The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

The optimization of isolation media used in immunomagnetic separation methods for the detection of Escherichia coli O157 in foods

AIMS: To compare media used in immunomagnetic separation (IMS) techniques for the isolation of Escherichia coli O157 from food. METHODS AND RESULTS: Foods, both naturally contaminated and spiked, with low numbers (< 1 g(-1)) of stressed E. coli O157 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at 30, 37, 40 and 42 degrees C. Following immunomagnetic separation, beads were plated on a range of selective agars. CONCLUSION: BPW supplemented with vancomycin (8 mg l(-1)) incubated at 42 degrees C, followed by IMS and subsequent plating of immunobeads onto cefixime tellurite sorbitol MacConkey agar plus either Rainbow or CHROMagar agars, proved optimum for the recovery of spiked, stressed E. coli O157 in minced beef, cheese, apple juice and pepperoni. The same protocol was optimum for recovery from naturally-contaminated minced beef and cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum protocol would increase isolation rates of E. coli O157 from foods.     

Methods for the isolation of water-borne Escherichia coli O157

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

Direct inoculation into media containing bile salts and antibiotics is unsuitable for the detection of acid/salt stressed Escherichia coli O157:H7

The efficiency of selective enrichment broths for the recovery of low numbers of acid/salt stressed Escherichia coli O157:H7 was determined. Stressed cultures were diluted to low levels and recovered in tryptone soya broth with added bile salts, to make modified tryptone soya broth, and buffered peptone water with various combinations of antibiotic supplementation including novobiocin, acriflavine and a mixture of vancomycin, cefsulodin and cefixime (VCC) at 37 °C and 42 °C. Significantly fewer stressed cells, in some cases as little as 0·3% of the starting population, were recovered by all the selective enrichment broths containing bile salts or VCC antibiotics compared to the non-selective controls. The use of such enrichments to recover low numbers of stressed E. coli O157:H7 may result in failure to detect the organism. Parallels with salmonella methodology are made and the need for a non-selective pre-enrichment stage in E. coli O157:H7 methods discussed.     

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.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

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.     

Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique

AIMS: Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to magnetic beads to bind and extract cells from enrichment broths followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain variation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/CT-SMAC plates. METHODS AND RESULTS: Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. CONCLUSIONS: Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. SIGNIFICANCE AND IMPACT OF THE STUDY: If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present.     

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.     

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.     

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures

A.R. BENNETT, S. MACPHEE AND R.P. BETTS. 1996. The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti- E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY OF HEAT-STRESSED ESCHERICHIA COLI O157:H7

Tryptone soya agar (TSA) and three selective media, BCM^1M O157:H7(+) agar (BCM), modified eosin methylene blue agar (MEMB), and sorbitol MacConkey agar (SMAC) were evaluated for recovery of two strains of E. coli O157:H7 (salami and cider isolates) heated at 56, 58, and 60C for up to 60 min in tryptone soya broth (TSB). TSA and MEMB were equally effective at recovery of heat-stressed (56, 58, and 60C) E . coli O 157:H7 and superior to SMAC and BCM (P 0.05). When heated at 56 and 58C, recovery of E. coli O157:H7 on MEMB and TSA was not significantly different (P > 0.05); recovery was poorer on SMAC, followed by BCM (P 0.05). There was no significant difference in recovery of E. coli O157:H7 on BCM and SMAC when strains were heated at 60C (P > 0.05).     

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.     

Isolation of vero cytotoxin-producing Escherichia coli O157 from wild birds

In a survey of wild birds (mainly gulls), 0.9% of the bacterial isolates from faecal samples at an urban landfill site and 2.9% of bacterial isolates from faecal samples on intertidal sediments in Morecambe Bay were Vero cytotoxin-producing Escherichia coli O157. Isolation procedures employing commonly used cultural methods were hindered by the selection of a large number of false positives. The only procedure which resulted in the isolation of E. coli O157 from bird faecal samples was: enrichment (18 h) in a selective tryptone soya broth followed by filtration using hydrophobic grid membranes and growth on Chromagar O157. The majority of isolates selected as potential E. coli O157 by characteristic growth on Chromagar O157 could be eliminated by subsequent growth on CT-SMAC or CR-SMAC. This second identification (characterization) stage reduced the number of potential E. coli O157 requiring further confirmation by typing methods (serotype and Vero cytotoxin) by more than 70%.     

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef

AIM: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. METHODS AND RESULTS: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41.5 degrees C in tryptone soya broth supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l(-1))] and supplemented with cefixime (50 microg ml(-1)) and potassium tellurite (2.5 mg l(-1)). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 microg ml(-1)), cefsulodin (5 mg l(-1)) and vancomycin (8 mg l(-1)). Minced beef samples were inoculated with a number of strains of E. coli O26 (n=9) and O111 (n=8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. CONCLUSIONS: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef.     

Long-term survival of Escherichia coli O157 on pasture following an outbreak associated with sheep at a scout camp

AIMS: To monitor the decay of E. coli O157 in soil (loamy sand) on a scout campsite following an outbreak in humans. METHODS AND RESULTS: Samples of soil and sheep faeces were collected from the campsite and tested for the presence of E. coli O157 by immunomagnetic separation (IMS) after enrichment in buffered peptone water + vancomycin at 42 degrees C for 6 h. Enumeration of target was carried out by direct plating onto sorbitol MacConkey agar plates supplemented with cefixime and tellurite (CTSMAC) incubated at 37 degrees C for 24 h. Low numbers (< 100 g(-1)) were estimated by the most probable number (3-tube MPN) technique. CONCLUSIONS: Survival was observed for 15 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: A number of laboratory studies have followed the decay of E. coli O157 in soil, animal faeces and water. This study follows (for the first time) the decay of the organism in soil after an outbreak associated with sheep. It demonstrates the long-term persistence of the organism in the environment and the results will be potentially important in performing risk assessments for both human and animal infection.     

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.     

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.     

Combined use of an immunomagnetic separation method and immunoblotting for the enumeration and isolation of Escherichia coli O157 in wastewaters

AIMS: The detection of Escherichia coli O157:H7 in environmental samples is a human concern. The high persistence of this serotype in the environment suggests that contaminated animal wastewater could act as a potential reservoir. Nevertheless, the high levels of background microflora and cell damage because of environmental stress hamper the isolation of this pathogen without using enrichment methods. This study develops a method for the detection of E. coli and investigates its prevalence in animal and human wastewaters. METHODS AND RESULTS: Incubation of the sample for 1 h 30 min at 37 degrees C in peptone water supplemented with vancomycin and cefsulodin, enhanced the recovery of bacteria whilst ensuring that no growth occurred. Subsequently, a combination of immunomagnetic separation, cefixime-tellurite-sorbitol MacConkey (CT-SMAC) plating and immunoblotting with specific O157 antibodies allowed the detection, enumeration and isolation of E. coli O157 strains in human, swine and cattle wastewaters, which presented values of 0.2, 0.4, and 1.0 log10 ml(-1) units, respectively. Some of the isolates carried genes coding for Shiga toxins, intimin and enterohemolysin. CONCLUSIONS: Escherichia coli O157 is commonly present in animal and human wastewaters. The developed method reduced the high rate of false positives reported for other technical approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The confirmation of serotype by specific immunomethods is necessary to prevent false-positive detection and incorrect enumeration.     

Detection and recovery rates achieved using direct plate and enrichment/immunomagnetic separation methods for Escherichia coli O157:H7 in minced beef and on bovine hide

AIMS: To assess the detection and recovery rates achieved with commonly used cultural methods for the enumeration and recovery of Escherichia coli O157:H7 from minced beef and bovine hide. METHODS AND RESULTS: Minced beef and bovine hide were inoculated with varying concentrations (log(10) 1.58-2.58 CFU g(-1) and log(10) 2.42-4.49 CFU 100 cm(2) respectively) of E. coli O157:H7 and recovered using a direct plate method or an enrichment/immunomagnetic separation (IMS) method and then plated onto SMAC or SMAC-CT in both cases. The direct plate method detected the pathogen consistently from minced beef samples with an average recovery of 69.2-91.2%. From faecal material on the bovine hide the recovery of the pathogen ranged from 1.80 to 64.5% with fresh faeces depending on the inocula while from dried faeces on hide the results ranged from no recovery at all to 25.1%. Enrichment/IMS recovered E. coli O157:H7 at all inocula levels tested in minced beef while the pathogen was only detected consistently at an average inocula level of log(10) 2.73 CFU 100 cm(2) from fresh faeces and log(10) 4.49 CFU 100 cm(2) from dried faeces on bovine hide. CONCLUSIONS: The direct count enumeration method for E. coli O157:H7 underestimated the numbers of pathogens present. The enrichment/IMS procedure consistently detected the pathogen from minced beef but did not always detect E. coli O157:H7 from faeces on bovine hide. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall this study highlights that any microbial data, used in either predictive microbiology or risk assessment, must take account of the sensitivity and associated performance of the methods employed, in order to make an accurate reflection of the true microbiology of the examined sample.