.int). However, in some geographic areas, non-O157:H7 STEC infections are considered to be at least as important as E. coli O157:H7 infections, but they are often underdiagnosed (21, 46). In spite of diverse virulence characteristics, one common trait of pathogenic STEC strains could be resistance to the gastric acidity in humans. Indeed, it has been suggested that acid resistance of E. coli O157:H7 is negatively correlated with the infectious dose required for this organism to cause disease in humans (17).Healthy cattle and other ruminants appear to be the main reservoir of STEC strains. However, colonization of the cattle gastrointestinal tract (GIT) by STEC seems to be a transient event, with a mean duration of 14 days to 1 month (4, 8, 38). The site of STEC persistence and proliferation in the GIT depends on the STEC strain and seems to vary from one individual to another. Some previous studies identified the rumen as the primary site of colonization (8), whereas other studies referred to the cecum, the colon, or the rectum (10, 18, 23, 32, 42). Although STEC strains adhere in vitro to bovine colonic mucosa, forming the characteristic attaching and effacing lesions (35), they are very rarely associated with tissues in animal carriers and are generally isolated from the digesta (8). STEC does not, therefore, seem to colonize the gut mucosa, except for the anorectal mucosa, which has been described as the preferred colonization site for O157:H7 strains but not for non-O157:H7 strains (24, 32). During their transit through the ruminant GIT, STEC strains encounter various acidic conditions. Volatile fatty acid (VFA) concentrations are high in the rumen of grain-fed animals, and the pH may vary from 5.0 to 6.5. In these conditions, VFAs are in the undissociated form and can freely enter the bacterial cells, dissociate, and acidify the cytosol. In hay-fed animals, less fermentation occurs in the rumen, and the pH remains between 6.5 and 7. In the abomasum, STEC encounters strongly acidic conditions, regardless of the diet, due to the presence of mineral acids, resulting in a pH below 3. Then the pH increases from the proximal part to the distal part of the small intestine, and in the cecum and the colon STEC encounters more neutral pH conditions.All STEC strains are not equally resistant to acidic conditions (2, 9, 30, 45). Therefore, it could be hypothesized that acid-resistant (AR) STEC survives and persists better in the GIT of ruminants than acid-sensitive (AS) STEC. Acid resistance mechanisms can be induced during exposure to a moderately acidic environment (12, 26, 41). The rumen contents of a grain-fed animal could be such an environment favorable for the induction of acid resistance in STEC. While the diet does not seem to affect the acid resistance of an E. coli O157:H7 strain (19), grain feeding increases the number of acid-resistant generic coliforms (15, 19), either by inducing acid resistance mechanisms in the rumen or by selecting acid-resistant E. coli strains during passage through the abomasum. Hence, generic coliforms behave differently than E. coli O157:H7 in ruminants (19), and the potential ecological advantage conferred by acid resistance to non-O157:H7 STEC strains for persistence in the ruminant GIT has never been investigated.Inhibition of STEC proliferation in the ruminant gut may be mediated through probiotic supplementation. Several studies have demonstrated the capacity of certain lactic acid bacteria or yeast to reduce E. coli O157:H7 counts in vitro (1, 34) or in vivo (5, 40). The mechanisms of action of probiotics are not well characterized but could involve competition for nutrients and adhesion sites in the GIT, an increase in the VFA concentration and a decrease in the pH, production of antimicrobial molecules, or interference with quorum-sensing signaling (27-29). However, the impact of probiotics on non-O157:H7 STEC has been poorly investigated (36). Although not all non-O157:H7 STEC strains are pathogenic, limiting their carriage by ruminants should decrease the risk of food-borne illness. The impact of probiotics and of the physicochemical conditions of the rumen digesta on the survival of non-O157:H7 STEC strains or on induction of acid resistance mechanisms could have significant implications for farm management practices and food safety.The purpose of this work was to investigate whether the level of acid resistance, determined using an in vitro assay, confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. Moreover, we evaluated the potential of probiotics to limit STEC survival and induction of acid resistance in the ruminant GIT.     

Effect of forage or grain diets with or without monensin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10(10) CFU/animal) made resistant to nalidixic acid (Nal(r)). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal(r) E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal(r) E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Potential To Reduce Escherichia coli Shedding in Cattle Feces by Using Sainfoin (Onobrychis viciifolia) Forage, Tested In Vitro and In Vivo

There is a growing concern about the presence of pathogens in cattle manure and its implications on human and environmental health. The phytochemical-rich forage sainfoin (Onobrychis viciifolia) and purified phenolics (trans-cinnamic acid, p-coumaric acid, and ferulic acid) were evaluated for their ability to reduce the viability of pathogenic Escherichia coli strains, including E. coli O157:H7. MICs were determined using purified phenolics and acetone extracts of sainfoin and alfalfa (Medicago sativa), a non-tannin-containing legume. Ground sainfoin or pure phenolics were mixed with fresh cattle feces and inoculated with a ciprofloxacin-resistant strain of E. coli, O157:H7, to assess its viability at −20°C, 5°C, or 37°C over 14 days. Forty steers were fed either a sainfoin (hay or silage) or alfalfa (hay or silage) diet over a 9-week period. In the in vitro study, the MICs for coumaric (1.2 mg/ml) and cinnamic (1.4 mg/ml) acids were 10- to 20-fold lower than the MICs for sainfoin and alfalfa extracts. In the inoculated feces, the −20°C treatment had death rates which were at least twice as high as those of the 5°C treatment, irrespective of the additive used. Sainfoin was less effective than coumaric acid in reducing E. coli O157:H7 Cip^r in the inoculated feces. During the animal trial, fecal E. coli numbers declined marginally in the presence of sainfoin (silage and hay) and alfalfa silage but not in the presence of hay, indicating the presence of other phenolics in alfalfa. In conclusion, phenolic-containing forages can be used as a means of minimally reducing E. coli shedding in cattle without affecting animal production.