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.     

Detection of Escherichia coli O157:H7 in raw meat by immunomagnetic separation and multiplex PCR

The aim of this research was to elaborate fast and sensitive method ofdetection of E. coli O157:H7 in food samples. Raw ground meat obtained from retail was artificially inoculated with low numbers of E. coli O157:H7. 18 h enrichment culture allowed pathogenic bacteria to multiply to the levels detectable in multiplex PCR. Immunomagnetic separation with magnetic beads coated with an antibody against E. coli O157:H7 were used to concentrate target bacteria and to separate PCR inhibitors. A portion of the bacterial suspension was used in a multiplex PCR to amplify eae (attaching and effacing) gene, stx (shiga toxin) genes and 90 kbp plasmid. The sensitivity of E. coli O157:H7 detection method was shown to be 1 cfu per 25 g of food sample. The total analysis can be completed within 24 h, whilst traditional methods involves enrichment, direct plating and confirmation tests with entire time at least 3 days.     

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.     

Occurrence of Escherichia coli O157:H7 in faeces, skin and carcasses from sheep and goats in Ethiopia

Aims:  To determine the occurrence and proportion of Escherichia coli O157:H7 in faeces, skin swabs and carcasses before and after washing, from sheep and goats in Ethiopia.
Method and Results:  Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx ₁, stx ₂ and eae genes. Escherichia coli O157:H7 was isolated from faeces (4·7%), skin swabs (8·7%) and carcasses before washing (8·1%) and after washing (8·7%) and on water samples (4·2%). The proportion of carcasses contaminated with E. coli O157:H7 was strongly associated with those recovered from faecal and skin samples. Both stx ₁ and stx ₂ genes were identified from one E. coli O157:H7 isolate from a goat carcass.
Conclusions:  Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E.   coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations.
Significance and Impact of the Study:  Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins.     

Fate of Escherichia coli O157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 x 10(3) in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.     

Comparison of multiplex PCR,and an immunochromatographic method sensitivity for the detection of Escherichia coli O157:H7 in minced beef

In this study, the sensitivities of multiplex PCR and an immuno-chromatographic methods to detect Escherichia coli O157:H7 in minced beef were compared. The detection of Escherichia coli O157:H7 in minced beef inoculated with 1-100 cells of this bacterium was possible after enrichment of culture and subsequent analysis by either of the two methods. Enrichment conditions were eight hours of incubation at 37 degrees C or 42 degrees C in a non-selective medium (Buffered Peptone Water). Multiplex PCR analysis was performed using three primer sets with analysis by gel electrophoresis. The Quix immuno-chromatographic assay which is a new kit being marketed by New Horizons Diagnostics, Columbia, MD, was used for immunological analysis of the enriched broths.The sensitivity of both tests was similar. The results depended on the concentration of the specific bacterium in the culture since the influence of the proportion of other bacteria to the E. coli O157:H7 was not observed. The data suggests that either method or used together, when coupled with an enrichment technique, could provide a rapid mean to detect the presence of this pathogen in minced meat samples.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.     

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.     

Extraction of lithium from spodumene by bioleaching

A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.     

Comparison of primers for the detection of pathogenic Escherichia coli using real-time PCR

AIMS: To evaluate PCR primers for the detection of pathogenic Escherichia coli in a real-time PCR assay and determine their utility in produce irrigation water testing. METHODS AND RESULTS: Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce amplification products for several pathogenic E. coli serotypes from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 10(0) and 10(1) CFU rxn(-1) in sterile water respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 10(1) and between 10(2) and 10(3) CFU rxn(-1) in diluted sprout irrigation water respectively. CONCLUSIONS: The primer set coneaeA was able to produce an amplification product from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: The necessity of a dissociation analysis to distinguish positive samples from those with fluorescence of random dsDNA generation for real-time PCR in a complex background was established.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Adsorption of endogenous polyphenols relieves the inhibition by fruit juices and fresh produce of immuno-PCR detection of Escherichia coli O157:H7

The immuno-polymerase chain reaction (PCR) approaches facilitate rapid (8 h) detection of Escherichia coli O157:H7 in contaminated dairy products and ground beef samples with detection sensitivities approaching 1 colony forming unit (cfu) g-1 ml-1. However, no PCR products were obtained when the method was applied to identify E. coli O157:H7 in tainted apple juice. Enzyme-linked immuno-assay (ELISA) results suggested non-specific binding of endogenous polyphenols (ubiquitous in plant products) to antibodies present on the surface of the immunobeads, making the latter unavailable for capturing the target bacteria Treatment of the test sample, prior to IMS, with a synthetic fining agent, polyvinylpyrrolidone, restored the full function and sensitivity of the immuno-PCR. The study demonstrates the suitability of the improved method as a generic strategy for rapid screening of fruit juices and plant produce for E. coli O157:H7.     

EVALUATION OF 5'NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

Two 5'nuclease-based PCR methods (PCR-LS-50B and PCR-7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR-7200 method was able to detect E. coli O157:H7 at ± 10² CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR-7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR-LS-50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR-based detection techniques for E. coli O157:H7, however, the 5'nuclease-based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.     

Same-day detection of Escherichia coli O157:H7 from spinach by using electrochemiluminescent and cytometric bead array biosensors

Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.     

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.     

An adapted ImmunoMagnetic cell separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces

Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-microm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-microm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-microm diameter Dynal bead.