Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.     

Growth of starved Escherichia coli O157 cells in selective and non-selective media.

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter-sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non-selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non-selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non-starved cells from overnight cultures with an initial density of less than 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a "resuscitation" of the cells with non-selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.     

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.     

Rapid and specific detection of escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 in ground beef, beef trim, and produce by loop-mediated isothermal amplification

Escherichia coli O157 and six additional serogroups of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (wzx or wzy) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 10(3) to 10(4) CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing.     

Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.     

Methods for the detection and isolation of Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) are an important cause of haemorrhagic colitis and the diarrhoea-associated form of the haemolytic uraemic syndrome. Of the numerous serotypes of E. coli that have been shown to produce Shiga toxin (Stx), E. coli O157:H7 and E. coli O157:NM (non-motile) are most frequently implicated in human disease. Early recognition of STEC infections is critical for effective treatment of patients. Furthermore, rapid microbiological diagnosis of individual patients enables the prompt notification of outbreaks and implementation of control measures to prevent more cases. Most human infections caused by STEC have been acquired by the consumption of contaminated foods, especially those of bovine origin such as undercooked ground beef and unpasteurized cows' milk, and by person-to-person contacts. To identify the reservoirs of STEC and the routes of transmission to man, sensitive methods are needed as these pathogens may only be present in food, environmental and faecal samples in small numbers. In addition, sensitive and rapid detection methods are necessary for the food industry to ensure a safe supply of foods. Sensitive methods are also needed for surveillance programmes in risk assessment studies, and for studies on survival and growth of STEC strains. Cultural methods for the enrichment, isolation and confirmation of O157 STEC are still evolving. Several selective enrichment media have been described, of which modified tryptone soy broth with novobiocin and modified E. coli broth with novobiocin, seem to be the most appropriate. These media are minimally-selective broths that give a somewhat limited differential specificity favouring isolation of O157 STEC, as opposed to other Gram-negative bacteria, in the sample. An incubation temperature of 41-42 degrees C further enhances selectivity. The occurrence of heat-, freeze-, acid- or salt-stressed STEC in foods means that it is important to be able to detect cells that are in a stressed state, as STEC generally have a very low infectious dose, and injured cells mostly retain their pathogenic properties. For the isolation of stressed O157 STEC, pre-enrichment in a non-selective broth is necessary. The most widely used plating medium for the isolation of typical sorbitol-non-fermenting strains of STEC of serogroup O157 is sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC). As some STEC strains are sensitive for tellurite and/or are sorbitol-fermenting, the use of a second isolation medium, such as one of the newer chromogenic media, is recommended. Immunomagnetic separation (IMS) following selective enrichment, and subsequent spread-plating of the concentrated target cells onto CT-SMAC agar, appears to be the most sensitive and cost-effective method for the isolation of E. coli O157 from raw foods. IMS increases sensitivity by concentrating E. coli O157 relative to background microflora, which may overgrow or mimic O157 STEC cells on selective agars. While cultural isolation of O157 STEC from foods and faeces is time-consuming, labour-intensive and hence, costly, rapid immunological detection systems have been developed which significantly reduce the analysis time. These methods include enzyme-linked immunosorbent assays (ELISAs), colony immunoblot assays, direct immunofluorescent filter techniques, and several immunocapture techniques. Both polyclonal and monoclonal antibodies specific for the O and H antigens are used for these methods. Many of these test systems are able to detect less than one O157 STEC cell g(-1) of raw meat after overnight enrichment. Presumptive results are available after just one day, but need to be completed with the isolation of the organisms. The primary use of these procedures is therefore to identify food and faecal samples that possibly contain O157 STEC.     

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.     

Modeling and Predicting the Simultaneous Growth of Escherichia coli O157:H7 and Ground Beef Background Microflora for Various Enrichment Protocols

The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37°C or 40°C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40°C) did not inhibit BM growth, and incubation at 40°C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.     

Modeling and predicting the simultaneous growth of Escherichia coli O157:H7 and ground beef background microflora for various enrichment protocols

The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37 degrees C or 40 degrees C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40 degrees C) did not inhibit BM growth, and incubation at 40 degrees C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.     

Light Scattering Sensor for Direct Identification of Colonies of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145 and O157

Background

Shiga-toxin producing Escherichia coli (STEC) have emerged as important foodborne pathogens, among which seven serogroups (O26, O45, O103, O111, O121, O145, O157) are most frequently implicated in human infection. The aim was to determine if a light scattering sensor can be used to rapidly identify the colonies of STEC serogroups on selective agar plates.

Methodology/Principal Findings

Initially, a total of 37 STEC strains representing seven serovars were grown on four different selective agar media, including sorbitol MacConkey (SMAC), Rainbow Agar O157, BBL CHROMagarO157, and R&F E. coli O157:H7, as well as nonselective Brain Heart Infusion agar. The colonies were scanned by an automated light scattering sensor, known as BARDOT (BActerial Rapid Detection using Optical scattering Technology), to acquire scatter patterns of STEC serogroups, and the scatter patterns were analyzed using an image classifier. Among all of the selective media tested, both SMAC and Rainbow provided the best differentiation results allowing multi-class classification of all serovars with an average accuracy of more than 90% after 10–12 h of growth, even though the colony appearance was indistinguishable at that early stage of growth. SMAC was chosen for exhaustive scatter image library development, and 36 additional strains of O157:H7 and 11 non-O157 serovars were examined, with each serogroup producing unique differential scatter patterns. Colony scatter images were also tested with samples derived from pure and mixed cultures, as well as experimentally inoculated food samples. BARDOT accurately detected O157 and O26 serovars from a mixed culture and also from inoculated lettuce and ground beef (10-h broth enrichment +12-h on-plate incubation) in the presence of natural background microbiota in less than 24 h.

Conclusions

BARDOT could potentially be used as a screening tool during isolation of the most important STEC serovars on selective agar plates from food samples in less than 24 h.     

A chromogenic plating medium for isolating Escherichia coli O157:H7 from beef

There was no significant difference (P > 0.05) in the percentages of Escherichia coli O157:H7 cells recovered on BCM O157:H7 (+) agar (69.7%) and MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid (MSA-BCIG) (76.8%) vs Tryptic soy agar. Three E. coli O157:H7 strains (ATCC 35150, 43890 and 43894) were separately inoculated into raw ground beef at low (mean 0.32 cfu g-1) and high (mean 3.12 cfu g-1) levels. Using the United States Department of Agriculture (USDA) m-EC + novobiocin enrichment broth, BCM O157:H7 (+) medium surpassed MSA-BCIG agar with overall percentage sensitivities for BCM O157:H7 (+) of 92.1 and 94.4 compared with 52.6 and 84.7 for MSA-BCIG at low and high levels, respectively. A comparison of BCM O157:H7 (+) and MSA-BCIG agars using naturally contaminated beef samples was made utilizing presumptively positive enrichment broths previously identified by rapid methods. The E. coli O157:H7 cells in these broths were concentrated with Dynabeads anti-E. coli O157 before inoculating the agars. The respective percentage sensitivity and specificity values were 90.0 and 78.5 for BCM O157:H7 (+) and 70.0 and 46.4 for MSA-BCIG. Thus, under identical pre-plating conditions, BCM O157:H7 (+) medium displayed a greater sensitivity than MSA-BCIG for detecting E. coli O157:H7 in artificially inoculated beef, and both greater sensitivity and specificity upon examining naturally contaminated beef samples.     

Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar

The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.     

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Detection and Long-Term Existence of Shiga Toxin (Stx)-Producing Escherichia coli in Sheep

The isolation and characterization of Shiga-like toxin (Stx)-producing Escherichia coli (STEC) from sheep are described. The distribution of stx genes in E. coli isolates was detected by PCR. When brain heart infusion (BHI) broth and novobiocin supplemented m-EC broth (N-mEC) were used as enrichment culture for the isolation of STEC, N-mEC, compared to BHI, showed clearly lower efficiency. Finally, 5 STEC isolates from 4 sheep were isolated and characterized by biochemical and genetical analysis. All of them were confirmed by ELISA and Vero cell cytotoxicity assay for the production of Stx. Moreover, some strains carried hemolysin and eaeA genes and harbored large plasmids. Based on their plasmid profiles, antibiotic patterns and PCR-based DNA fingerprinting analysis using random amplified polymorphic DNA (RAPD), all isolates were different from each other. Three of the isolates were identified to belong to serogroups O2, O153 and O165, respectively, and the STEC strains belonging to these serogroups had been isolated from STEC outbreaks in humans. Four months after the first isolation in July 1997, STEC from sheep #1 was isolated again. A new isolate, HI-11, was identified as STEC O2: Hnt. Simultaneously, 2 STEC, which were genetically and phenotypically different from each other, were isolated from the same sheep at intervals of 4 months. These results demonstrate that sheep may be an important animal for studying human STEC infections, and that further epidemiological surveys on STEC are necessary.     

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens capable of causing hemolytic-uremic syndrome. STEC O157:H7 strains cause the majority of severe disease in the United States; however, there is a growing concern for the amount and severity of illness attributable to non-O157 STEC. Recently, the Food Safety and Inspection Service (FSIS) published the intent to regulate the presence of STEC belonging to serogroups O26, O45, O103, O111, O121, and O145 in nonintact beef products. To ensure the effective control of these bacteria, sensitive and specific tests for their detection will be needed. In this study, we identified single nucleotide polymorphisms (SNPs) in the O-antigen gene cluster that could be used to detect STEC strains of the above-described serogroups. Using comparative DNA sequence analysis, we identified 22 potentially informative SNPs among 164 STEC and non-STEC strains of the above-described serogroups and designed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) assays to test the STEC allele frequencies in an independent panel of bacterial strains. We found at least one SNP that was specific to each serogroup and also differentiated between STEC and non-STEC strains. Differences in the DNA sequence of the O-antigen gene cluster corresponded well with differences in the virulence gene profiles and provided evidence of different lineages for STEC and non-STEC strains. The SNPs discovered in this study can be used to develop tests that will not only accurately identify O26, O45, O103, O111, O121, and O145 strains but also predict whether strains detected in the above-described serogroups contain Shiga toxin-encoding genes.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

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.