Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Characterization of Shiga toxin‐producing Escherichia coli isolated from dairy cows in Argentina

Aims: To feno‐genotypically characterize the Shiga toxin‐producing Escherichia coli (STEC) population in Argentinean dairy cows. Methods and Results: From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52%stx2 and 37%stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty‐five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29%stx2, saa, ehxA. One hundred and fifty‐six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2⁺, eae⁺, ehxA⁺ virulence pattern. Conclusions: We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. Significance and Impact of the study: The non‐O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.     

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.     

A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10⁴ CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.     

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use

AIMS: To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. METHODS AND RESULTS: First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. CONCLUSIONS: Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. SIGNIFICANCE AND IMPACT OF THE STUDY: We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.     

Shiga toxin-producing Escherichia coli O171:H25 strain isolated from a patient with haemolytic uraemic syndrome

Shiga toxin-producing Escherichia coli (STEC) strains of O157:H7 serotype are a predominant cause of haemolytic uraemic syndrome (HUS) worldwide, but strains of non-O157 serotypes can also be associated with serious disease. Some of them are associated with outbreaks of HUS, others with sporadic cases of HUS, and some with diarrhoea but not with outbreaks or HUS. A large number of STEC serotypes isolated from ruminants and foods have never been associated with human disease. In this study we characterize a STEC strain belonging to serotype O171:H25 that is responsible for a case of HUS. This strain has a single Shiga toxin gene encoding Stx2 toxin, and hlyA gene, but is eae-negative.     

Methods available for the detection of Escherichia coli O157 in clinical,food and environmental samples

Because of the potential severity of infections caused by Escherichia coli O157 it is important that the most sensitive laboratory methods are used both for outbreak investigation and surveillance. Selective culture of E. coli O157 remains the detection method of choice, particularly in investigation of outbreaks where strains isolated from various sources may need to be compared by various typing methods. Strains of E. coli O157 do not normally ferment sorbitol, whereas many other serogroups of E. coli do, and sorbitol MacConkey agar, or modified forms of this medium, have become widely used for their isolation. Detection of small numbers of E. coli O157 may be facilitated by enrichment culture which may include a recovery period during which selective agents are not added to the medium. Immunomagnetic separation of E. coli O157 after enrichment culture enhances sensitivity still further and has the potential to be fully automated. Alternatives to culture include immunoassays and PCR, both of which are available as commercial detection kits. The last 15 years has seen many advances in detection of E. coli O157 and has been accompanied by a plethora of reports in the scientific literature. However, it is an area which is continually developing and we are still far away from a universally accepted method for this purpose. This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Prevalence of Stx Phages in Environments of a Pig Farm and Lysogenic Infection of the Field <Emphasis Type="Italic">E. coli</Emphasis> O157 Isolates with a Recombinant Converting Phage

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.     

Inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by cinnamon extracts in carrot-kinnow mandarin blends

We investigated the antimicrobial efficacy of cinnamon extracts in laboratory prepared Kinnow-mandarin carrot blends challenged with Escerichia coli O157:H7. Freshly squeezed carrot and kinnow-mandarin juices were mixed to obtain a typical blend, inoculated with E. coli O157:H7 cultures at 10² CFU/mL with and without cinnamon extracts (0.3%) and stored at 4, 8 and 28°C for up to 10 hours. Counts on tryptic soy agar (TSA) selective medium (Mac conkey sorbitol agar) and thin agar layer (TAL) were determined at every 2 hour. The TAL method was used for recovery of sublethally injured cells. Inactivation of E. coli O157:H7 in blends containing 0.3% cinnamon extracts were observed, with killing effects being more pronounced at 28°C, similar trends were evident with blends stored under refrigeration conditions (4 and 8°C).The decrease in counts were attributed to several factors namely, pH, storage temperature and addition of cinnamon. The results of our study indicate that cinnamon extracts could be used as an effective, natural antimicrobial for assuring consumer safety at the point of preparation of carrot-kinnow mandarin blends, which is a popular, nutritional beverage consumed in India.     

Sensitivity of Shiga toxin-producing Escherichia coli (STEC) strains for colicins under different experimental conditions

Twenty Escherichia coli strains producing well-characterised colicins were tested for their inhibitory activity against five Shiga toxin-producing E. coli (STEC) strains using different media under aerobic and anaerobic conditions. The five STEC strains used were of serotype O26, O111, O128, O145 and O157:H7 which are frequently isolated serotypes associated with disease in humans. The main route of infection for humans is through the eating of badly cooked or handled beef. The major reservoir for STEC strains in cattle is the rumen. To mimic the situation in the rumen of cattle, overlay assays were also performed under anaerobic conditions in the presence of 30% rumen fluid. Colicins E1, E4, E8-J, K and S4 are most active against STEC strains under anaerobic conditions in the absence or presence of rumen fluid. These colicins will be used in future experiments with the aim to eradicate the presence of STEC in cattle.     

Serogroup distribution and virulence characteristics of sorbitol‐negative Escherichia coli from food and cattle stool

Aims: To (i) study the serogroup distribution and virulence characteristics of non‐sorbitol‐fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re‐examine the true sorbitol and β‐d ‐glucuronidase (GUD) reactions of sorbitol‐negative (Sor^?) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157. Methods and Results: One hundred and thirty Sor^?E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite–supplemented SMAC (CT‐SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O‐rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O‐rough and OUT strains were enterohaemolytic. Further, 39·2% and 63·1% of Sor^? isolates from CT‐SMAC fermented sorbitol in phenol red broth and hydrolysed 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O‐rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157. Conclusions: Sor^?E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14·6% of the isolates. Many of these nonclinical Sor^? strains were potentially pathogenic. Nearly 39% of these Sor^?E. coli from CT‐SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth. Significance and Impacts of the Study: Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.     

A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples

Background

Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains.

Methods and Findings

Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity.

Conclusions

The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.     

Effect of organic acids on inhibition of Escherichia coli O157:H7 colonization in gnotobiotic mice associated with infant intestinal microbiota

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 became carriers. In this study, we analysed the mechanisms of E. coli O157:H7 elimination using GB-3 and GB-4. When GB-3 and GB-4 mice were challenged with E. coli O157:H7, the E. coli O157:H7 population was reduced in the caecum of GB-3 when compared to that in the GB-4 caecum, although the numbers of E. coli O157:H7 in the small intestine were not significantly different between these two groups of gnotobiotic mice. The lag time of E. coli O157:H7 growth in a 50% GB-3 caecal suspension increased when compared to that in a GB-4 caecal suspension. Acetate and lactate were detected in the GB-3 caecal contents, and acetate and propionate in those from GB-4. Although E. coli O157:H7 growth was not suppressed when it was cultured in anaerobic broth supplemented with these organic acids, the motility of E. coli O157:H7 was suppressed when it was cultured on semi-solid agar supplemented with the combination of acetate and lactate. These results indicate that the organic acid profile in the caecum is an important factor related to the elimination of E. coli O157:H7 from the intestine.     

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment

Aims: To develop a real‐time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin‐producing E. coli (STEC) serotypes classified in seropathotype C. Methods and Results: The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan^® minor groove binder probe specific for fliC‐H21 were designed and used in a 5′‐nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC‐H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony‐forming units per 25 g of ground beef was detected after overnight enrichment. Conclusions: The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. Significance and Impact of the Study: The real‐time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.     

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.     

Heterogeneity in Induction Level,Infection Ability,and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.     

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.     

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.     

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.