Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers

The influence of treating Escherichia coli O157:H7 cells labeled with an enhanced green fluorescent protein (EGFP) plasmid with 20 microg/ml active chlorine, 100 mg/ml hydrogen peroxide, and 80 mg/ml acetic acid on fluorescence intensity was determined. In addition, fluorescent staining methods to differentiate viable and dead E. coli O157:H7 cells on the cuticle of Red Delicious cv. apples following treatment with water or 200 microg/ml active chlorine were evaluated. Suspensions of E. coli O157:H7 EGFP+ cells were exposed to chemical treatment solutions for 0, 30, 60, 120, or 300 s before populations (log10 cfu/ml) were determined by surface plating, and fluorescence intensities of suspensions and individual cells were measured using spectrofluorometry and confocal scanning laser microscopy (CSLM), respectively. The relative fluorescence intensity of suspensions and individual cells changed upon exposure to various treatments. Results indicate that the use of EGFP to tag E. coli O157:H7 may not be appropriate for investigations seeking to microscopically differentiate viable and dead cells on produce following surface treatment with sanitizers. SYTOX Orange and SYTOX Green nucleic acid stains fluorescently labeled dead E. coli O157:H7 cells attached to apple cuticles more intensely than did propidium iodide. A cross-signal occurred between CSLM photomultipliers when examining tissues treated with SYTOX Orange to detect dead cells and antibody labeled with Alexa Fluor 488 to detect total (dead and viable) cells. Because of the possibility of cross-signal resulting in an overestimation of the number of dead cells on apples and, perhaps, other produce treated with these stains, SYTOX Green is preferred to detect dead cells and antibody labeled with Alexa Fluor 594 is preferred to detect the total number of cells on apple surfaces following treatment with sanitizers. The performance of SYTOX Green in combination with Alexa Fluor 594 to detect dead and total cells of E. coli O157:H7 on other produce remains to be determined.     

Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection

Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157: H7 for 24+/-1 h at 4 degrees C. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.     

Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 microm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.     

Survival differences of Escherichia coli O157:H7 strains in apples of three varieties stored at various temperatures

Differences in survival and growth among five different Escherichia coli O157:H7 strains in three apple varieties were determined at various temperatures. Jonathan, Golden Delicious, and Red Delicious apples were wounded and inoculated with E coli O157:H7 strains C7929 (apple cider isolate), 301C (chicken isolate), 204P (pork isolate), 933 (beef isolate), and 43890 (human isolate) at an initial level of 6 to 7 log CFU/g. The inoculated apples were stored at a constant temperature of 37, 25, 8, or 4 degrees C or at 37 degrees C for 24 h and then at 4 degrees C, and bacterial counts were determined every week for 28 days. By day 28, for Jonathan apples at 25 degrees C, the apple isolate counts were significantly higher than the chicken and human isolate counts. At 4 degrees C for 28 days, the human isolate inoculated into Jonathan, Golden Delicious, and Red Delicious apples was present in significantly smaller numbers than the other strains. The apple isolate survived significantly better at 4 degrees C, yielding the highest number of viable cells. By days 21 and 28, for apples stored at 37 degrees C for the first 24 h and then at 4 degrees C, the counts of viable E. coli O157:H7 apple and human isolates were 6.8 and 5.8 log CFU/g at the site of the wound, whereas for apples kept at 4 degrees C for the duration of storage, the respective counts were 5.6 and 1.5 log CFU/g. Our study shows that E. coli O157:H7 strains responded differentially to their ability to survive in these three apple varieties at 25 or 4 degrees C and produced higher viable counts when apples were temperature abused at 37 degrees C for 24 h and then stored at 4 degrees C for 27 days.     

Survival of Escherichia coli O157:H7 and Salmonella Muenchen on apples as affected by application of commercial fruit waxes

Survival of Escherichia coli O157:H7, Salmonella Muenchen, and yeasts and molds on apples as affected by application of five commercial apple waxes was investigated. Red Delicious cv. apples at 21 degrees C were spot inoculated with E. coli O157:H7 and S. Muenchen and spray coated with waxes. Apples sprayed with water served as controls. Apples were dried at either 21 or 55 degrees C for 2 min before subjecting to microbiological analysis after storage for 0, 1, 3, 6, and 12 weeks at 2 or 21 degrees C. Drying temperature did not significantly influence populations of E. coli O157:H7 and S. Muenchen. Waxing reduced populations E. coli O157:H7 and S. Muenchen by up to 1.48 log10 cfu/apple. Compared to untreated apples, treatment of apples with water or waxes resulted in significant (P < or = 0.05) reductions in populations of E. coli O157:H7 and S. Muenchen during storage at 2 degrees C. Reductions on waxed apples stored at 21 degrees C were not as marked compared to reductions on waxed apples stored at 2 degrees C. With the exception of one wax, drying temperature did not significantly influence populations of yeasts and molds. Mold populations were less affected by wax applications than were yeasts, and were detected in higher numbers on apples treated with three of the five waxes compared to populations recovered from untreated control apples. None of the waxes evaluated can be relied upon to kill or remove E. coli O157:H7 and Salmonella on apples.     

Control of browning and microbial growth on fresh-cut apples by sequential treatment of sanitizers and calcium ascorbate

ABSTRACT:  This study investigated the efficacy of different sanitizers, including acidic electrolyzed water (AEW), peroxyacetic acid (POAA), and chlorine, on the inactivation of Escherichia coli O157:H7 on fresh-cut apples. The effects of the sanitizers and sequential treatments of AEW or POAA followed by calcium ascorbate (CaA) on browning inhibition and organoleptic qualities of fresh-cut apples stored under different package atmospheres at 4 °C were also evaluated. Changes in package atmosphere composition, product color, firmness, total aerobic bacterial counts, yeast and mold counts, and sensory qualities were examined at 0, 4, 8, 11, and 21 d. Among all sanitizer treatments, POAA and AEW achieved the highest reduction on E. coli O157:H7 populations. The sequential treatment of AEW followed by CaA (AEW-CaA) achieved the best overall dual control of browning and bacterial growth on fresh-cut apple wedges. Package atmospheres changed significantly over time and among package materials. Packages prepared with films having an oxygen transmission rate (OTR) of 158 had significantly lower O₂ and higher CO₂ partial pressures than those prepared with 225 OTR films and the Ziploc™ bags. The effect of package atmospheres on the browning of apples is more pronounced on AEW, POAA, and POAA-CaA-treated apple wedges than on AEW-CaA-treated samples.     

Survival of enterohemorrhagic Escherichia coli O157:H7 in bovine feces applied to lettuce and the effectiveness of chlorinated water as a disinfectant.

Bovine feces are a potential vehicle for transmitting enterohemorrhagic Escherichia coli O157:H7 to humans. A study was undertaken to determine survival characteristics of E. coli O157:H7 on iceberg lettuce using 0.1% peptone water and bovine feces as carriers for inocula. Four levels of inoculum, ranging from 10(0) to 10(5) CFU of E. coli O157:H7 per g of lettuce, were applied. Populations surviving on lettuce stored at 4 degrees C were monitored for up to 15 days. Regardless of the type of carrier, viable cells of E. coli O157:H7 were detected on lettuce after 15 days, even when the initial inoculum was 10(0) to 10(1) CFU/g. Spray treatments of lettuce with 200 ppm chlorine solution or deionized water were equally effective in killing or removing E. coli O157:H7 from lettuce. Holding lettuce for 5 min after spray treatment was not more effective in reducing populations than holding for 1 min before rinsing with water. Prevention of contamination of lettuce with bovine feces that may harbor E. coli O157:H7 as well as other infectious microorganisms is essential to minimizing the risk of illness. The development of sanitizers more efficacious than chlorine for the removal of pathogens from raw fruits and vegetable is needed.     

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.     

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.     

Reduction of Escherichia coli O157:H7 counts on whole fresh apples by treatment with sanitizers

The objectives of this study were to determine if washing of whole apples with solutions of three different sanitizers (peroxyacetic acid, chlorine dioxide, or a chlorine-phosphate buffer solution) could reduce a contaminating nonpathogenic Escherichia coli O157:H7 population by 5 logs and at what sanitizer concentration and wash time such a reduction could be achieved. Sanitizers were tested at 1, 2, 4, 8, and 16 times the manufacturer's recommended concentration at wash times of 5, 10, and 15 min. Whole, sound Braeburn apples were inoculated with approximately 1 x 108 or 7 x 106 CFU per apple, stored for 24 h, then washed with sterile water (control) or with sanitizers for the prescribed time. Recovered bacteria were enumerated on trypticase soy agar. Washing with water alone reduced the recoverable population by almost 2 logs from the starting population; this can be attributed to physical removal of organisms from the apple surface. No sanitizer, when used at the recommended concentration, reduced the recovered E. coli population by 5 logs under the test conditions. The most effective sanitizer, peroxyacetic acid, achieved a 5-log reduction when used at 2.1 to 14 times its recommended concentration, depending on the length of the wash time. The chlorine-phosphate buffer solution reduced the population by 5 logs when used at 3 to 15 times its recommended concentration, depending on wash time. At no concentration or wash time tested did chlorine dioxide achieve the 5-log reduction.     

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.     

Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach

Escherichia coli O157:H7 contaminated spinach has recently caused several outbreaks of human illness in the USA and Canada. However, to date, there has been no study demonstrating an effective way to eliminate E. coli O157:H7 in spinach. Therefore, this study was conducted to investigate the effect of chemical sanitizers alone or in combination with packaging methods such as vacuum and modified atmosphere packaging (MAP) on inactivating E. coli O157:H7 in spinach during storage time. Spinach inoculated with E. coli O157:H7 was packaged in four different methods (air, vacuum, N(2) gas, and CO(2) gas packaging) following treatment with water, 100 ppm chlorine dioxide, or 100 ppm sodium hypochlorite for 5 min at room temperature and stored at 7+/-2 degrees C. Treatment with water did not significantly reduce levels of E. coli O157:H7 in spinach. However, treatment with chlorine dioxide and sodium hypochlorite significantly decreased levels of E. coli O157:H7 by 2.6 and 1.1 log(10)CFU/g, respectively. Levels of E. coli O157:H7 in samples packaged in air following treatments grew during storage time, whereas levels were maintained in samples packaged in other packaging methods (vacuum, N(2) gas, and CO(2) gas packaging). Therefore there were significant differences (about 3-4 log) of E. coli O157:H7 populations between samples packed in air and other packaging methods following treatment with chemical sanitizers after 7 days storage. These results suggest that the combination of treatment with chlorine dioxide and packaging methods such as vacuum and MAP may be useful for improving the microbial safety of spinach against E. coli O157:H7 during storage.     

Direct microscopic observation of lettuce leaf decontamination with a prototype fruit and vegetable washing solution and 1% NaCl-NaHCO3

Efficacy of a prototype, food-grade alkaline surfactant washing solution and 1% NaCl-NaHCO3 (pH 10.0) against Escherichia coli O157:H7 cells on lettuce leaves was evaluated. Lettuce was inoculated with 10(9) CFU/ml of E. coli O157:H7 for 24 +/- 1 h at 4 degrees C. Samples were rinsed and treated with the prototype washing solution containing lauryl sodium sulfate or NaCl-NaHCO3 for 3 min at 22 degrees C. Viability of E. coli O157:H7 cells was examined by plate counts at the surface and cut edge, and by confocal scanning microscopic (CSLM) observation of samples stained with Sytox green and Alexa 594 conjugated antibody against E. coli O157:H7 at intact leaf surface, stomata, and damaged tissue (0 to 10, 30 to 40, and 0 to 40 microm from the cut surface). Although both treatments caused significant log reductions in CFU at the surface and cut edge, log reductions were greater for the prototype washing solution (0.7 to 1.1 log CFU/cm2) than for NaCl-NaHCO3 (0.2 to 0.4 log CFU/cm2) (P < 0.05). Percentage of viability determined by CSLM for prototype washing solution was significantly greater at 30 to 40 microm from cut surfaces than at 0 to 10 and 0 to 40 microm from cut surfaces and intact surfaces (P < 0.05). Stomata provided moderate protection. NaCl-NaHCO3 was less effective than the prototype washing solution, and high percentages of E. coli O157:H7 cells remained viable at all sites except at the surface. The percent viabilities determined by CSLM were not significantly different from those determined by plate counts for NaCl-NaHCO3 treatment (P > 0.05). However, CSLM indicated significantly greater percent viability than plate counts for lettuce treated with the prototype washing solution (P < 0.05). Surfactant-containing washing solutions warrant additional testing for decontamination of fresh produce.     

Influence of extended acid stressing in fresh beef decontamination runoff fluids on sanitizer resistance of acid-adapted Escherichia coli O157:H7 in biofilms

This study evaluated resistance to sanitizing solutions of Escherichia coli O157:H7 cells forming biofilms on stainless steel coupons exposed to inoculated meat decontamination runoff fluids (washings). A previously acid-adapted culture of a rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated in unsterilized or sterilized combined hot-water (85 degrees C) and cold-water (10 degrees C) (50/50 [vol/vol]) composite water (W) washings (pH 6.29 to 6.47) and in W washings mixed with 2% acetic acid (pH 4.60 to 4.71) or in 2% lactic acid W washings (pH 4.33 to 4.48) at a ratio of 1/99 (vol/vol). Stainless steel coupons (2 by 5 by 0.08 cm) were submerged in the inoculated washings and stored for up to 14 days at 15 degrees C. Survival of E. coli O157:H7 was determined after exposure (0 to 60 s for cells in suspension and 0 to 300 s for attached cells) to two commercial sanitizers (150 ppm peroxyacetic acid and 200 ppm quaternary ammonium compound) at 2, 7, and 14 days. E. coli O157:H7 attached more rapidly to coupons submerged in washings containing the natural flora than to those without. The attached cells were more resistant to the effects of the sanitizers than were the cells in suspension, and survival was highest in the presence of the natural flora. Attached cells in the presence of dilute acid washings were more sensitive to subsequent sanitizer treatments than were cells generated in the presence of W washings. Under the conditions of this study, cells of E. coli O157:H7 in W washings were more sensitive to acidic (peroxyacetic acid) than to alkaline (quaternary ammonium) sanitizers during storage. These results suggest that meat processing plants that apply no decontamination or that use only water washings of meat should consider using acidic sanitizers to enhance biofilm removal. Plants that apply both water and acidic washings may create a sublethal acid-stressing environment in the runoff fluids, sensitizing biofilm cells to subsequent sanitizing treatments.     

Evaluation of inoculation method and inoculum drying time for their effects on survival and efficiency of recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes inoculated on the surface of tomatoes

A study was undertaken to evaluate methods for applying inoculum and to examine the effect of inoculum drying time on survival and recovery of foodborne pathogens inoculated onto the surface of raw, ripe tomatoes. Five-strain mixtures of Escherichia coli O157:H7, Salmonella, or Listeria monocytogenes were applied to tomatoes by dip, spot, or spray inoculation methods. Inocula were dried for 1 or 24 h at 22 degrees C before tomatoes were treated with water (control) or chlorine (200 micrograms/ml). Significantly (alpha = 0.05) larger populations (CFU per tomato) of E. coli O157:H7 and Salmonella were recovered from dipinoculated tomatoes than from spot- or spray-inoculated tomatoes. This difference was attributed to larger numbers of cells adhering to tomatoes subjected to dip inoculation. Populations of E. coli O157:H7 and Salmonella recovered from spot- and spray-inoculated tomatoes containing the same initial number of cells were not significantly different. Significantly different L. monocytogenes population sizes were recovered from inoculated tomatoes (dip > spot > spray). Populations of pathogens recovered from tomatoes were significantly larger when inocula were dried for 1 h compared with 24 h. Significant differences (water > chlorine) were observed in the sizes of populations for all pathogens recovered from tomatoes treated with chlorine, regardless of inoculation method or drying time. Results indicate that inoculation method, drying time, and treatment affect survival and/or recovery of foodborne pathogens inoculated onto the surface of tomatoes. We recommend that spot inoculation with a drying time of 24 h at 22 degrees C be used with standard methods to determine the efficacy of chlorine and other sanitizers for killing foodborne pathogens on tomatoes.     

Survival and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method of inoculation, time between inoculation and analysis, and treatment with chlorinated water

The effects of method for applying inoculum and of drying time after inoculation on survival and recovery of foodborne pathogens on iceberg lettuce and parsley were studied. Five-strain mixtures of Escherichia coli O157:H7, Salmonella, or Listeria monocytogenes were applied to lettuce and parsley by dip, spot, or spray inoculation methods. Inocula were dried for 2 h at 22 degrees C or for 2 h at 22 degrees C and then 22 h at 4 degrees C before being treated with water (control) or chlorine (200 microg/ml). Significantly higher populations (CFU per lettuce or parsley sample) of E. coli O157:H7 and Salmonella (alpha = 0.05) were recovered from dip-inoculated produce than from spot- or spray-inoculated produce. This difference was attributed to larger numbers of cells adhering to lettuce and parsley subjected to dip inoculation. Populations of E. coli O157:H7 and Salmonella recovered from lettuce inoculated by spot and spray methods were not significantly different, but populations recovered from spot-inoculated parsley were significantly higher than those recovered from spray-inoculated parsley, even though the number of cells applied was the same. Significantly different numbers of L. monocytogenes were recovered from inoculated lettuce (dip > spray > spot); populations recovered from dip-inoculated parsley were significantly higher than those recovered from spot- or spray-inoculated parsley, which were not significantly different from each other. Populations of pathogens recovered from lettuce and parsley after drying inoculum for 2 h at 22 degrees C were significantly higher than or equal to populations recovered after drying for 2 h at 22 degrees C and then for 22 h at 4 degrees C. Significant differences (water > chlorine) were observed in populations of all pathogens recovered from treated lettuce and parsley, regardless of inoculation method and drying time. It is recommended that spot inoculation with a drying time of 2 h at 22 degrees C followed by 22 h at 4 degrees C be used to determine the efficacy of chlorine and other sanitizers in killing foodborne pathogens on lettuce and parsley.     

Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and Non-O157 Strains and Their Tolerance to Sanitizers Commonly Used in the Food Processing Environment

Shiga toxin-producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride-based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination.     

Growth and survival of Escherichia coli O157:H7 on fresh-cut apples in modified atmospheres at abusive temperatures

The effects of reduced-O2 and elevated-CO2 modified atmospheres (MAs) and abusive temperatures on the growth and survival of E. coli O157:H7, yeast, and molds and on changes in the visual quality of fresh-cut apples were evaluated. High-CO1 and low-O2 (> or = 15% and < 1%, respectively) atmospheres inhibited the growth of the pathogen on apple slices at 15 and 20 degrees C. However, the population of the pathogen increased by 1 log cycle after 2 weeks of storage in air. The high-CO2 MA resulted in the inhibition of yeast and mold growth, less browning, and better visual quality than did air and ambient-CO2 atmospheres. The results of this study confirm that E. coli O157:H7 can grow on apple slices in air. These results also show that these organisms survive but are inhibited in MAs with high CO2 levels at abusive temperatures. An MA can increase the shelf life of fresh-cut apples by improving retention of visual quality and inhibiting yeast and molds. Thus, contamination of minimally processed apples with E. coli O157:H7 can be a safety issue for both air- and MA-packaged cut apples.     

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.     

Influence of inoculation method, spot inoculation site, and inoculation size on the efficacy of acidic electrolyzed water against pathogens on lettuce

The influence of bacterial inoculation methods on the efficacy of sanitizers against pathogens was examined. Dip and spot inoculation methods were employed in this study to evaluate the effectiveness of acidic electrolyzed water (AcEW) and chlorinated water (200 ppm free available chlorine) against Escherichia coli O157:H7 and Salmonella spp. Ten pieces of lettuce leaf (5 by 5 cm) were inoculated by each method then immersed in 1.5 liters of AcEW, chlorinated water, or sterile distilled water for 1 min with agitation (150 rpm) at room temperature. The outer (abaxial) and inner (adaxial) surfaces of the lettuce leaf were distinguished in the spot inoculation. Initial inoculated pathogen population was in the range 7.3 to 7.8 log CFU/g. Treatment with AcEW and chlorinated water resulted in a 1 log CFU/g or less reduction of E. coli O157:H7 and Salmonella populations inoculated with the dip method. Spot inoculation of the inner surface of the lettuce leaf with AcEW and chlorinated water reduced the number of E. coli O157:H7 and Salmonella by approximately 2.7 and 2.5 log CFU/g, respectively. Spot inoculation of the outer surface of the lettuce leaf with both sanitizers resulted in approximately 4.6 and 4.4 log CFU/g reductions of E. coli O157:H7 and Salmonella, respectively. The influence of inoculation population size was also examined. Each sanitizer could not completely eliminate the pathogens when E. coli O157:H7 and Salmonella cells inoculated on the lettuce were of low population size (10(3) to 10(4) CFU/g), regardless of the inoculation technique.