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.     

Heat resistance of Escherichia coli O157:H7 in apple juice.

The objective was to determine the effect of cider composition on the heat resistance of Escherichia coli O157:H7. The average D52 value in a model Empire apple juice was 18 min with a z value of 4.8 degrees C. Increasing the Brix from 11.8 to 16.5 degrees had no effect on thermal resistance, while increasing L-malic acid from 0.2 to 0.8%, or reducing the pH from 4.4 to 3.6 sensitized the cells to heat. The greatest effect on heat resistance was afforded by the preservatives benzoic and sorbic acids: D50 values in ciders containing 1,000 mg/l were 5.2 min in the presence of sorbic acid and only 0.64 min in the presence of benzoic acid. Commercial apple juice concentrates yielded lower numbers of survivors than single-strength juices even though their higher sugar concentrations of about 46 degrees Brix increased heat resistance.     

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.     

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.     

Heat inactivation of Escherichia coli O157:H7 in apple juice exposed to chlorine

Exposure of Escherichia coli O157:H7 to chlorine before heat treatment results in increased production of heat shock proteins. Current heating regimens for pasteurizing apple cider do not account for chlorine exposure in the wash water. This research determined the effect of sublethal chlorine treatment on thermal inactivation of E. coli O157:H7. D58-values were calculated for stationary-phase cells exposed to 0.6 mg/liter of total available chlorine and unchlorinated cells in commercial shelf-stable apple juice (pH 3.6). D58-values for unchlorinated and chlorine-exposed cells in buffer were 5.45 and 1.65 min, respectively (P < 0.01). Death curves of chlorine-exposed and unchlorinated cells in apple juice were not completely linear. Unchlorinated cells heated in apple juice exhibit a 3-min delay before onset of linear inactivation. Chlorine treatment eliminated this shoulder, indicating an overall loss of thermotolerance. The linear portion of each curve represented a small fraction of the total population. D58-values calculated from these populations are 0.77 min for unexposed cells and 1.19 min for chlorine-exposed cells (P = 0.05). This indicates that a subpopulation of chorine-treated cells is possibly more resistant to heat because of chlorine treatment. The effect of chlorine treatment, however, is insignificant when compared with the effect of losing the shoulder. This is illustrated by the time required to kill the initial 90% of the cell population. This is observed to be 3.14 min for unchlorinated versus 0.3 min for chlorine-exposed cells (P < 0.001). These observations indicate that current heat treatments need not be adjusted for the effect of chlorine treatment.     

Phosphate buffer increases recovery of Escherichia coli O157:H7 from frozen apple juice

It is common practice to dilute food products in 0.1% peptone before microbiological analysis. However, this diluent may not be appropriate for detection of injured organisms present in acidic foods. Shelf-stable unclarified apple juice (pH 3.6) was inoculated with approximately 1 x 10(7) CFU/ml of Escherichia coli O157:H7 and held at 23 +/- 2 degrees C (control) or frozen to -20 +/- 2 degrees C for 24 h to induce injury before sampling. Unfrozen or frozen and thawed juice was diluted 1:1 or 1:10 in 0.1% (wt/vol) peptone (pH 6.1) or 0.1 M phosphate buffer (pH 7.2). Juice samples were plated onto tryptic soy agar with 0.1% (wt/vol) sodium pyruvate (TSAP) to measure survival or onto sorbitol MacConkey agar (SMA) to indicate injury. Counts on TSAP or SMA were the same for control samples held in peptone or phosphate buffer for up to 45 min. However, populations of E. coli in frozen and thawed samples declined rapidly upon dilution in 0.1% peptone. Within 20 min, E. coli underwent a >1-log10 CFU/ml reduction in viability as measured on TSAP and a >2-log10 CFU/ml reduction to below the limit of detection (1.6 or 2.3 log10 CFU/ml) on SMA. In contrast, populations of E. coli in frozen and thawed samples diluted in phosphate buffer did not decrease significantly on TSAP and decreased by <0.6 log CFU/ml on SMA during a 45-min holding period. The acidity of apple juice appears to interfere with the recovery of freeze-thaw-injured E. coli O157:H7 during sampling. Using 0.1 M phosphate buffer (pH 7.2) as a diluent results in superior recovery of these organisms on both selective and nonselective plating media.     

Detection of Escherichia coli O157 in foods by a novel polymyxin-based enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) system was developed using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for Escherichia coli O157 lipopolysaccharide (LPS) antigens. Extracts from cell suspensions were reacted with polymyxin-coated microwells followed by immunoenzymatic detection of captured LPS antigens using a commercially available anti-E. coli O157 antibody-peroxidase conjugate and a 3,3',5,5'-tetramethylbenzidine substrate. The polymyxin ELISA was highly sensitive and specific for E. coli strains bearing the O157 antigen. When this ELISA was combined with enrichment, results were in complete agreement with those of standard culture techniques for the detection of this pathogen in a variety of artificially inoculated and naturally contaminated foods. The polymyxin ELISA is a simple and inexpensive assay for E. coli O157 with a 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.     

基于噬菌体特异性的新型荧光酶联方法检测食品中大肠埃希菌O157∶H7

目的对一种基于噬菌体特异性的检测食品中大肠埃希菌O157∶H7的新型荧光酶联方法(VIDASECPT)进行了评价。方法利用VIDASECPT、VIDASECO、BAXO157三种快速方法检测不同浓度大肠埃希菌O157∶H7标准菌株的生理盐水悬浮液,并对VIDASECPT和常规培养法检测食品中大肠埃希菌O157∶H7进行了比对。结果 VIDASECPT检测大肠埃希菌O157∶H7的检出限为104 CFU/ml,并且不受大肠埃希菌背景菌的干扰,其他两种快速方法的检出限均为105 CFU/ml,其中VIDASECO易受到背景菌的干扰;检测不同食品基质中大肠埃希菌O157∶H7,当大肠埃希菌O157∶H7的浓度为104 CFU/ml时,VIDASECPT与常规培养法无统计学差异。结论 VIDASECPT方法检测大肠埃希菌O157∶H7具有快速、灵敏、抗干扰性强的特点。     

Inactivation of Escherichia coli O157:H7 and Escherichia coli 8739 in apple juice by pulsed electric fields.

The effect of high voltage pulsed electric field (PEF) treatment on Escherichia coli O157:H7 and generic E. coli 8739 in apple juice was investigated. Fresh apple juice samples inoculated with E. coli O157:H7 and E. coli 8739 were treated by PEF with selected parameters including electric field strength, treatment time, and treatment temperature. Samples were exposed to bipolar pulses with electric field strengths of 30, 26, 22, and 18 kV/cm and total treatment times of 172, 144, 115, and 86 micros. A 5-log reduction in both cultures was determined by a standard nonselective medium spread plate laboratory procedure. Treatment temperature was kept below 35 degrees C. Results showed no difference in the sensitivities of E. coli O157:H7 and E. coli 8739 against PEF treatment. PEF is a promising technology for the inactivation of E. coli O157:H7 and E. coli 8739 in apple juice.     

Fourier transform infrared spectroscopy, detection and identification of Escherichia coli O157:H7 and Alicyclobacillus strains in apple juice

Fourier Transform Infrared (FT-IR) spectroscopy (4000-400 cm(-1)) combined with multivariate statistical methods were used to identify and detect Escherichia coli O157:H7 from Alicyclobacillus spp. recovered from apple juice. Four treatments and a control in triplicate experiments (N=3) were studied; the first three treatments of pasteurized apple juice were inoculated with E. coli O157:H7 ATCC 35150, Alicyclobacillus acidoterrestris 1016 and Alicyclobacillus spp. C-Fugi-6 respectively. The fourth treatment was a 1:1 (v:v) mixed culture of both A. acidoterrestris 1016 and Alicyclobacillus spp. C-Fugi-6. The control was uninoculated pasteurized apple juice. The second derivative transformation and loadings plot over the range of 1800-900 cm(-1) highlighted the most distinctive variations among bacterial spectra. Loadings 1 and 2 were distinctively representative of the bacterial spectral data and accounted for 73% of the total variability. Treatments were noticeably segregated with distinct clustering by principal component analysis (PCA). Using soft independent modeling of class analogy (SIMCA) analysis, 88.3% of (E. coli O157:H7 ATCC 35150) spectra, 75.0% of (A. acidoterrestris 1016) spectra, 88.3% of (Alicyclobacillus spp. C-Fuji-6) spectra, and 80.0% of the mixed culture of both Alicyclobacillus strains spectra were correctly classified. Using the spectral features of bacterial cellular constituents such as nucleic acids, proteins, phospholipids, peptidoglycan, and lipopolysaccharides from examined bacterial cells, pure and mixed cultures of Alicyclobacillus spp. cells, and the pathogenic E. coli cells could be detected in apple 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.     

Effect of vanillin on the fate of Listeria monocytogenes and Escherichia coli O157:H7 in a model apple juice medium and in apple juice

The effects of vanillin on the fates of Listeria monocytogenes and Escherichia coli O157:H7 at pH values between 3.5 and 4.5 were verified in a model apple juice (MAJ) medium and in apple juice incubated at 4 or 15 degrees C. Viable E. coli O157:H7 cells were recovered from MAJ for up to 10 days, but L. monocytogenes did not survive at pH 3.5. Supplementation with 40 mm vanillin exerted a lethal effect that was species, concentration, pH and temperature dependant. E. coli O157:H7 was more sensitive to vanillin than L. monocytogenes, and viable cells could not be recovered after 2 days incubation at either temperature. L. monocytogenes and E. coli O157:H7 were inoculated (10(5) cfu/ml) in pH adjusted (pH 4.00) or unadjusted (pH 3.42) juice from Granny Smith apples that was supplemented with 40 mm vanillin. Neither species were recovered after 3 days incubation at 4 or 15 degrees C. These findings indicate that vanillin could be useful as a preservative for minimally processed apple products.     

Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk

Lateral flow assay (LFA) has been applied in many fields due to its relative ease of use and cost-effectiveness. However, it has low sensitivity and its applications are limited. Probe materials play a significant role in improving the detection efficiency and sensitivity of LFA. In this study, by using concave palladium-platinum (Pd-Pt) nanoparticles as a nanozyme probe, we developed a sensitive LFA based on the sandwich format for qualitative and quantitative detection of Escherichia coli O157:H7. The sensitivity of the LFA was improved by applying the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate onto the test line where the nanozyme was accumulated in the presence of analytes. The nanozyme showed high catalytic performance toward TMB and greatly enhanced the signal intensity of the test line. The sensitivity of the nanozyme-based LFA was 9.0 × 10² cfu/mL in milk, which was 111-fold higher than that of traditional colloidal gold-based LFA. The proposed method has remarkable potential in the detection of various pathogens in real samples.     

The effects of freezing and thawing on the survival of Escherichia coli O157:H7 in apple juice

Unpasteurized apple juice, adjusted to pH 3.6 to 7.0 was inoculated (10(7) CFU/ml) with single strains of E. coli O157:H7 to evaluate the effect of frozen storage on the viability of this organism. Samples were stored under frozen conditions (-20+/-2 degrees C) for up to 16 days. Cell populations were determined at regular intervals by plating onto tryptic soy agar with added pyruvate (TSAP) or onto sorbitol MacConkey agar (SMA). Populations in the neutralized juice remained unchanged during frozen storage. Populations in non-neutralized juice decreased by 1-3 log10 CFU/ml depending on the strain tested and the pH of the juice. The greatest population decrease was observed with the first freeze/thaw cycle of frozen storage (24 h) and a slow decline in survival occurred thereafter. Injury was observed after 2 weeks of storage when juice pH was at or below pH 4.2. When samples were subjected to multiple freeze/thaw cycles, loss of viability and injury increased with each freeze/thaw cycle.     

Inactivation of Salmonella typhimurium and Escherichia coli O157:H7 in apple juice by a combination of nisin and cinnamon

Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10(4) CFU/ml and stored at 5 and 20 degrees C. Counts on tryptic soy agar (TSA), selective medium (xylose Lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E. coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days. The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells. The pathogens were gradually inactivated by the acidic pH of apple juice. Nisin and cinnamon greatly contributed to the inactivation. The killing effect was more marked at 20 degrees C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E. coli O157:H7. Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature. The TAL method was as effective as TSA in recovering injured cells of the pathogens. The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E. coli O157:H7 in apple juice and so enhances the safety of the product.     

原料乳中大肠杆菌O157:H7的快速检测

建立了一种快速检测原料乳中大肠杆菌O157:H7的PCR技术.该方法利用过滤富集菌体后的PCR技术来检测原料乳中大肠杆菌O157:H7,先对人工污染大肠杆菌O157:H7的原料乳进行离心脱脂,然后添加EDTA-2Na获得澄清乳液,最后通过0.45 μm微膜过滤收集菌体,整个过程只需6 h左右即可完成.检测灵敏度高达10-mL-1.这种方法在传统检测方法的基础上做了有效改进,使得原料乳中的大肠杆菌O157:H7的检测能够快速、准确、灵敏的进行.     

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.     

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in apple juice with gaseous ozone

This research was initiated to assess the efficacy of gaseous ozone for inactivation Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in apple juice. Juice samples with solids content of 18, 36, and 72 °Brix inoculated with a culture cocktail of three foodborne pathogens were treated with gaseous ozone at a flow rate of 3.0 L/min and an ozone generation rate of 0.10, 0.90, 3.51, and 5.57 g/h for 0.5, 1, 5, and 10 min, respectively. The inactivation kinetics of gaseous ozone on foodborne pathogens conformed to the Weibull model. The time required to achieve a 5 log reduction (t_5d) was estimated using the parameters of the Weibull model. The t_5d increased with increasing solids content of apple juice. The ozone generation rate did not impart a significant effect (p > 0.05) on t_5d. Gaseous ozone is effective at inactivating foodborne pathogens in apple juice but the efficacy is dependent on the solids content of the juice sample.     

High pressures in combination with antimicrobials to reduce Escherichia coli O157:H7 and Salmonella Agona in apple juice and orange juice

The effect of high pressure processing in conjunction with the chemical antimicrobials, dimethyl dicarbonate (DMDC), hydrogen peroxide, cinnamic acid, potassium sorbate, and sodium benzoate (NaB) on E. coli O157:H7 strain E009 and Salmonella enterica serovar Agona was investigated in apple juice and orange juice, respectively. Juices were inoculated with approximately 10(6) CFU/ml and subjected to pressures of 550 MPa (E. coli O157:H7 samples) and 400 MPa (Salmonella Agona samples) for 2 min at 6 degrees C (initial temperature). Populations of each pathogen were determined before pressurization, immediately after pressurization, and after samples had been held after treatment for 24 h at 4 degrees C. The most effective treatment for E. coli O157:H7, as determined by plating immediately after pressurization, was 125 ppm of DMDC, which caused a >4.98-log reduction. Other treatments that were significantly different from the sample with no added antimicrobial were 62.5 ppm of DMDC, 300 ppm of hydrogen peroxide, and 500 ppm of NaB, which produced 4.97-, 5.79-, and 3.91-log total reductions, respectively. After 24 h at 4 degrees C, E. coli O157:H7 was undetectable in all treatment groups (and controls). In samples inoculated with Salmonella, the most effective treatment was 62.5 ppm of DMDC, which produced a 5.96-log decrease immediately after pressure treatment. The results for 1,000 ppm of NaB, which produced a 3.26-log decrease, also were significantly different from those for the sample containing no antimicrobials. After 24 h at 4 degrees C, all samples with added antimicrobials had near or more than a 5-log total reduction of Salmonella Agona.     

Sensitive fluorescence ELISA with streptavidin scaffolded DNA tetrads for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7 poses a threat to humans. Traditional ELISA is not a sensitive method for the detection of E. coli O157:H7. Here, an efficient method was designed for improving the load capacity of alkaline phosphatase (ALP) with streptavidin scaffolded DNA tetrad (SS-DNAt). With more ALP, more ascorbic acid 2-phosphate was catalyzed to ascorbic acid that was used to synthesize fluorescence poly adenine-thymine–templated copper nanoclusters. Based on SS-DNAt, fluorescence ELISA was successfully proposed for improving the sensitivity for detection of E. coli O157:H7 in milk samples. The method showed a linear range of 10⁴ to 10⁶ cfu/mL. The limit of detection of fluorescence ELISA was 3.75 × 10³ cfu/mL and 6.16-fold better than that of traditional ELISA. The recovery of the fluorescence ELISA was 86.7 to 93.6% with the coefficient of variation of 5.6 to 10.5% in milk. This method could be used to detect hazardous material in food.