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.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.     

Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection

Many rapid tests have been developed for the detection of Escherichia coli O157:H7 from complex matrices such as food and water. However, many of these methods rely on traditional culture steps for confirmation, which can take an extra 24-48 h. The fiber optic biosensor has been used to rapidly detect pathogens from complex matrices. In this paper, we demonstrate a method using a rapid biosensor assay, recovery through a short enrichment, and PCR to detect and confirm the presence of at least 10(3) CFU/ml of E. coli O157:H7 in a sample in less than 10 h.     

Rapid PCR confirmation of E. coli O157:H7 after evanescent wave fiber optic biosensor detection

Escherichia coli O157:H7 is an enteric pathogen of public health importance, which is monitored by several government agencies. Many rapid detection tests have been developed to identify foodstuff and water supplies contaminated by E. coli O157:H7. However, these methods can be time consuming (24-48 h) due to the need to culture the bacteria to confirm detection results. Fiber optic biosensors can rapidly detect pathogens from complex matrices, yet confirmation tests can take up to 10h to complete. In addition, fiber optic biosensors can also be used to reduce the impact of PCR inhibitors present in complex matrices such as food and water. This paper presents methodologies to reduce the time necessary for confirmation from 10 to about 2 h, by direct PCR of bacteria from the fiber optic waveguides without the need for culture or enrichment steps.     

Detection of Escherichia coli O157 in French food samples using an immunomagnetic separation method and the VIDASTME. coli O157

C. VERNOZY-ROZAND, C. MAZUY, S. RAY-GUENIOT, S. BOUTRAND-LOEï, A. MEYRAND AND y. richard. 1997. Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS^TM E. coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples. A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined. Four poultry, one raw sausage and one ground beef sample were found to be positive for E. coli O157 by both methods. Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E. coli .     

The impact of E. coli O157 on the food industry

The relatively recent emergence of Escherichia coli O157 as a foodborne pathogen has had a significant impact on the food industry. This serovar possesses a number of undesirable characteristics that combine to make it one of the most serious threats to food safety in recent years. The widespread and sporadic occurrence in the gastrointestinal tracts of cattle, sheep, man and many other species, sometimes in the absence of any disease, and the complex nature of its ecology mean that it is not feasible to easily eradicate this serovar. In order to control and minimise foodborne disease by these organisms, it has been necessary to change primary production, processing, retailing and consumer-handling practices. Despite these new control measures, foodborne disease outbreaks caused by these microorganisms continue to occur and are accompanied by an increase in the number of cases of illness in many countries. This revised version was published online in November 2006 with corrections to the Cover Date.     

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

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.     

Growth of Escherichia coli O157 in poorly fermented laboratory silage: a possible environmental dimension in the epidemiology of E. coli O157

Laboratory silages, inoculated with either c. 1000 cfu g-1, an atoxigenic Escherichia coli O157 or a toxigenic E. coli O157 isolate, were made in plastic bags which permitted limited aerobic spoilage. Replicate bags of each treatment were opened at weekly intervals after incubation at 20 degrees C. In all silages the fermentation was slow and aerobic spoilage with visible moulding ocurred at the tie ends after 7 d. In all the aerobically spoiled silages Enterobacteriaceae reached over 107 cfu g-1 within 1 week. The E. coli in control silages increased from barely detectable levels to 104 cfu g-1 within 13 d; over the same period both strains of E. coli O157 increased from 103 to 107 cfu g-1. The increases in the poorly fermented interior of the silage bags were initially similar but declined slightly as the pH fell. It is suggested that faecal contamination of grass followed by poor silage management may be a factor in the persistence of E. coli O157 carriage in ruminants.     

Detection of pathogen Escherichia coli O157:H7 using self-excited PZT-glass microcantilevers

Composite self-excited PZT-glass cantilevers (5 and 3 mm in length, 1.8 and 2.0 mm wide) were fabricated and their resonance characteristics were determined in air and at 1 mm liquid immersion. In air, resonance occurred at 65.8 and 63.4 kHz for the two cantilevers used in this paper. Monoclonal antibody (MAb) specific to the pathogen Escherichia coli (E. coli) O157:H7 was immobilized at the cantilever glass tip, and then exposed to pathogen in the concentration range of 7x10(2) to 7x10(7)bacteria/mL. Resonance of the second mode decreased due to pathogen attachment in accordance with a proposed kinetic model. The specific attachment rate constant was found to be 3x10(-9) to 5x10(-9) min-1 (cell/mL)-1. Exposure to a mixed population containing both a pathogenic and non-pathogenic strain showed that the antibody-immobilized cantilever is highly selective, thus demonstrating its usefulness for detecting water-borne pathogens.     

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

Monitoring biosensor capture efficiencies: development of a model using GFP-expressing Escherichia coli O157:H7

One of the known limitations for biosensor assays is the high limit of detection for target cells within complex samples (e.g., Escherichia coli at 10(4) to 10(5) CFU/mL) due to poor capture efficiencies. Currently, researchers can only estimate the cell capture efficiency necessary to produce a positive signal for any type of biosensor using either cumbersome techniques or regression modeling. To solve this problem, green fluorescent protein (GFP) transformed E. coli O157:H7 was used to develop a novel method for directly and easily measuring the cell capture efficiency of any given biosensor platform. For demonstration purposes, E. coli-GFP was assayed on both fiber optic and planar waveguide biosensor platforms. Cells were enumerated using an epifluorescent microscope and digital camera to determine the number of cells captured on the surfaces. Conversion algorithms were used with these digital images to determine the cell density of entire waveguide surface areas. For E. coli-GFP, the range of cell capture efficiency was between 0.4 and 1.2%. This indicates that although the developed model works for calculating cell capture, there is still need for significant improvements in capture methods themselves, to increase the capture efficiency and thereby lower detection limits. The use of GFP-transformed target cells and cell capture efficiency calculations can facilitate the development and optimization processes by allowing direct enumeration of new biosensor design configurations and sample processing strategies.     

Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E. coli O157

Misuse of biocides has encouraged the emergence of resistance and cross-resistance in certain strains. This study investigated resistance of triclosan-adapted Escherichia coli K-12 and E. coli O55 to antimicrobial agents and compared these to E. coli O157:H7. Cross-resistance in E. coli K-12 and E. coli O55 was observed however to a lesser extent than in E. coli O157:H7. Triclosan-adapted E. coli K-12 demonstrated cross-resistance to chloramphenicol, whereas triclosan-adapted E. coli O55 exhibited resistance to trimethoprim. In comparison, E. coli O157:H7 was resistant to chloramphenicol, tetracycline, amoxicillin, amoxicillin/clavulanic acid, trimethoprim, benzalkonium chloride and chlorohexidine suggesting strain specific rather than general resistance mechanisms.     

The history and evolution of Escherichia coli O157 and other Shiga toxin-producing E. coli

Shiga toxin-producing Escherichia coli (STEC) O157 is a formidable human pathogen with the capacity to cause large outbreaks of gastrointestinal illness. The known virulence factors of this organism are encoded on phage, plasmid and chromosomal genes. There are also likely to be novel, as yet unknown virulence factors in this organism. Many of these virulence factors have been acquired by E. coli O157 by transfer from other organisms, both E. coli and non-E. coli species. By examination of biochemical and genetic characteristics of various E. coli O157 strains and the relationships with other organisms, an evolutionary pathway for development of E. coli O157 as a pathogen has been proposed. E. coli O157 evolved from an enteropathogenic E. coli ancestor of serotype O55:H7, which contained the locus of enterocyte effacement containing the adhesin intimin. During the evolutionary process, Shiga toxins, the pO157 plasmid and other characteristics which enhanced virulence were acquired and other functions such as motility, sorbitol fermentation and β-glucuronidase activity were lost by some strains. It is likely that E. coli O157 is constantly evolving, and changes can be detected in genetic patterns during the course of infection. A variety of mechanisms may be responsible for the development of the virulent phenotype that we see today. Such changes include uptake of as yet uncharacterised virulence factors, possibly enhanced by a mutator phenotype, recombination within virulence genes to produce variant genes with different properties, loss of large segments of DNA (black holes) to enhance virulence and possible adaptation to different hosts. Although little is known about the evolution of non-O157 STEC it is likely that the most virulent clones evolved in a similar manner to E. coli O157. This revised version was published online in November 2006 with corrections to the Cover Date.