The use of Fourier transform infrared spectroscopy to differentiate Escherichia coli O157:H7 from other bacteria inoculated into apple juice

Fourier transform infrared spectroscopy (FT-IR) can discriminate Escherichia coli O157:H7 ATCC 35150 from other bacteria: E. coli ATCC 25522, Bacillus cereus ATCC 10876, and Listeria innocua ATCC 51742 inoculated in to apple juice. Spectra of bacterial suspensions (ca. 10(9) cfu/ml in 0.9% NaCl) on Anodisc (aluminum oxide) filters were tested. Unique FT-IR vibrational combination bands from mid-IR active components of bacterial cells are present in the "fingerprint region" at wavenumbers between 1500 and 800 cm(-1). Principal component analysis (PCA) revealed clear segregations between different bacterial strains. Also, soft independent modeling of class analogy (SIMCA) correctly classified E. coli O157:H7 ATCC 35150 from E. coli ATCC 25522 at an 82% confidence level; whereas a 77% confidence level was obtained when using SIMCA to classify E. coli O157:H7 from three other bacterial strains.     

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.     

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.     

Evaluation of Escherichia coli O157:H7 in apple juice with Cornus fruit (Cornus officinalis Sieb. et Zucc.) extract by conventional media and thin agar layer method

Escherichia coli O157:H7 survival in apple juice supplemented with Cornus fruit (Cornus officinalis Sieb. et Zucc.) extract was studied. Inoculated samples with or without Cornus fruit extract were kept at 21 and 7 degrees C. Microbial analysis was conducted on days 0, 1, 3, 5, and 7. MacConkey sorbitol agar (MSA), tryptic soy agar (TSA), and thin agar layer (TAL) medium were used to compare the recovery of bacteria stressed under combination treatment. Influence of temperature, storage time, and Cornus fruit on survival of cells was evaluated. The most dramatic reduction of E. coli O157:H7 was observed in apple juice with Cornus fruit extract at 21 degrees C. At 7 degrees C, E. coli O157:H7 was reduced by 2.3logcfu/ml in the apple juice with Cornus fruit extract compared to the control sample on day 7. TAL and TSA were more efficient than MSA. Cornus fruit extract can be used in combination with temperature and storage time controls to inactivate E. coli O157:H7 in apple juice. This study has shown that TAL is a viable method of recovering and differentiating injured microorganisms and apple juice supplemented with Cornus fruit has potential as a value-added beverage with antimicrobial effects and potential health benefits.     

Aqueous extracts of yerba mate (Ilex paraguariensis) as a natural antimicrobial against Escherichia coli O157:H7 in a microbiological medium and pH 6.0 apple juice

Ilex paraguariensis is popularly used in the preparation of a tea infusion (yerba mate), most commonly produced and consumed in the South American countries of Uruguay, Paraguay, Argentina, and Brazil. In this study, aqueous extracts of commercial tea, derived from the holly plant species I. paraguariensis were evaluated for their ability to inhibit or inactivate Escherichia coli O157:H7 in a microbiological medium and modified apple juice. Dialyzed, lyophilized aqueous extracts were screened for antimicrobial activity against E. coli O157:H7 strains ATCC 43894 and 'Cider' in tryptic soy broth (TSB) and apple juice (adjusted to pH 6.0 to allow for growth of the bacterium). A mixture of the two strains was used as the inoculum when apple juice was used as the medium. MBCs were determined to be ca. 5 and 10 mg/ml for ATCC 43894 and 'Cider', respectively, in TSB. Higher concentrations of the extract were required to inactivate E. coli O157:H7 in pH-adjusted apple juice. An approximate 4.5-log reduction was observed for E. coli O157:H7 treated with 40 mg/ml extract. It was concluded that aqueous extracts from commercial yerba mate have potential to be used as antimicrobials in foods and beverages against pathogenic E. coli O157:H7.     

Transmission electron microscopy study of enterohemorrhagic Escherichia coli O157:H7 in apple tissue

We investigated the ability of enterohemorrhagic Escherichia coli O157:H7 to spread in wounded apple tissue by transmission electron microscopy. Red Delicious apples were wounded with an artist knife (7 mm depth) and either inoculated with 10 microl per wound of decimally diluted E. coli O157:H7 or submerged into E. coli O157:H7 suspended in sterile distilled water and then stored at 37 degrees C for 24 h. Transmission electron microscopy showed E. coli O157:H7 formed bacterial aggregates near the apple cell walls, and single cells were in close proximity to the apple cell wall surfaces and to plasma membranes. E. coli O157:H7 presence caused degradation of plasma membranes and release of the cytoplasm contents of the apple cortical cells into the central vacuole. Apple tissue turgor pressure tests showed that the apple cells infected with E. coli O157:H7 isolates were more likely to rupture than the control noninoculated apple cells. E. coli O157:H7 cells grown in apple tissue showed the formation of granules and vesicles within the bacterial cytoplasma and separation of the plasma membranes. Our study shows that E. coli O157:H7 can grow and survive in the apple tissue environment by causing degradation of the apple cellular components.     

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.     

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.     

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.     

Rapid detection of Escherichia coli O157:H7 inoculated in ground beef,chicken carcass,and lettuce samples with an immunomagnetic chemiluminescence fiber-optic biosensor

A biosensor was evaluated with regard to its usefulness in the rapid detection of Escherichia coli O157:H7 inoculated in ground beef, chicken carcass, and romaine lettuce samples. The biosensor consisted of a chemiluminescence reaction cell, a fiber-optic light guide, and a luminometer linked to a personal computer in conjunction with immunomagnetic separation. The samples inoculated with E. coli O157:H7 were first centrifuged and suspended in buffered peptone water and then incubated with anti-E. coli O157 antibody-coated magnetic beads and horseradish peroxidase (HRP)-labeled anti-E. coli O157 antibodies to form antibody-coated bead-bacterium-HRP-labeled antibody sandwich complexes. Finally, the sandwich complexes were separated from the samples in a magnetic field and reacted with luminol in the reaction cell. The number of E. coli O157:H7 cells was determined by collecting the HRP-catalyzed chemiluminescence signal from the bead surface through a fiber-optic light guide and measuring the signal with a luminometer. The chemiluminescence biosensor was specific for E. coli O157:H7 in samples containing other bacteria, including Salmonella Typhimurium, Campylobacter jejuni, and Listeria monocytogenes. The chemiluminescence signal was linear on a log scale from 10(2) to 10(5) CFU of E. coli O157:H7 per ml in samples. Detection could be completed within 1.5 h without any enrichment. The detection limits for ground beef, chicken carcass, and lettuce samples were 3.2 x 10(2), 4.4 x 10(2), and 5.5 x 10(2) CFU of E. coli O157:H7 per ml, respectively.     

Optimization of a fluorescence sandwich enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 in apple juice

Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 10(4) to 10(6) CFU/ml, whereas that for spiked phosphate-buffered saline was 10(6) to 10(8) CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.     

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.     

Thermal resistance parameters for Shiga toxin-producing Escherichia coli in apple juice

The purpose of the present study was to determine the heat resistance of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison to E. coli O157:H7 in single-strength apple juice without pulp. The thermal parameters for stationary-phase and acid-adapted cells of E. coli strains from serogroups O26, O45, O103, O111, O121, O145, and O157:H7 were determined by using an immersed coil apparatus. The most heat-sensitive serotype in the present study was O26. Stationary-phase cells for serotypes O145, O121, and O45 had the highest D(56°C)-value among the six non-O157 serotypes studied, although all were significantly lower (P < 0.05) than that of E. coli O157:H7. At 60°C E. coli O157:H7 and O103 demonstrated the highest D-values (1.37 ± 0.23 and 1.07 ± 0.03 min, respectively). The D(62°C) for the most heat-resistant strain belonging to the serotype O145 was similar (P > 0.05) to that for the most resistant O157:H7 strain (0.61 ± 0.17 and 0.60 ± 0.09 min, respectively). The heat resistance for stationary-phase cells was generally equal to or higher than that of acid-adapted counterparts. Although E. coli O157:H7 revealed D-values similar to or higher than the individual six non-O157 STEC serotypes in apple juice, the z-values for most non-O157 STEC tested strains were greater than those of E. coli O157:H7. When data were used to calculate heat resistance parameters at a temperature recommended in U.S. Food and Drug Administration guidance to industry, the D(71.1°C) for E. coli O157:H7 and non-O157 STEC serotypes were not significantly different (P > 0.05).     

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.     

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.     

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.     

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

The aim was to describe the inactivation kinetics of Escherichia coli O157:H7 suspended in apple juice after pulsed electric fields (PEF) and a subsequent storage under refrigeration. Escherichia coli O157:H7 showed a great PEF resistance in apple juice, when survivors were evaluated immediately after PEF. However, PEF-treated cells exhibited a great sensitivity to a subsequent holding in apple juice for 3 days. For instance, although a PEF treatment of 80 pulses at 35.0 kV/cm inactivated less than 0.5 log₁₀ cell cycles, the maintenance of the samples up to 3 days at 4C caused an inactivation of 5.0 log₁₀ cycles. An equation based on the Weibullian-like distribution accurately described the kinetics of cell inactivation.

PRACTICAL APPLICATIONS

The storage time influences the pulsed electric fields (PEF) inactivation of Escherichia coli O157:H7 cells suspended in apple juice. The potential of Weibullian-like distributions to describe survival curves with deviations in their linearity has allowed us to obtain an equation that accurately describes the complete PEF survival profile of E. coli in apple juice, when survivors were evaluated immediately after PEF and also after a subsequent storage under refrigeration. These results underline the possibility of applying PEF to pasteurize acidic foods by taking into account the postprocessing effect of the acidity of the product.     

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.     

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.