H7 Cells Research Articles

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10 5 cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10 4 cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.

Analysis of Intracellular pH in Escherichia coli O157:H7 to Determine the Effect of Chlorine Dioxide Decontamination

The aim of this study was to investigate the response of Escherichia coli O157:H7 when exposed to different concentrations of sanitation agent chlorine dioxide (ClO2) by determining intracellular pH (pHi). For this purpose, fluorescence ratio imaging microscopy was used together with pH-sensitive, ratiometric green fluorescent protein that was introduced in E. coli O157:H7 cells. Along with pHi, colony counts were determined during the treatment with ClO2. Results revealed several post-treatment subpopulations with different physiological states, as judged by their pHi. The fraction of cells with no pH gradient increased, and the colony count decreased as the concentration of ClO2 increased.

Evaluation of different micro/nanobeads used as amplifiers in QCM immunosensor for more sensitive detection of E. coli O157:H7

Micro/nanobeads with different materials (magnetic, silica and polymer) and different sizes (diameters from 30nm to 970nm) were investigated for their use as amplifiers in a quartz crystal microbalance (QCM) immunosensor for more sensitive detection of Escherichia coli O157:H7. The micro/nanobeads were conjugated with anti-E. coli antibodies. E. coli O157:H7 cells were first captured by the first antibody immobilized on the electrode surface, and then micro/nanobeads labeled secondary antibodies attached to the cells, and finally the complexes of antibody–E. coli–antibody modified beads were formed. The results showed that antibody-labeled beads lead to signal amplification in both the change in frequency (ΔF) and the change in resistance (ΔR). Since the penetration depth of the oscillation-induced shear-waves for a ∼8MHz crystal is limited to 200nm, the interpretation of how the signal is amplified by the adsorbed particles was represented in terms of the coupled-oscillator theory. The amplification is not sensed in terms of increase in mass on the sensor surface. Amplification is sensed as a change in bacterial resonance frequency when the spheres adsorb to the bacteria. The change in the values of ΔF caused by different micro/nanobeads (amplifiers) attaching on target bacterial cells is indicative of the ratio between the resonance frequency of the absorbed bacterial-particle complex (ωs), and the resonance frequency of the crystal (ω).

Cross-protective effects of temperature, pH, and osmotic and starvation stresses in Escherichia coli O157:H7 subjected to pulsed electric fields in milk

The effect of exposure of Escherichia coli O157:H7 to prior stress on the effectiveness of pulsed electric field (PEF) treatment of milk was studied. Cells were exposed to a variety of temperatures (7–45 °C), pH (4.0–7.0), osmotic conditions (0–10% NaCl), starvation (100 h at 25 °C), and cold shock (5 °C for 250 h). Stress responses were explained by the Gompertz and Weibull models with similar goodness-of-fit. Using these models, conditions that induced adequate stress were identified and used to pre-condition E. coli O157:H7 cells. The inactivation of the bacterial cells in milk by PEF was characterized by downward concavity with differences in mean time of resistance between cells exposed to temperature, acid or osmotic stress. Application of the Weibullian–Log-Logistic model indicated that, at higher field strengths, inactivation occurred at lower onset temperatures, and this was related to an increase in heat dissipation.

Maintenance of Safety and Quality of Refrigerated Ready‐to‐Cook Seasoned Ground Beef Product (Meatball) by Combining Gamma Irradiation with Modified Atmosphere Packaging

Meatballs were prepared by mixing ground beef and spices and inoculated with E. coli O157:H7, L. monocytogenes, and S. enteritidis before packaged in modified atmosphere (3% O₂ + 50% CO₂ + 47% N₂) or aerobic conditions. The packaged samples were irradiated at 0.75, 1.5, and 3 kGy doses and stored at 4 °C for 21 d. Survival of the pathogens, total plate count, lipid oxidation, color change, and sensory quality were analyzed during storage. Irradiation at 3 kGy inactivated all the inoculated (approximately 10⁶ CFU/g) S. enteritidis and L. monocytogenes cells in the samples. The inoculated (approximately 10⁶ CFU/g) E. coli O157:H7 cells were totally inactivated by 1.5 kGy irradiation. D¹⁰-values for E. coli O157:H7, S. enteritidis, and L. monocytogenes were 0.24, 0.43, and 0.41 kGy in MAP and 0.22, 0.39, and 0.39 kGy in aerobic packages, respectively. Irradiation at 1.5 and 3 kGy resulted in 0.13 and 0.36 mg MDA/kg increase in 2-thiobarbituric acid-reactive substances (TBARS) reaching 1.02 and 1.49 MDA/kg, respectively, on day 1. Irradiation also caused significant loss of color and sensory quality in aerobic packages. However, MAP effectively inhibited the irradiation-induced quality degradations during 21-d storage. Thus, combining irradiation (3 kGy) and MAP (3% O₂ + 50% CO₂ + 47% N₂) controlled the safety risk due to the potential pathogens and maintained qualities of meatballs during 21-d refrigerated storage. Combined use of gamma irradiation and modified atmosphere packaging (MAP) can maintain quality and safety of seasoned ground beef (meatball). Seasoned ground beef can be irradiated at 3 kGy and packaged in MAP with 3% O₂ + 50% CO₂ + 47% N₂ gas mixture in a high barrier packaging materials. These treatments can significantly decrease risk due to potential pathogens including E. coli O157:H7, L. monocytogenes, and S. enteritidis in the product. The MAP would reduce the undesirable effects of irradiation on quality, and extend the shelf life of the product for up to 21 d at 3 °C.

Down-regulation of hepatitis B virus replication by heparin sulfate-D-glucosaminyl-3-O-sulfotransferase 3B1

To investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication. HepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference. Southern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity. HS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.

Improved Detection of Viable Escherichia coli O157:H7 in Milk by Using Reverse Transcriptase-PCR

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at <TEX>$65^{\circ}C$</TEX> for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

Role of O-antigen on the Escherichia coli O157:H7 cells hydrophobicity, charge and ability to attach to lettuce.

Environmental factors encountered during growing and harvesting may contribute to Escherichia coli O157:H7 contamination of lettuce. Limited nutrients and extended exposure to water may cause E. coli O157:H7 to shed its O antigen. Absence of the O157-polysaccharide antigen could affect the cell's physicochemical properties (hydrophobicity and cell charge) and ultimately influence its attachment to surfaces. The objectives of this study were to evaluate the effect of the E. coli O157:H7 O-antigen on the cell's overall hydrophobicity, charge and ability to attach to cut edge and whole leaf iceberg lettuce surfaces. Three strains of E. coli O157:H7 (86-24 wild type; F-12, mutant lacking the O-antigen and pRFBE, plasmid for O157 gene reintroduced) were examined for their hydrophobicity, overall charge and ability to attach to lettuce. Overall, E. coli O157:H7 attached at higher levels to cut surfaces over whole leaf surfaces (P=0.008) for all strains and treatments. Additionally, the strain lacking the O-antigen (F12)-attached significantly less to lettuce (P=0.015) than the strains expressing the antigen (WT and pRFBE). Cells lacking the O antigen (strain F-12) were also significantly more hydrophobic than strains 86-24 or pRFBE (P≤0.05). Surface charge differed among the strains tested (P≤0.05); however, it did not appear to influence bacterial attachment to lettuce surfaces. The charge was not fully restored in the pRFBE strain (expression of O-antigen reintroduced), therefore, no conclusions can be made pertaining to the effect of charge on attachment in this study. Results indicate that E. coli O157:H7 cells which lack the O-antigen have greater hydrophobicity and attach at lower concentrations than cells expressing the O-antigen, to iceberg lettuce surfaces.

Efficacy of adding detergents to sanitizer solutions for inactivation of Escherichia coli O157:H7 on Romaine lettuce.

Numerous Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh lettuce. The development of effective and easily implemented wash treatment could reduce such incidents. The purpose of this study was to evaluate the addition of food-grade detergents to sanitizer solutions for inactivation of E. coli O157:H7 on Romaine lettuce. Freshly-cut leaves of Romaine lettuce were dip-inoculated to achieve a final cell concentration of 7.8±0.2 log CFU/g, air-dried for 2h, and stored overnight at 4 °C. Leaves were then washed for 2 min in an experimental short chain fatty acid formulation (SCFA) or in one of the following solutions with or without 0.2% dodecylbenzenesulfonic acid or 0.2% sodium 2-ethyl hexyl sulfate: 1) deionized water; 2) 100 ppm chlorine dioxide; 3) 100 ppm chlorine; and 4) 200 ppm chlorine. Following wash treatment, samples were blended in neutralizing buffer (1:3) and surface plated on the selective media CT-SMAC. The efficacy of wash treatments, with or without the detergents, in inactivating E. coli O157:H7 cells on lettuce leaves were not significantly different. The most effective wash solution was SCFA, which was capable of reducing E. coli O157:H7 populations by more than 5 log CFU/g. The rest of the wash treatments resulted in a population reduction of less than 1 log CFU/g. The effectiveness of SCFA surpasses that of other sanitizer treatments tested in this study and requires further research to optimize treatments to preserve lettuce quality. Conventional detergents did not enhance the efficacy of any of the wash treatments tested during this study.

Distinct Acid Resistance and Survival Fitness Displayed by Curli Variants of Enterohemorrhagic Escherichia coli O157:H7

Curli are adhesive fimbriae of Enterobacteriaceae and are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagic Escherichia coli O157:H7. The relative proportions of curli-producing variants (C(+)) and curli-deficient variants (C(-)) in an E. coli O157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C(+) and C(-) subpopulations occurred mostly in response to starvation and was unidirectional from C(-) to C(+); in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C(+) variants grew to higher concentrations in nutrient-limited conditions than C(-) variants, whereas C(-) variants were significantly more acid resistant than C(+) variants. This difference in acid resistance does not appear to be linked to the curli fimbriae per se, since a csgA deletion mutant in either a C(+) or a C(-) variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants of E. coli O157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in an E. coli O157:H7 population may provide a survival strategy in which C(+) variants are selected in a nutrient-limited environment, whereas C(-) variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.

Thermal inactivation of acid, cold, heat, starvation, and desiccation stress-adapted Escherichia coli O157:H7 in moisture-enhanced nonintact beef.

This study was conducted to compare thermal inactivation of stress-adapted and nonadapted Escherichia coli O157:H7 in nonintact beef moisture enhanced with different brine formulations and cooked to 65°C. Coarsely ground beef was mixed with acid, cold, heat, starvation, or desiccation stress-adapted or nonadapted rifampin-resistant E. coli O157:H7 (eight-strain mixture, 5 to 6 log CFU/g) and a brine solution for a total moisture enhancement level of 10%. The brine treatments included distilled water (control), sodium chloride (0.5% NaCl) plus sodium tripolyphosphate (0.25% STP), or NaCl + STP combined with cetylpyridinium chloride (0.2% CPC), lactic acid (0.3% LA), or sodium metasilicate (0.2% SM). The treated meat was extruded into bags (15 cm diameter), semifrozen (-20°C for 4.5 h), and cut into 2.54-cm (1-in.)-thick portions. Samples were individually vacuum packaged, frozen (-20°C for 42 h), and tempered at 4°C for 2.5 h before cooking. Partially thawed (-1.8 ± 0.4°C) samples were pan broiled to an internal temperature of 65°C. Pathogen counts of partially thawed (before cooking) samples moisture enhanced with brines containing CPC, LA, or SM were 0.7 to 1.1, 0.0 to 0.4, and 0.2 to 0.4 log CFU/g, respectively, lower than those of the control. Compared with microbial count reductions obtained after pan broiling of beef inoculated with nonadapted E. coli O157:H7 cells, count reductions during cooking of meat inoculated with cold and desiccation stress-adapted, acid stress-adapted, and heat and starvation stress-adapted cells indicated sensitization, cross protection, and no effect, respectively, of these stresses on the pathogen during subsequent exposure to heat. Among all stressed cultures, CPC-treated samples (0.8 to 3.6 log CFU/g) and LA-treated samples (0.8 to 3.5 log CFU/g) had the lowest numbers of E. coli O157:H7 survivors after cooking.

Effects of culture conditions on the subsequent heat inactivation of E. coli O157:H7 in apple juice

Escherichia coli O157:H7 cells were grown in various acidic growth media including an acidogenic nutrient broth (NB) supplemented with 1% glucose (NBG) and NB acidified to pH 4.5 with 0.25% citric or malic acids. The pH in NBG continuously decreased, reaching a final pH of 4.46 after 28 h. The pH in the control culture (NB) did not significantly change throughout the incubation. When heated in apple juice at 55 °C, cells grown in NBG had the significantly highest D55 value (250 ± 39.67 s). Thermal inactivation rates established for cells grown in NB, citric acid- and malic acid-acidified NBs were 51.39 ± 1.34, 25.46 ± 1.21, and 45.42 ± 0.36 s, respectively. When compared with the D55 values of cells previously exposed to combinations of environmental stress factors, cells grown in NBG were also found to be significantly heat resistant and were deemed appropriate to be used in heat challenge studies.

Survival of Escherichia coli O157:H7 in Cucumber Fermentation Brines

Bacterial pathogens have been reported on fresh cucumbers and other vegetables used for commercial fermentation. The Food and Drug Administration currently has a 5-log reduction standard for E. coli O157:H7 and other vegetative pathogens in acidified pickle products. For fermented vegetables, which are acid foods, there is little data documenting the conditions needed to kill acid resistant pathogens. To address this knowledge gap, we obtained 10 different cucumber fermentation brines at different stages of fermentation from 5 domestic commercial plants. Cucumber brines were used to represent vegetable fermentations because cabbage and other vegetables may have inhibitory compounds that may affect survival. The 5-log reduction times for E. coli O157:H7 strains in the commercial brines were found to be positively correlated with brine pH, and ranged from 3 to 24 d for pH values of 3.2 to 4.6, respectively. In a laboratory cucumber juice medium that had been previously fermented with Lactobacillus plantarum or Leuconostoc mesenteroides (pH 3.9), a 5-log reduction was achieved within 1 to 16 d depending on pH, acid concentration, and temperature. During competitive growth at 30 °C in the presence of L. plantarum or L. mesenteroides in cucumber juice, E. coli O157:H7 cell numbers were reduced to below the level of detection within 2 to 3 d. These data may be used to aid manufacturers of fermented vegetable products determine safe production practices based on fermentation pH and temperature. Disease causing strains of the bacterium E. coli may be present on fresh vegetables. Our investigation determined the time needed to kill E. coli in cucumber fermentation brines and how E. coli strains are killed in competition with naturally present lactic acid bacteria. Our results showed how brine pH and other brine conditions affected the killing of E. coli strains. These data can be used by producers of fermented vegetable products to help assure consumer safety.

Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance.

A surface plasmon resonance (SPR) immunosensor was developed for the detection of E. coli O157:H7 by means of a new subtractive inhibition assay. In the subtractive inhibition assay, E. coli O157:H7 cells and goat polyclonal antibodies for E. coli O157:H7 were incubated for a short of time, and then the E. coli O157:H7 cells which bound antibodies were removed by a stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using a BIAcore 3000 biosensor. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 104 to 3.0 × 108 cfu/mL with a detection limit of 3.0 × 104 cfu/mL. Compared with direct SPR by immobilizing antibodies on the chip surface to capture the bacterial cells and ELISA for E. coli O157:H7 (detection limit: both 3.0 × 105 cfu/mL in this paper), the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein interaction, which has the potential for providing a practical alternative for the monitoring of E. coli O157:H7 and other pathogens.

Determination of free chlorine concentrations needed to prevent Escherichia coli O157:H7 cross-contamination during fresh-cut produce wash.

This study was conducted to investigate the effect of free chlorine concentrations in wash water on Escherichia coli O157:H7 reduction, survival, and transference during washing of fresh-cut lettuce. The effectiveness of rewashing for inactivation of E. coli O157:H7 on newly cross-contaminated produce previously washed with solutions containing an insufficient amount of chlorine also was assessed. Results indicate that solutions containing a minimum of 0.5 mg/liter free chlorine were effective for inactivating E. coli O157:H7 in suspension to below the detection level. However, the presence of 1 mg/liter free chlorine in the wash solution before washing was insufficient to prevent E. coli O157:H7 survival and transfer during washing because the introduction of cut lettuce to the wash system quickly depleted the free chlorine. Although no E. coli O157:H7 was detected in the wash solution containing 5 mg/liter free chlorine before washing a mix of inoculated and uninoculated lettuce, low numbers of E. coli O157:H7 cells were detected on uninoculated lettuce in four of the seven experimental trials. When the prewash free chlorine concentration was increased to 10 mg/liter or greater, no E. coli O157:H7 transfer was detected. Furthermore, although rewashing newly cross-contaminated lettuce in 50 mg/liter free chlorine for 30 s significantly reduced (P = 0.002) the E. coli O157:H7 populations, it failed to eliminate E. coli O157:H7 on lettuce. This finding suggests that rewashing is not an effective way to correct for process failure, and maintaining a sufficient free chlorine concentration in the wash solution is critical for preventing pathogen cross-contamination.

Development of a Sensitive Detection Method for Stressed E. coli O157:H7 in Source and Finished Drinking Water by Culture-qPCR

A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.

Development of oscillation method for reducing foodborne pathogens on lettuce and spinach

In this study, the efficacy of an oscillator for reducing the numbers of foodborne pathogens on lettuce and spinach was tested. A cocktail of three strains each of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes cells and of Bacillus cereus spores was inoculated onto lettuce and spinach leaves and followed by oscillation at 10 Hz and 20 Hz for up to 30 s. After treatment of inoculated lettuce leaf with an oscillator at 20 Hz for 30 s, 2.58, 2.82, 2.21 and 2.22 Log 10 CFU/g reductions were obtained with the cells of S. typhimurium, E. coli O157:H7 and L. monocytogenes and the spores of B. cereus, respectively. In the case of the oscillation treatment of spinach leaf, 2.89, 3.73, 2.46 and 2.25 Log 10 CFU/g reductions of those pathogens were achieved under the same condition. Statistically significant reductions were observed after oscillation treatment at 20 Hz for 5–10 s. The oscillation treatment at 10 Hz led to slightly less reductions of the pathogens tested as compared to the treatment at 20 Hz. In conclusion, the oscillation method developed shows to be highly efficacious in reducing foodborne pathogens on lettuce and spinach leaves.

Magnetic Nanoparticle Based Magnetophoresis for Efficient Separation of E. coli O157:H7

In this research, a magnetophoretic separation device was designed, fabricated, and tested using magnetic nanoparticles (MNPs) for more efficiently separating target foodborne pathogens. This magnetophoretic separation device consisted of a rectangular channel with a steel rod on its top, both of which were immersed in a homogenous magnetic field generated by two permanent magnets. A sample containing foodborne pathogens was incubated with biotin conjugated antibody (Ab) and then streptavidin-coated MNPs with a diameter of 30 nm to form MNPs-Ab-cell complexes, the magnetic cells. The magnetized steel rod concentrated the magnetic field and generated a high magnetic field gradient, which could separate the magnetic cells from a background fluid without need of repeated washing. First, a mathematical model was developed in order to understand and predict the performance of the separation device. Secondly, the magnetophoretic separation device was experimentally tested using Escherichia coli O157:H7 as a model bacterium. The results showed that 97.5% of the E. coli O157:H7 cells (4.4 103 CFU mL-1) could be separated from a phosphate buffered saline (PBS) solution at a flow rate of 14.4 L min-1. In addition, a separation efficiency of 85.6% was achieved during the separation of E. coli O157:H7 from a mixture of E. coli O157:H7 (4.6 103 CFU mL-1) and Listeria innocua (5.7 103 CFU mL-1) in a PBS solution. The magnetophoretic separation device has great potential for broad applications in the preparation or pretreatment of a large volume of food, agricultural, and environmental samples in conjunction with a rapid biodetection method, such as biosensors, PCR, or ELISA, to more sensitively detect bacterial pathogens.

Effect of slightly acidic electrolyzed water for inactivating Escherichia coli O157:H7 and Staphylococcus aureus analyzed by transmission electron microscopy.

The use of different available chlorine concentrations (ACCs) of slightly acidic electrolyzed water (SAEW; 0.5 to 30 mg/liter), different treatment times, and different temperatures for inactivating Escherichia coli O157:H7 and Staphylococcus aureus was evaluated. The morphology of both pathogens also was analyzed with transmission electron microscopy. A 3-min treatment with SAEW (pH 6.0 to 6.5) at ACCs of 2 mg/liter for E. coli O157:H7 and 8 mg/liter for S. aureus resulted in 100% inactivation of two cultures (7.92- to 8.75-log reduction) at 25°C. The bactericidal activity of SAEW was independent of the treatment time and temperature at a higher ACC (P > 0.05). E. coli O157:H7 was much more sensitive than S. aureus to SAEW. The morphological damage to E. coli O157:H7 cells by SAEW was significantly greater than that to S. aureus cells. At an ACC as high as 30 mg/liter, E. coli O157:H7 cells were damaged, but S. aureus cells retained their structure and no cell wall damage or shrinkage was observed. SAEW with a near neutral pH may be a promising disinfectant for inactivation of foodborne pathogens.

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.