Efficacy of a post enrichment acid treatment for isolation of Escherichia coli O157:H7 from alfalfa sprouts

The enrichment, detection and isolation procedure in the current US FDA BAM have been shown effective for Escherichia coli O157:H7 in a wide variety of foods. Recently reported modifications to the enrichment protocol, including post-enrichment immunomagnetic separation (IMS) procedures have improved sensitivity of the method for alfalfa sprouts. However, cultural isolation on selective agar plates still presents a challenge in this food matrix.     

Optimization and evaluation of a modified enrichment procedure combined with immunomagnetic separation for detection of E. coli O157:H7 from artificially contaminated alfalfa sprouts

Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with alfalfa sprouts. Detection of the organism in sprouts by standard cultural methods can be difficult due to the high background microflora. The objective of this study was to develop and optimize an enrichment protocol with and without post-enrichment immunomagnetic separation (IMS) for the rapid detection by real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure, enriching samples in modified buffered peptone water with pyruvate and at 37 °C for 5 h, followed by the addition of acriflavin, cefsulodin and vancomycin (mBPWp + ACV) and static incubation at 42 °C gave poor results for both PCR detection and isolation for alfalfa sprouts artificially contaminated at 0.2 cfu/g. The addition of post-enrichment IMS improved detection but not isolation. This procedure was modified and optimized by changing to mBPWp with cefsulodin and vancomycin at 42 °C and shaking for 24 h with and without IMS prior to PCR detection and cultural isolation. Using the resulting protocol we were able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated at 0.2 cfu/g. This was validated for five strains of E. coli O157:H7. Isolation was 84% without added post-enrichment IMS and 100% with IMS. The optimized procedure was effective for detection and isolation of E. coli O157:H7 from this difficult food matrix.     

Survival of Escherichia coli O157:H7, O111:H- and O26:H11 in artificially contaminated chocolate and confectionery products

To date, the survival of Escherichia coli O157:H7 and other verocytotoxin-producing E. coli (VTEC) in chocolate and other confectionery products has not been fully established, unlike Salmonella, which have been responsible for occasional outbreaks of infection linked to contaminated chocolate and related products, although none of these outbreaks have been related to products produced in the United Kingdom. The United Kingdom Biscuit, Cake, Chocolate and Confectionery Alliance commissioned this study to obtain information on the decline and potential survival of E. coli, particularly verocytotoxin-producing strains, in reduced aw confectionery products chocolate, biscuit cream and mallow. These products were artificially contaminated with high (4 log10 cfu/g) and low (2 log10 cfu/g) levels of E. coli O157:H7, O111:H- and O26:H11 and their survival, as affected by storage temperature (10, 22 and 38 degrees C), was monitored over 12 months. Preliminary studies to establish the best inoculation and recovery procedures indicated that differences between counts on selective and non-selective media used were not sufficiently different to influence the outcome of this study. Irrespective of sample type, rapid decline was observed in products stored at 38 degrees C and increased survival occurred in products stored at 10 degrees C. In chocolate (average aw 0.40), these bacteria were detected for up to 43 days in samples stored at 38 degrees C. At 22 degrees C they survived for up to 90 days and in product stored at 10 degrees C they could still be detected after 366 days storage. In biscuit cream (average aw 0.75) they survived for 2 days at 38 degrees C, 42 days at 22 degrees C and 58 days at 10 degrees C. Whilst mallow (aw ca. 0.73) was not stored at 38 degrees C, these bacteria could still be detected in samples stored for up to 113 and 273 days at 22 and 10 degrees C, respectively. The observed prolonged survival of these bacteria under conditions of reduced aw and lowered storage temperature in this study is supported by previous studies with Salmonella and E. coli O157:H7 in other foods. In the same way that Salmonella bacteria can survive for long periods, in excess of 12 months, in chocolate, this study provides evidence that E. coli, including pathogenic strains, can also survive for similar periods of time. Assuming the routes of transmission are similar, controls currently used by the confectionery industry to prevent contamination by Salmonella should also be effective against E. coli, including VT-producing strains, providing that all raw materials have been suitably processed, stored and handled before and during manufacture.     

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.     

Comparison of enrichment procedures for isolation of Escherichia coli O157:H7 from ground beef and radish sprouts

Enrichment procedures using Tryptic soy broth (TSB), modified TSB (mTSB), modified E. coli broth with novobiocin (mEC+n), mTSB (without novobiocin) with vancomycin, cefixime and cefsulodin (mTSB-VCC), or TSB with cefixime, tellurite and vancomycin (TSB-CTV) were evaluated by determining the rate of successful isolation of fifteen Escherichia coli O157:H7 strains from inoculated broth containing ground beef or radish sprout extract. E. coli O157:H7 tended to be isolated more efficiently after enrichment with TSB, mTSB and mEC+n than with the other broths. In order to identify the most efficient enrichmemt condition using these broths, E. coli O157:H7 were inoculated into 25 g ground beef or radish sprouts, which were then homogenized in 225 ml broth and incubated static at 37°C or 42°C for 6 h or 18 h. Attempts were made to isolate the inoculated bacteria by plating method in combination with the immunomagnetic separation method. The most effective enrichment condition was incubation in mTSB or mEC+n at 42°C for 18 h for ground beef, and in mEC+n at 42°C for 18 h for radish sprouts.     

Comparison of Escherichia coli O157:H7 enrichment in spiked produce samples

Two strains of Escherichia coli O157:H7 were spiked into six varieties of produce at approximately 0.5 CFU g(-1). Samples were enriched by using the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) method and by using an experimental method incorporating acid shock. Target colonies were detectable on selective agars after 30 of 48 analyses with BAM enrichment and 48 of 48 analyses with acid enrichment. Real-time PCR screening of 24-h enrichment broths revealed the presence of the diagnostic stx1 or stx2 genes after 27 of 48 analyses with BAM enrichment and 42 of 48 analyses with acid enrichment. The efficiency of the enrichment varied with strain and type of produce spiked but overall was better with the experimental enrichment method. Modifications of both the acid enrichment and BAM enrichment methods also were tested. The acid method with a modified incubation temperature consistently yielded high rates of recovery (> 10(8) CFU ml(-1)), with no instances in which target cells could not be detected. Modification of the BAM procedure did not reproducibly improve enrichment efficiency.     

Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

Evaluation of increased incubation temperature and cefixime-tellurite treatment for the isolation of Escherichia coli O157:H7 from minced beef

To improve enrichment and isolation of Escherichia coli O157:H7, this study evaluated increased incubation temperature and cefixime-tellurite (CT) on five strains of each of the following bacteria, E. coli, Hafnia alvei, Enterobacter spp., Citrobacter freundii and E. coli O157:H7, and two strains of E. coli O157:nH7. These were grown in pure culture in LST broth with varying cefixime-tellurite concentrations. A range of incubation temperatures from 37 to 46 degrees C was investigated for the inhibition of cohabitant microorganisms. Minced beef, spiked with E. coli O157:H7 and cohabitant microorganisms was investigated. Increased incubation temperature (42 degrees C) and treatment with half of the prescribed amount of cefixime-tellurite by BAM for SMAC agar in enrichment step were the most effective in selectively growing E. coli O157:H7. The results show that E. coli O157:H7 is more resistant to these two conditions than the other cohabitant bacteria.     

Sensitive detection of Escherichia coli O157:H7 by conventional plating techniques

The direct detection and estimation of concentration of Escherichia coli O157:H7 down to 1 CFU/g of cheese was achieved by conventional plating techniques. Cheese was manufactured with unpasteurized milk inoculated with E. coli O157: H7 at 34 +/- 3 CFU/ml. The numbers of E. coli O157:H7 were monitored during cheese ripening by plating on sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) and on CT-O157:H7 ID medium. Using the pour plate method, E. coli O157:H7 colonies could easily be distinguished from non-O157:H7 colonies on CT-O157:H7 ID medium but not on CT-SMAC. Higher numbers of E. coli O157:H7 were detectable with O157:H7 ID medium. Latex agglutination and PCR were used to confirm the identification of typical E. coli O157:H7 colonies, and nontypical colonies as not being E. coli O157:H7. As few as 1 CFU/g of cheese could be detected. E. coli O157:H7 also was detected in deliberately contaminated milk at concentrations as low as 4 CFU/10 ml.     

Reduction of Escherichia coli O157:H7 and Salmonella typhimurium in artificially contaminated alfalfa seeds and mung beans by fumigation with ammonia.

Sprouts eaten raw are increasingly perceived as hazardous foods because they have been vehicles in outbreaks of foodborne disease, often involving Escherichia coli O157:H7 and Salmonella Typhimurium. Although the source of these pathogens has not been established, it is known that the seeds usually are already contaminated at the time sprouting begins. Earlier studies had shown that ammonia was lethal to these same pathogens in manure, so it seemed reasonable to determine whether ammonia was effective against them when associated with seeds to be used for sprouting. Experimentally contaminated (10(8) to 10(9) CFU/g) and dried seeds, intended for sprouting, were sealed in glass jars in which 180 or 300 mg of ammonia/liter of air space was generated by action of ammonium sulfate and sodium hydroxide. Samples were taken after intervals up to 22 h at 20 degrees C. Destruction of approximately 2 to 3 logs was observed with both bacteria associated with alfalfa seeds, versus 5 to 6 logs with mung beans. Greater kills are apparently associated with lower initial bacterial loads. Germination of these seeds was unaffected by the treatment. It appears that this simple treatment could contribute significantly to the safety of sprout production from alfalfa seeds and mung beans.     

Evaluation of standard enrichment broths for recovery of healthy and chlorine-injured Escherichia coli O157:H7 cells in kimchi

Food Science and Biotechnology - This study aimed to evaluate three standard enrichment broth preparations for the recovery of healthy and chlorine-injured E. coli O157:H7 cells in kimchi. The...     

Treatment of Escherichia coli O157:H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidizing water

Electrolyzed oxidizing water is a relatively new concept that has been utilized in agriculture, livestock management, medical sterilization, and food sanitation. Electrolyzed oxidizing (EO) water generated by passing sodium chloride solution through an EO water generator was used to treat alfalfa seeds and sprouts inoculated with a five-strain cocktail of nalidixic acid resistant Escherichia coli O157:H7. EO water had a pH of 2.6, an oxidation-reduction potential of 1150 mV and about 50 ppm free chlorine. The percentage reduction in bacterial load was determined for reaction times of 2, 4, 8, 16, 32, and 64 min. Mechanical agitation was done while treating the seeds at different time intervals to increase the effectiveness of the treatment. Since E. coli O157:H7 was released due to soaking during treatment, the initial counts on seeds and sprouts were determined by soaking the contaminated seeds/sprouts in 0.1% peptone water for a period equivalent to treatment time. The samples were then pummeled in 0.1% peptone water and spread plated on tryptic soy agar with 5 microg/ml of nalidixic acid (TSAN). Results showed that there were reductions between 38.2% and 97.1% (0.22-1.56 log(10) CFU/g) in the bacterial load of treated seeds. The reductions for sprouts were between 91.1% and 99.8% (1.05-2.72 log(10) CFU/g). An increase in treatment time increased the percentage reduction of E. coli O157:H7. However, germination of the treated seeds reduced from 92% to 49% as amperage to make EO water and soaking time increased. EO water did not cause any visible damage to the sprouts.     

Evaluation of a novel enrichment procedure for the isolation of Escherichia coli O157 from naturally contaminated raw beef,lamb and mixed meat products

The aim of this study was to compare enrichment at 41·5°C for 24 h in a prototype enrichment broth developed by Oxoid, UK (sTSB), with enrichment at 37°C for 6 h in buffered peptone water supplemented with vancomycin, cefixime and cefsulodin (BPW-VCC) for isolation of Escherichia coli O157 from naturally contaminated raw beef, lamb and mixed meat products.A total of 120 meat samples from previous surveillance studies, 91 of which were previously positive and 29 previously negative for E. coli O157, were used for the study. All had been stored at −70°C for between 19 and 58 months since first examination. Of the 91 previously positive samples, 67 were positive after enrichment in sTSB and 42 were positive after enrichment in BPW-VCC;E.coli O157 was not recovered from 20 previously positive samples by either method. Of the 29 previously negative samples, three were positive by enrichment in sTSB and two were positive by enrichment in BPW-VCC;E.coli O157 was not recovered from 25 previously negative samples by either method.Of the 71 isolates obtained from the meat samples, only 53 had the same toxin genotype and plasmid profile as the E.coli O157 isolated from the sample originally. On four occasions, the strain isolated originally, the strain isolated from re-culture in BPW-VCC and the strain isolated from sTSB were all different. The only feasible explanation for these differences is that two or more different strains of E.coli O157 were present in the original sample. Results of strain typing ofE.coli O157 isolated from food samples, particularly during an outbreak investigation, should therefore be interpreted with caution.     

Development of a medium for differentiation between Escherichia coli and Escherichia coli O157:H7.

A new medium (Escherichia coli O157:H7 medium: EOH) was developed for differentiation between E. coli and E. coli O157:H7. The EOH medium was compared with sorbitol MacConkey agar (SMAC), which is the most popular medium to enumerate E. coli O157:H7. Several combinations of 35 dyes were evaluated to develop the new medium. Indigo carmine (0.03) g/liter) and phenol red (0.036 g/liter) were found as the best combination for differentiation between E. coli O157:H7 and E. coli and added to the basal agar medium (SMAC medium excluding neutral red and crystal violet) for EOH medium. On the dark blue EOH medium, E. coli produced a yellow color with clear zone, whereas E. coli O157:H7 produced a red color without clear zone. For differentiation between E. coli and E. coli O157:H7, EOH has much better potential than SMAC. Furthermore. the red color produced by normal E. coli in SMAC may mask the light gray color produced by E. coli O157: H7, whereas the yellow color with clear zone did not mask the red color without clear zone in the EOH medium. The recovery numbers of E. coli O157:H7 from inoculated ground beef, pork, and turkey were not significantly different between SMAC and EOH media (P > 0.05). The recovery rates of heat- and cold-injured E. coli O157:H7 also were not significantly different (P > 0.05).     

免疫磁分离法高效富集牛奶中大肠杆菌O157:H7

目的建立一种免疫磁分离(immunomagnetic separation,IMS)方法高效富集大肠杆菌O157:H7。方法合成一种核壳型的纳米磁珠(magnetic nanobeads, MNBs),并基于制备的MNBs构建了IMS。通过优化制备免疫MNBs时抗体浓度, IMS过程免疫MNBs的用量和孵育时间,构建了高效的IMS方法。结果构建的IMS方法能够在35 min内完成牛奶中大肠杆菌O157:H7的高效富集,当大肠杆菌O157:H7浓度低于10~5 CFU/m L时,捕获效率高于93.4%,当菌浓度达到10~7CFU/mL,捕获效率仍大于50%。结论该方法简单高效,可被广泛应用于其他食源性致病菌检测的样品前处理。     

Experimental use of a gas sensor-based instrument for differentiation of Escherichia coli O157:H7 from non-O157:H7 Escherichia coli field isolates

The rapid and economical detection of human pathogens in animal and food production systems would enhance food safety efforts. An instrument based on gas sensors coupled with an artificial neural network (ANN) was developed for the detection of and differentiation between laboratory isolates of Escherichia coli O157:H7 and non-O157:H7 E. coli. The purpose of this study was to use field isolates of E. coli to further evaluate the sensor system. This gas sensor-based, computer-controlled detection system was used to monitor gas emissions from 12 isolates of E. coli O157:H7 and 8 non-O157:H7 E. coli isolates. A standard concentration of each isolate was grown in 10 ml of nutrient broth at 37 degrees C for 16 h, and gas sampling was carried out every 5 min. Readings were continuously plotted to generate gas signatures. A back-propagation ANN algorithm was used to interpret the gas patterns. By analysis of the response of the ANN, the sensitivity and specificity of the instrument were calculated. Detectable differences between the gas signatures of the E. coli O157:H7 isolates and the non-O157:H7 isolates were observed. The instruments degree of sensitivity was high for E. coli O157:H7 isolates, but a lower degree of accuracy was observed for non-O157:H7 isolates because of increased strain variation. The sensitivity of the detection system was improved by the normalization of the data generated from the gas sensors. Because of its ability to detect differences in gas patterns, this instrument has a broad range of potential food safety applications.     

Reduction of Salmonella spp. and strains of Escherichia coli O157:H7 by gamma radiation of inoculated sprouts

There have been several recent outbreaks of salmonellosis and infections with Escherichia coli O157:H7 linked to the consumption of raw sprouts. Use of ionizing radiation was investigated as a means to reduce or to totally inactivate these pathogens, if present, on the sprouts. The radiation D value, which is the amount of irradiation in kilograys for a 1-log reduction in cell numbers, for these pathogens was established using a minimum of five doses at 19 +/- 1 degrees C. Before inoculation, the sprouts were irradiated to 6 kGy to remove the background microflora. The sprouts were inoculated either with Salmonella spp. cocktails made with either meat or vegetable isolates or with E. coli O157:H7 cocktails made with either meat or vegetable isolates. The radiation D values for the Salmonella spp. cocktails on sprouts were 0.54 and 0.46 kGy, respectively, for the meat and vegetable isolates. The radiation D values for the E. coli O157:H7 cocktails on sprouts were 0.34 and 0.30 kGy, respectively, for the meat and vegetable isolates. Salmonella was not detected by enrichment culture on sprouts grown from alfalfa seeds naturally contaminated with Salmonella after the sprouts were irradiated to a dose of 0.5 kGy or greater. Ionizing radiation is a process that can be used to reduce the population of pathogens on sprouts.     

Irradiation to kill Escherichia coli O157:H7 and Salmonella on ready-to-eat radish and mung bean sprouts

A study was carried out to evaluate the effectiveness of ionizing radiation in eliminating Escherichia coli O157:H7 and Salmonella on commercial ready-to-eat radish and mung bean sprouts and to assess the chemical and physical quality of these sprouts. The use of ionizing radiation was investigated as a means of reducing or totally inactivating these pathogens, if present, on the sprouts. Treatment of mung bean and radish sprouts with a dose of 1.5 and 2.0 kGy, respectively, significantly reduced E. coli O157:H7 and Salmonella to nondetectable limits. The total vitamin C content was gradually reduced with the increase in irradiation dose (P < 0.0001). However, the effect of storage interval on the loss of vitamin C was nonsignificant for radish sprouts and significant for mung bean sprouts (P < 0.04). The color, firmness, and overall visual quality of the tested sprouts were acceptable when effective doses were applied to both radish and mung bean sprouts. Therefore, ionizing radiation could be useful in reducing the population of pathogens on sprouts and yet retain acceptable quality parameters.     

Isolation of Escherichia coli O157:H7 from foods in Greece

The presence of Escherichia coli O157:H7 in various foods of animal origin was surveyed in northwestern Greece. Six hundred samples of unpasteurized cows', ewes' and goats' milk, raw minced meat, uncooked frozen beef hamburgers, sandwiches (containing ham or turkey, mixed vegetable salad with mayonnaise and lettuce), fresh traditional Greek pork sausages and swine intestines appropriate for traditional Greek kokoretsi were assayed for E. coli serogroup O157:H7 using the standard cultural method and the immunomagnetic separation technique. The pathogen was detected in 1 out of 100 (1.0%) samples of ewes' milk, 1 out of 75 (1.3%) fresh sausages and 1 out of 50 (2.0%) swine intestines prepared for kokoretsi. The isolated strains were nonsorbitol fermenters, MUG-negative, O157 agglutinating, verotoxin-producing and carried both VT1 and VT2 genes. The three isolated strains were tested for antibiotic resistance and were found to be susceptible to eight antimicrobial agents (ampicillin, chloramphenicol, kanamycin, nalidixic acid, norfloxacin, streptomycin, sulfamethoxazole-trimethoprim and tetracycline).