Modified immunoliposome sandwich assay for the detection of Escherichia coli O157:H7 in apple cider

Detection of Escherichia coli O157:H7 in fruit juices such as apple cider is necessary for diagnosis of infection and epidemiological investigations. However, inhibitors in the apple cider, such as endogenous polyphenols and acids, often decrease the sensitivity of PCR assays and immunoassays, thus routinely requiring laborious cell separation steps to increase the sensitivity. In the current study, polyethylene glycol (PEG)-derivatized liposomes encapsulating sulforhodamine B were tagged with anti-E. coli O157:H7 antibodies and used in an immunoliposome sandwich assay for the detection of E. coli O157:H7 in apple cider. Even without prior separation, this assay can detect E. coli O157:H7 in apple cider samples inoculated with as few as 1 CFU/ml after an 8-h enrichment period. The lower limit of detection in pure cultures without enrichment was 7 x 10(3) CFU/ml (280 CFU/40-microl sample). PEGylated immunoliposomes are suitable as an analytical reagent for the detection of E. coli O157:H7 in fruit juices containing polyphenols.     

Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature

Inactivation of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice treated with ozone was evaluated. A five-strain mixture of E. coli O157:H7 or a five-serovar mixture of Salmonella was inoculated (7 log CFU/ml) into apple cider and orange juice. Ozone (0.9 g/h) was pumped into juices maintained at 4 degrees C, ambient temperature (approximately 20 degrees C), and 50 degrees C for up to 240 min, depending on organism, juice, and treatment temperature. Samples were withdrawn, diluted in 0.1% peptone water, and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on tryptic soy agar (TSA), sorbitol MacConkey agar, hemorrhagic coli agar, and modified eosin methylene blue agar; recovery of Salmonella was compared on TSA, bismuth sulfite agar, and xylose lysine tergitol 4 (XLT4) agar. After treatment at 50 degrees C, E. coli O157:H7 populations were undetectable (limit of 1.0 log CFU/ml; a minimum 6.0-log CFU/ml reduction) after 45 min in apple cider and 75 min in orange juice. At 50 degrees C, Salmonella was reduced by 4.8 log CFU/ml (apple cider) and was undetectable in orange juice after 15 min. E. coli O157:H7 at 4 degrees C was reduced by 4.8 log CFU/ml in apple cider and by 5.4 log CFU/ml in orange juice. Salmonella was reduced by 4.5 log CFU/ml (apple cider) and 4.2 log CFU/ml (orange juice) at 4 degrees C. Treatment at ambient temperature resulted in population reductions of less than 5.0 log CFU/ml. Recovery of E. coli O157:H7 and Salmonella on selective media was substantially lower than recovery on TSA, indicating development of sublethal injury. Ozone treatment of apple cider and orange juice at 4 degrees C or in combination with mild heating (50 degrees C) may provide an alternative to thermal pasteurization for reduction of E. coli O157:H7 and Salmonella in apple cider and orange juice.     

Destruction of Escherichia coli O157:H7 by vanillic acid in unpasteurized juice from six apple cultivars

The behavior of Escherichia coli O157:H7 in Granny Smith, Gala, Empire, McIntosh, Red Delicious, and Golden Delicious apple juice with or without supplementation with 5 or 10 mM vanillic acid was examined over a storage period of 7 days at 4 and 15 degrees C. The consequences of supplementation on sensory difference and preference were also determined by triangle testing. Juices made from the six apple cultivars had pH values ranging between pH 3.13 and 3.92. Vanillic acid exerted a concentration, pH, and time-dependent lethal effect toward E. coli O157:H7 in unpasteurized apple juice. Supplementation with 10 mM vanillic acid led to a 5-logarithm reduction in populations after 7 days at both temperatures, but sensory analysis revealed significant differences from and preference for unsupplemented juices. Supplementation with 5 mM vanillic acid accelerated death of E. coli O157:H7, but population reductions ranged from 5 log CFU/ml in low pH juices to none in high pH juices, particularly at 4 degrees C. No sensory difference or preference was detected in two of the six juices at this level of supplementation.     

Enzyme-linked immunomagnetic electrochemical detection of live Escherichia coli 0157:H7 in apple juice

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli O157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 x 10(3) cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.     

Direct detection of E. Coli O157:H7 in selected food systems by a surface plasmon resonance biosensor

The Spreeta^TM, surface plasmon resonance (SPR)-based biosensor, was used to detect Escherichia coli O157:H7 spiked in milk, apple juice and ground beef extract using specific antibodies. In the Spreeta^TM biosensor light from an LED is reflected off a gold surface, and the angle and intensity corresponding to the SPR minimum is measured and represented as a refractive index (RI) change corresponding to the antigen-antibody coupling at the sensor surface. Milk, apple juice, and ground beef patties spiked with E. coli O157:H7, at varying concentrations, were injected on the sensor surface immobilized with antibodies against the pathogen at a rate of 1 ml/min for a total of 2 min. The change in RI due to the binding of O157:H7 corresponding to each concentration was computed as an average of three replications over a 2 min interaction period. Assays were conducted at near real-time and results obtained after about 30 min of sample injection. Sensitivity of the E. coli O157:H7 assay was 10²-10³ colony forming unit (CFU)/ml. The biosensor assay was also specific to E. coli O157:H7 as other organisms (E. coli K12 and Shigella) did not produce any appreciable change in the sensogram. Further experiments are needed to establish well-defined methods for detecting other food-borne pathogens in more complex and specific food matrices.     

Effect of storage in juice with or without pulp and/or calcium lactate on the subsequent survival of Escherichia coli O157:H7 in simulated gastric fluid

A study was conducted to evaluate the effect of storing Escherichia coli O157:H7 in fruit or vegetable juices with or without pulp and/or calcium lactate, on the bacterial resistance to a simulated gastric fluid (SGF, pH 1.5). Apple, carrot, orange, and tomato juices containing pulp or freed from pulp by filtration were used in this study. Calcium lactate at about 1.4 g/l was added to juices to obtain calcium supplemented juices. Juices with or without pulp and/or calcium lactate were inoculated with E. coli O157:H7 and then were stored at 7 degrees C for 0, 1, 2, or 4 days. The acid resistance of cells stored in juices with or without pulp and/or calcium lactate was determined by incubating in SGF for 90 or 240 min at 37 degrees C. Cells stored in apple juice for 4 days, carrot juice for 2 days, and orange juice for 4 days with pulp only had greater acid resistance, while all cells stored in tomato juice with pulp had greater acid resistance than cells stored in juice without pulp. The D-values of cells stored in supplemented apple and orange juices with calcium lactate declined 1.7-3.5 fold, whereas D-values of cells stored in supplemented tomato juice decreased by about 1.4-fold when compared to cells stored in juice without calcium lactate after exposure in SGF. These results indicate that storing E. coli O157:H7 in juices with pulp had little or no effect on the acid resistance of cells during subsequent exposure in SGF. Calcium lactate supplemented into juices could dramatically decrease the ability of E. coli O157:H7 to survive in SGF, possibly reducing the risk of foodborne illness by juice products.     

Inhibition of Shiga Toxin 2 (Stx2) in Apple Juices and its Resistance to Pasteurization

Abstract: In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P < 0.05) in the densities or molecular weights between Stx2 in either PBS or apple juices. These results suggest that Stx2 may be reversibly bound to small molecular weight constituents in the juice. The Stx2 toxin was not inactivated on exposure to heat programs (63 °C for 30 min, 72 °C for 15 s, 89 °C for 1 s) commonly used to pasteurize apple juice, but lost all activity when exposed to 100 °C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin. Practical Application: This study explores the possibility of using naturally occurring antioxidative substances, in this case high phenolic apples juices, to inactivate Shiga toxin (Stx2) produced by E. coli O157:H7 in contaminated foods.     

Effect of low-temperature, high-pressure treatment on the survival of Escherichia coli O157:H7 and Salmonella in unpasteurized fruit juices

The destructive effect of high pressure (615 MPa) combined with low temperature (15 degrees C) on various strains of Escherichia coli O157:H7 and various serovars of Salmonella in grapefruit, orange, apple, and carrot juices was investigated. The three-strain cocktail of E. coli O157:H7 (SEA13B88, ATCC 43895, and 932) was found to be most sensitive in grapefruit juice (8.34-log reduction) and least in apple juice (0.41-log reductions) when pressurized at 615 MPa for 2 min at 15 degrees C. Correspondingly, no injured survivor was detected in grapefruit and carrot juices under similar treatment conditions. No Salmonella spp. were detected in a 2-min pressure treatment (615 MPa, 15 degrees C) of grapefruit and orange fruit juices. Except for Enteritidis, all four serovars tested in the present study have viability loss of between 3.92- and 5.07-log reductions when pressurized in apple juice at 615 MPa for 2 min at 15 degrees C. No injured cells were recovered from grapefruit and orange juices, whereas the same treatment demonstrated reduction in numbers of Salmonella serovars Agona and Muenchen in apple juices and to a lesser extent with Typhimurium, Agona, and Muenchen in carrot juice. The present study demonstrated that low-temperature, high-pressure treatment has the potential to inactivate E. coli O157:H7 strains and different Salmonella spp. in different fruit juices.     

High-pressure resistance variation of Escherichia coli O157:H7 strains and Salmonella serovars in tryptic soy broth, distilled water, and fruit juice

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7 and five serovars of Salmonella enterica was investigated in tryptic soy broth, sterile distilled water, and commercially sterile orange juice (for Salmonella) and apple cider (for E. coli). Samples were subjected to high-pressure processing treatment at 300 and 550 MPa for 2 min at 6 degrees C. Samples were plated onto tryptic soy agar directly after pressurization and after being held for 24 h at 4 degrees C. At 300 MPa, little effect was seen on E. coli O157:H7 strains, while Salmonella serovars varied in resistance, showing reductions between 0.26 and 3.95 log CFU/ml. At 550 MPa, E. coli O157:H7 strains exhibited a range of reductions (0.28 to 4.39 log CFU/ml), while most Salmonella populations decreased beyond the detection limit (> 5-log CFU/ml reduction). The most resistant strains tested were E. coli E009 and Salmonella Agona. Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water. E. coli O157:H7 cultures held for 24 h at 4 degrees C after treatment at 550 MPa showed a significant log decrease as compared with cultures directly after treatment (P < or = 0.05), while Salmonella serovars did not show this significant decrease (P > 0.05). All Salmonella serovars tested in orange juice treated at 550 MPa for 2 min at 6 degrees C and held for 24 h showed a > 5-log decrease, while E. coli O157:H7 strains require a higher pressure, higher temperature, longer pressurization, or a chemical additive to achieve a 5-log decrease.     

Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat

This work approaches the possibility of combining mild heat treatments with citrus fruit essential oils (EOs) to improve the effectiveness of heat treatments and thus to reduce treatment intensity. Concentrations between 10 and 200μL/L of lemon, mandarin, or orange EO were tested at 54°C for 10min in laboratory media, determining that 200μL/L of each EO was necessary to achieve a 5 log(10) reduction of the initial Escherichia coli O157:H7 concentration. A relationship could be established between sublethally injured cells after the heat treatment and inactivated cells after the combined process. In apple juice, the synergism in the inactivation of E. coli O157:H7 when adding 200μL/L of lemon EO might suppose a reduction in the treatment temperature (of 4.5°C) or in the treatment time (by 5.7 times) within the range of temperature assayed (54-60°C). Addition of 75μL/L of lemon EO was determined to achieve the same synergistic effect of the combined treatment when the initial inoculum was reduced from 3×10(7) to 3×10(4)CFU/mL. Since the addition of lemon EO did not decrease the hedonic acceptability of apple juice, the proposed combined treatment could be further studied and optimized for the production of new minimally processed juices.     

Antibacterial effect of monocaprylin on Escherichia coli O157:H7 in apple juice

The antibacterial effect of low concentrations of monocaprylin on Escherichia coli O157:H7 in apple juice was investigated. Apple juice alone (control) or containing 2.5 mM (0.055%) or 5 mM monocaprylin was inoculated with a five-strain mixture of E. coli O157:H7 at approximately 6.0 log CFU/ml. The juice samples were stored at 23 or 4 degrees C for 14 or 21 days, respectively, and the population of E. coli O157:H7 was determined on tryptic soy agar plates supplemented with 0.6% yeast extract. At both storage temperatures, the population of E. coli O157:H7 in monocaprylin-supplemented juice samples was significantly lower (P < 0.05) than that in the control samples. The concentration of monocaprylin and the storage temperature had a significant effect on the inactivation of E. coli O157:H7 in apple juice. Monocaprylin at 5 mM was significantly more effective than 2.5 mM monocaprylin for killing E. coli O157:H7 in apple juice. Inactivation of E. coli O157:H7 by monocaprylin was more pronounced in juice stored at 23 degrees C than in the refrigerated samples. Results of this study indicated that monocaprylin is effective for killing E. coli O157:H7 in apple juice, but detailed sensory studies are needed to determine the organoleptic properties of apple juice containing monocaprylin.     

Effect of citral on the thermal inactivation of Escherichia coli O157:H7 in citrate phosphate buffer and apple juice

Inactivation and sublethal injury of Escherichia coli O157:H7 cells induced by heat in citrate phosphate buffer and apple juice (both at pH 3.8) were studied, and the effect of a combined preservation treatment using citral and heat treatments was determined. Heat resistance of E. coli O157:H7 was similar in both treatment media; after 27 min at 54°C, 3 log units of the initial cell population was inactivated in both treatment media. However, under less harsh conditions a protective effect of apple juice was found. Whereas inactivation followed linear kinetics in the citrate phosphate buffer, when cells were treated in apple juice the survival curves were concave downward. Heat treatment caused a great degree of sublethal injury; 4 min at 54°C inactivated less than 0.5 log CFU/ml but sublethally injured more than 3 log CFU/ml. The addition of 18 and 200 ppm of citral to the treatment medium acted synergistically with heat at 54°C to inactivate 3 × 10(4) and 3 × 10(7) CFU/ml, respectively. Addition of citral thus reduced the time needed to inactivate 1 log unit of the initial E. coli O157:H7 population from 8.9 to 1.7 min. These results indicate that a combined process of heat and citral can inactivate E. coli O157:H7 cells and reduce their potential negative effects.     

Inactivation of Escherichia coli O157:H7 and Salmonella in Apple Cider and Orange Juice Treated with Combinations of Ozone, Dimethyl Dicarbonate, and Hydrogen Peroxide

ABSTRACT: Inactivation of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice treated with ozone in combination with antimicrobials was evaluated. E. coli O157:H7 or Salmonella was suspended in cider and orange juice, and ozone was pumped into juices (4°C) containing dimethyl dicarbonate (DMDC; 250 or 500 ppm) or hydrogen peroxide (300 or 600 ppm) for up to 90 min (study 1) or 60 min followed by 24-h storage at 4°C (study 2). Study 1: No combination of treatments resulted in a 5-log colony-forming units (CFU) /mL reduction of either pathogen. Study 2: All combinations of antimicrobials plus ozone treatments, followed by refrigerated storage, caused greater than a 5-log CFU/mL reduction, except ozone/DMDC (250 ppm) treatment in orange juice. Ozone treatment in combination with DMDC or hydrogen peroxide followed by refrigerated storage may provide an alternative to thermal pasteurization to meet the 5-log reduction standard in cider and orange juice.     

The survival of Escherichia coli O157:H7 in the presence of Penicillium expansum and Glomerella cingulata in wounds on apple surfaces

The survival of Escherichia coli O157:H7 in the presence of one of two plant pathogens, Penicillium expansum and Glomerella cingulata, in wounds on apples was observed during 14 days storage at room temperature (RT) and at 4 degrees C. The aim of this work was to determine if changes in apple physiology caused by the proliferation of fungal decay organisms would foster the survival of E. coli O157:H7. Trials were performed where (A) plant pathogens (4 log10 spores) were added to apple wounds 4 days before the wounds were inoculated with E. coli O157:H7 (3 log10 CFU g(-1) apple) (both RT and 4 degrees C storage), (B) plant pathogens and E. coli O157:H7 were added on the same day (both RT and 4 degrees C storage), and (C) E. coli O157:H7 was added 2 days (RT storage) and 4 days (4 degrees C storage) before plant pathogens. In all trials E. coli O157:H7 levels generally declined to <1 log10 at 4 degrees C storage, and in the presence of P. expansum at 4 degrees C or RT. However, in the presence of G. cingulata at RT E. coli O157:H7 numbers increased from 3.18 to 4.03 log10 CFU g(-1) in the apple wound during trial A, from 3.26 to 6.31 log10 CFU g(-1) during trial B, and from 3.22 to 6.81 log10 CFU g(-1) during trial C. This effect is probably a consequence of the attendant rise in pH from 4.1 to approximately 6.8, observed with the proliferation of G. cingulata rot. Control apples (inoculated with E. coli O157:H7 only) were contaminated with opportunistic decay organisms at RT during trials A and B, leading to E. coli O157:H7 death. However, E. coli O157:H7 in control apples in trial C, where no contamination occurred, increased from 3.22 to 5.97 log10 CFU g(-1). The fact that E. coli O157:H7 can proliferate in areas of decay and/or injury on fruit highlights the hazards associated with the use of such fruit in the production of unpasteurized juice.     

INACTIVATION OF ESCHERICHIA COLI O157:H7 DURING THE STORAGE UNDER REFRIGERATION OF APPLE JUICE TREATED BY PULSED ELECTRIC FIELDS

The sensitivity of pulsed electric fields (PEF)‐treated E. coli O157:H7 cells to subsequent holding in apple juice has been evaluated. Escherichia coli O157:H7 cells in apple juice were resistant to PEF. A PEF treatment of 400 µs at any electrical field strength was not sufficient to inactivate one log₁₀cycle of cells. However, PEF injured a large proportion of E. coli O157:H7 cells that became sensitive to a subsequent storage under refrigeration in apple juice. The total lethal effect of the combined process depended on the electrical field strength and storage time. The combination of a PEF treatment at 25 kV/cm for 400 µs and a subsequent storage of the apple juice under refrigeration for 48 h allowed five log₁₀cycles of inactivation to be achieved. The combination of PEF and maintenance under refrigeration has been demonstrated to be an effective pasteurization method, by sufficiently reducing the presence of E. coli O157:H7 in apple juice in order to meet U.S. FDA recommendations.     

肠出血性大肠杆菌O157:H7的分子生物学检测及PCR检测

大肠杆菌一些特殊的血清型具有致病性,肠出血性大肠杆菌是大肠杆菌的一个亚型,主要致病菌株为O157:H7,可引起感染性腹泻,因能引起人类的出血性肠炎而得名。本文综述了分子生物学检测肠出血性大肠杆菌O157:H7的研究进展。分子生物学检测是利用抗原抗体特异性结合反应检测各种物质的分析方法,主要包括酶联免疫吸附法(ELISA)、胶体免疫金层析法以及免疫磁珠分离法(IMS)。PCR技术检测肠出血性大肠杆菌O157:H7,主要包括常规PCR检测、多重PCR检测以及实时荧光定量PCR检测。这两种方法灵敏度高、特异性强、操作简便、结果准确等优点,是检测肠出血性大肠杆菌O157:H7的常用方法。     

流式分析技术快速定量检测牛乳中大肠杆菌O157:H7

建立一种基于流式分析技术的快速定量检测牛乳中大肠杆菌O157:H7的方法。用偶联有异硫氰酸荧光素的大肠杆菌O157单克隆抗体对大肠杆菌O157:H7进行特异性标记,通过优化抗体反应条件,建立流式检测方法,然后对磷酸盐缓冲溶液（phosphate buffer saline,PBS）和人工污染牛乳样品中不同浓度的大肠杆菌O157:H7进行定量检测。本研究建立的流式检测方法的在PBS中的检测范围为2.57×103～1.12×108?CFU/mL,灵敏度达到2.57×103?CFU/mL。将所建立的流式检测方法应用于牛乳样品检测,当人工污染牛乳样品中大肠杆菌O157:H7的浓度在2.31×104～1.48×108?CFU/mL之间时,流式检测方法与平板计数方法检测结果基本一致,方法的灵敏度为2.31×104?CFU/mL,检测时间为35?min。该方法能快速、定量地检测出牛乳样品中的大肠杆菌O157:H7,在食源性致病菌的快速筛查和监控中具有重要的应用价值。     

Methods for recovering Escherichia coli O157:H7 from cattle fecal, hide, and carcass samples: sensitivity and improvements

The Meats Research Unit (MRU) methods, developed by MRU scientists of the U.S. Meat Animal Research Center, have been used to study the prevalence of Escherichia coli O157:H7 in cattle carcass, hide, and fecal samples. The sensitivity of these methods for recovery of injured E. coli O157:H7 cells from inoculated and uninoculated samples was determined, and potential improvements to these methods were evaluated. When using the conventional MRU methods, 91% of the pre-evisceration carcass samples tested positive for E. coli O157:H7 when inoculated with 5 to 10 CFU, 100% of hide samples tested positive for E. coli O157:H7 when inoculated with 30 to 50 CFU, and 96% of the fecal samples produced positive results when inoculated with 300 to 400 CFU per 10 g. The addition of a phosphate buffer to the tryptic soy broth enrichment improved recovery of E. coli O157:H7 from feces. Using the modified enrichment, 92% of the samples were identified as positive when inoculated with 10 to 30 CFU per 10 g. Substituting a commercially available wash buffer for the phosphate-buffered saline (PBS) plus Tween 20 wash buffer during immunomagnetic separation of hide samples improved recovery of the target organism at lower inoculum concentrations. When comparing uninoculated samples, substituting a PBS buffer plus a zwitterionic detergent for PBS plus Tween 20 also had a positive effect on recovery of E. coli O157:H7 from hide samples. Data presented here indicate that the MRU methods are highly effective at recovering injured E. coli O157:H7 from fecal, hide, and beef carcass samples; however, modifications can be added to increase the sensitivity.     

Combination of Carbon Dioxide and Cinnamon to Inactivate Escherichia coli O157:H7 in Apple Juice

ABSTRACT: Pasteurized apple juice with CO2 (0, 1, and 4%) and cinnamon (0 and 0.3%) was inoculated with Escherichia coli O157:H7 at 104 CFU/mL, and stored at 5 and 20 °C. Counts on nonselective and selective media, and thin agar layer (TAL; selective medium overlaid with nonselective medium) were determined at 1 h and 1, 3, 7, and 14 d. Inactivation was greater at 20 °C. Samples with 1 and 4% CO₂, alone and combined with cinnamon, presented < 0.7 log CFU/mL in 3 d. Counts in apple juice inoculated at 10² CFU/mL, a low-level E. coli O157:H7 contamination, were nondetectable at 3 d. The TAL method was as effective as nonselective medium to recover injured cells.