Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Biocontrol of Escherichia coli O157 with O157-specific bacteriophages.

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4 degrees C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4 degrees C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Veterinary significance of verocytotoxin-producing Escherichia coli O157

Verocytotoxin-producing Escherichia coli O157 is a serious pathogen in man that is carried by ruminants and has been isolated from some other animal species. Except in the very young of certain species and in greyhounds, the organism is not associated with disease in animals. Humans may be infected by ingestion of the organism through direct animal contact, from contaminated food or water or from the environment. Great efforts have been made to improve hygienic food production and handling, to protect water supplies and to give adequate advice to people handling animals. It is also essential to try to reduce the numbers of organisms shed by animals and, to do this, a clear understanding of the ecology of the organism is required. This revised version was published online in November 2006 with corrections to the Cover Date.     

Clinical significance of Verocytotoxin-producing Escherichia coli O157

In 1977, Konowalchuk and colleagues (Konowalchuk, J., Speirs, J.I. & Stavric, S. 1977 Infection and Immunity 18, 775–779) were the first to describe Verocytotoxin-producing strains of Escherichia coli or VTEC. The surveillance of infection caused by VTEC demonstrated strains of E. coli belonging to serogroup O157 as the main cause of human infection capable of causing haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Infection with O157 VTEC results in a range of disease manifestations including abdominal cramps, vomiting and fever. This frequently leads to cases with bloody diarrhoea and HC, and approximately 10% of patients develop HUS. The symptoms of disease caused by VTEC O157 have been well documented and the pathogenic mechanisms expressed by VTEC have been the focus of considerable attention. However, the role of putative pathogenic mechanisms in the pathogenesis of disease is not fully understood. The aim of this review is to consider the clinical aspects of infection with strains of VT-producing E. coli O157 in terms of the putative pathogenic mechanisms expressed by these bacteria. This revised version was published online in November 2006 with corrections to the Cover Date.     

Susceptibility of Escherichia coli O157 and non-O157 isolates to lactate

AIMS: To examine the effect of temperature on the antimicrobial efficacy of lactate and propionate against O157 and non-O157 Escherichia coli isolates. METHODS AND RESULTS: Lactate and, to a lesser extent, propionate effectively reduced viability at 37 degrees C. Ethanol enhanced this effect. Reducing the temperature to 20 or 5 degrees C caused an increase in survival in the presence of these organic acids with or without ethanol. At 20 degrees C the deltapH, membrane potential and intracellular lactate anion concentration were less than at 37 degrees C. CONCLUSIONS: The efficacy of lactate and propionate against E. coli O157 and non-O157 isolates is reduced at lower temperatures, perhaps due to the reduction in the deltapH, membrane potential and intracellular lactate anion concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the usefulness of organic acids as decontaminants for E. coli O157 is temperature dependent.     

Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Enhanced Acid Sensitivity of Pressure-Damaged Escherichia coli O157 Cells

Pressure-damaged Escherichia coli O157 cells were more acid sensitive than native cells and were impaired in pH homeostasis. However differences in acid sensitivity were not related to differences in cytoplasmic pH (pH_i). Cellular β-galactosidase was more acid labile in damaged cells. Sensitization to acid may thus involve loss of protective or repair functions.     

Survival of Escherichia coli O157 in abattoir waste products

AIMS: This study monitored survival and growth of Escherichia coli O157 in ovine and bovine abattoir waste. METHODS: Blood and gut contents were inoculated separately with cocktails of E. coli O157. Samples were stored aerophilically and microaerophilically at 5 degrees C, 15 degrees C and 30 degrees C to represent storage at different container depths and at extremes of UK ambient temperature. CONCLUSIONS: Results showed survival of E. coli O157 was irrespective of oxygen content with no significant differences observed between aerophilic and microaerophilic environments. Numbers of E. coli O157 in ovine and bovine gut contents showed no change when stored at 5 degrees C and increased 1-2 log(10) at 15 degrees C and 30 degrees C in 28 h. In ovine and bovine blood, irrespective of storage temperature, there was a 0.5-2 log(10) reduction or no change in numbers except in ovine blood stored at 30 degrees C where the fall in numbers was followed by a 3 log(10) increase. In aged (stored at 4 degrees C for 18 h before spiking) bovine blood there was no significant change in numbers at 5 degrees C while at 15 degrees C there was 2 log(10) rise after 48 h. At 30 degrees C there was an initial 1 log(10) decrease in numbers followed by a 1 log(10) rise over the following 40 h. SIGNIFICANCE AND IMPACT OF STUDY: Abattoir wastes may become contaminated from animals infected with Verocytotoxigenic E. coli O157 and in certain storage conditions these pathogens could significantly increase in numbers. There is need for care in abattoir waste disposal, not only for personnel subject to direct contact, but also in the prevention of cross contamination to adjacent land and water courses which could indirectly infect humans.     

Methods for the isolation of water-borne Escherichia coli O157

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.     

Methods available for the sub-typing of Escherichia coli O157

The epidemiological investigation of Escherichia coli O157 is complicated by the lack of heterogeneity between strains responsible for the majority of cases of infection. As a consequence it is difficult to reliably cluster together an outbreak strain and differentiate it from other sporadically occurring isolates. The methods available for the sub-typing of E. coli O157 vary in their speed, technical complexity, cost and ability to discriminate reliably between strains, with many of the recently developed methods targeting the genome to provide differentiation. No single typing method is individually superior, and ideally a combination of techniques should be employed depending on the level of discrimination required and time or resources available. The aim of this review is to consider the relative merits of the available typing methodologies with particular emphasis on those which may find application in a diagnostic laboratory. This revised version was published online in November 2006 with corrections to the Cover Date.     

Slugs: potential novel vectors of Escherichia coli O157

Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157.     

Cattle Water Troughs as Reservoirs of Escherichia coli O157

Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of E. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.     

Slugs: Potential Novel Vectors of Escherichia coli O157

Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157.     

Enhanced acid sensitivity of pressure-damaged Escherichia coli O157 cells

Pressure-damaged Escherichia coli O157 cells were more acid sensitive than native cells and were impaired in pH homeostasis. However differences in acid sensitivity were not related to differences in cytoplasmic pH (pH(i)). Cellular beta-galactosidase was more acid labile in damaged cells. Sensitization to acid may thus involve loss of protective or repair functions.     

Shedding of Escherichia coli O157:H7 in Dairy Cattle Housed in a Confined Environment following Waterborne Inoculation

A study of Escherichia coli O157:H7 transmission and shedding was conducted with bull calves housed in individual pens within a confined environment. For comparative purposes, the numbers and duration of E. coli O157:H7 shedding in naturally infected calves were monitored after a single purchased calf (calf 156) tested positive prior to inoculation. During the next 8 days, the calves in adjacent pens and a pen directly across a walkway from calf 156 began to shed this serotype O157:H7 strain. Five of the eight calves in this room shed this O157:H7 strain at some time during the following 8 weeks. The numbers of E. coli O157:H7 isolates shed in these calves varied from 60 to 10⁵ CFU/g of feces, and the duration of shedding ranged from 17 to >31 days. The genomic DNAs from isolates recovered from these calves were indistinguishable when compared by using XbaI digestion and pulsed-field gel electrophoresis. Inoculation of calves with 1 liter of water containing ca. 10³ to 10⁴ CFU of E. coli O157:H7/ml resulted in shedding in 10 of 12 calves (trial 1, 4 of 4 calves; trial 2, 6 of 8 calves). The inoculated calves shed the inoculation strain (FRIK 1275) as early as 24 h after administration. The duration of shedding varied from 18 to >43 days at levels from 10² to 10⁶ CFU/g of feces. The numbers of doses necessary to initiate shedding varied among calves, and two calves in trial 2 never shed FRIK 1275 after four doses (ca. 10⁶ CFU per dose). Results from this study confirm previous reports of animal-to-animal and waterborne dissemination of E. coli O157:H7 and highlight the need for an effective water treatment to reduce the spread of this pathogen in cattle.     

Loss of O157 O antigenicity of verotoxin-producing Escherichia coli O157:H7 surviving under starvation conditions

Verotoxin (VT)-producing Escherichia coli O157:H7 was culturable on agar media after being left in water for 21 months. However, there were a number of colonies which had lost O157 O antigenicity. These colonies produced VTs, which are pathogenic to humans. These observations suggest that the immunologic methods based on O157 O antigenicity are unable to detect and isolate VT-producing E. coli in foods and other environments if the organism has been under starvation conditions for a long period.     

Electrophoretic mobilities of Escherichia coli O157:H7 and wild-type Escherichia coli strains.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Loss of O157 O Antigenicity of Verotoxin-Producing Escherichia coli O157:H7 Surviving under Starvation Conditions

Verotoxin (VT)-producing Escherichia coli O157:H7 was culturable on agar media after being left in water for 21 months. However, there were a number of colonies which had lost O157 O antigenicity. These colonies produced VTs, which are pathogenic to humans. These observations suggest that the immunologic methods based on O157 O antigenicity are unable to detect and isolate VT-producing E. coli in foods and other environments if the organism has been under starvation conditions for a long period.