Comparison of microRNA expressions for the identification of chemical hazards in in vivo and in vitro hepatic injury models.

Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.

Behaviour of Aspergillus flavus and Fusarium graminearum on rice as affected by degree of milling, temperature, and relative humidity during storage.

We investigated the survival and growth patterns of Aspergillus flavus and Fusarium graminearum, as well as mycotoxin production, on Korean rice as affected by the degree of milling (rough, brown, and white rice) and storage conditions (21 °C/85% relative humidity [RH], 21 °C/97% RH, and 30 °C/85% RH). When rice was stored at 21 °C/85% RH, the population of A. flavus remained constant and aflatoxin was not produced, regardless of the degree of milling. At 21 °C/97% RH and 30 °C/85% RH, the populations of A. flavus increased significantly (P ≤ 0.05) and aflatoxins were produced. The highest population of A. flavus and highest amount of aflatoxin B1 were observed on brown rice stored at 21 °C/97% RH. For F. graminearum, when stored at 85% RH, the populations were reduced to less than a detectable level (5 CFU/g of rice) within 120 days and no deoxynivalenol (DON) was produced, regardless of the degree of milling and storage temperature. However, at 21 °C/97% RH, the population of F. graminearum increased significantly (P ≤ 0.05) and DON was produced on all types of rice. Findings from this study provide insights concerning storage conditions necessary to prevent growth and mycotoxin production by A. flavus and F. graminearum on Korean rice with different degrees of milling.

Bacillus cereus and Bacillus thuringiensis spores in Korean rice: prevalence and toxin production as affected by production area and degree of milling.

We determined the prevalence of and toxin production by Bacillus cereus and Bacillus thuringiensis in Korean rice as affected by production area and degree of milling. Rough rice was collected from 64 farms in 22 agricultural areas and polished to produce brown and white rice. In total, rice samples were broadly contaminated with B. cereus spores, with no effect of production area. The prevalence and counts of B. cereus spores declined as milling progressed. Frequencies of hemolysin BL (HBL) production by isolates were significantly (P ≤ 0.01) reduced as milling progressed. This pattern corresponded with the presence of genes encoding the diarrheal enterotoxins. The frequency of B. cereus isolates positive for hblC, hblD, or nheB genes decreased as milling progressed. Because most B. cereus isolates from rice samples contained six enterotoxin genes, we concluded that B. cereus in rice produced in Korea is predominantly of the diarrheagenic type. The prevalence of B. thuringiensis in rice was significantly lower than that of B. cereus and not correlated with production area. All B. thuringiensis isolates were of the diarrheagenic type. This study provides information useful for predicting safety risks associated with B. cereus and B. thuringiensis in rough and processed Korean rice.

Controlled fermentation of kimchi using naturally occurring antimicrobial agents.

Kimchi is a traditional Korean fermented food. Since it ferments continuously during distribution and storage, the extension of shelf life by preventing over-acidification is a major concern in the kimchi industry. One of the most frequently attempted ways to delay fermentation is to add naturally occurring antimicrobial agents. Many researchers have investigated ways to delay over-acidification by adding minor ingredients, fruits or fruit seed extracts, extracts of medicinal herbs, culinary herbs and spices, and other miscellaneous substances to kimchi. The addition of naturally occurring antimicrobial agents may enhance the acceptability of kimchi to consumers over a longer period of time but may also have a disadvantage in that it may cause changes in sensory quality, especially if added in large amounts. To avoid undesirable sensory changes, application of hurdle technologies (i.e., multifactor preservative systems) which involve using combinations of low amounts of various naturally occurring antimicrobial agents as ingredients should be explored with the goal of controlling fermentation. If synergistic or additive antimicrobial effects can be achieved using small amounts of a combination of natural agents, changes in sensory qualities will be minimized, thereby prolonging shelf life. Research findings summarized in this review provide a basis for developing effective hurdle technologies using naturally occurring antimicrobial agents to extend shelf life of kimchi and perhaps other types of traditional fermented foods.

Malignant peripheral nerve sheath tumor arising from the left ventricle.

We report a case of a 32-year-old female who underwent complete excision of a malignant left ventricular peripheral nerve sheath tumor. Malignant peripheral nerve sheath tumors in the mediastinum, including the pericardium, are rare. This case report describes a malignant peripheral nerve sheath tumor arising from the left ventricular origin for which complete excision was possible.

Inactivation of Escherichia coli O157:H7 on radish seeds by sequential treatments with chlorine dioxide, drying, and dry heat without loss of seed viability.

We developed and validated a treatment to inactivate Escherichia coli O157:H7 on radish seeds without decreasing seed viability. Treatments with aqueous ClO(2) followed by drying and dry-heat treatments were evaluated for efficacy to inactivate the pathogen. Conditions to dry radish seeds after treatment with water (control) or ClO(2) were established. When treated seeds with high water activity (a(w)) (>0.99) were stored at 45°C and 23% relative humidity (RH), the a(w) decreased to <0.30 within 24 h. Drying high-a(w) seeds before exposing them to dry-heat treatment (≥60°C) was essential to preserve seed viability. The germination rate of radish seeds which had been immersed in water for 5 min, dried at 45°C and 23% RH for 24 h, and heated at 70°C for 48 h or at 80°C for 24 h was not significantly decreased (P ≤ 0.05) compared to that of untreated radish seeds. Sequential treatments with ClO(2) (500 µg/ml, 5 min), drying (45°C, 23% RH, 24 h), and dry heating (70°C, 23% RH, 48 h) eliminated E. coli O157:H7 (5.9 log CFU/g) on radish seeds and, consequently, sprouts produced from them without decreasing the germination rate. These sequential treatments are recommended for application to radish seeds intended for sprout production.

Combined effects of chlorine dioxide, drying, and dry heat treatments in inactivating microorganisms on radish seeds.

We determined the combined effectiveness of ClO(2) (200 and 500 µg/ml, 5 min), air drying [25 °C, 40% relative humidity (RH), 2 h], and mild dry heat (55 °C, 23% RH, up to 48 h) treatments in killing total aerobic bacteria (TAB), Escherichia coli O157:H7, and molds and yeasts (MY) on radish seeds. A 5.1-log reduction in the number of TAB was achieved on radish seeds treated with 200 or 500 µg/ml ClO(2) followed by air drying for 2 h and dry heat treatment for 48 h or 24 h, respectively. When radish seeds were treated with 200 and 500 µg/ml ClO(2), air dried, and heat treated for 12 h and 6 h, respectively, the initial population of E. coli O157:H7 (5.6 log CFU/g) on seeds was reduced to an undetectable level (<0.8 log CFU/g). However, the pathogen was detected in 5-day-old sprouts. The reduction of MY (1.2-1.0 log CFU/g) on radish seeds under similar experimental conditions was not changed significantly during subsequent heat treatment up to 48 h. Results show that treating radish seeds with 500 µg/ml ClO(2), followed by air dried at 25 °C for 2 h and heat treatment at 55 °C for 36 h achieved a >5-log CFU/g reduction of TAB and E. coli O157:H7. These observations will be useful when developing effective strategies and practices to enhance the microbiological safety of radish sprouts.

Synergistic effect of chlorine dioxide and drying treatments for inactivating Escherichia coli O157:H7 on radish seeds.

Studies were done to determine whether calcium hypochlorite (Ca(OCl)(2)) and chlorine dioxide (ClO(2)) treatment followed by drying had a synergistic killing effect on microorganisms on radish seeds intended for sprout production. Uninoculated radish seeds and seeds inoculated with Escherichia coli O157:H7 were treated with water, Ca(OCl)(2) (free chlorine concentrations of 50 or 200 microg/ml), or ClO(2) (50 or 200 microg/ml) for 5 min and subsequently dried at 25 degrees C for up to 24 h. Populations of total aerobic bacteria (TAB), molds and yeasts (MY), and E. coli O157:H7 on the seeds treated with Ca(OCl)(2) were not significantly different (P = 0.05) than populations on seeds treated with ClO(2) at the same concentrations. However, populations of microorganisms on seeds treated with ClO(2) decreased more rapidly during drying. Treatment with ClO(2) (200 microg/ml) followed by drying caused reductions in TAB, MY, and E. coli O157:H7 of 3.1, 2.0, and 3.8 log CFU/g, respectively. When seeds were treated with water, Ca(OCl)(2) (50 or 200 microg/ml), and ClO(2) (50 microg/ml) and subsequently dried, reductions in TAB, MY, and E. coli O157:H7 were 0.2 to 2.0, 0.4 to 2.0, and 1.4 to 2.2 log CFU/g, respectively. Results indicate that inactivation of E. coli O157:H7 on radish seeds is greater after treatment with ClO(2) followed by drying than after treatment with Ca(OCl)(2) followed by drying, thus providing a synergistic treatment combination for reducing the safety risk associated with sprouts produced from these seeds.

Acetylcholinesterase activity in the brain of wild birds in Korea-2014 to 2016.

Acetylcholinesterase (AChE) activity level can be used as a diagnostic marker for anticholinesterase pesticide poisoning. In this study, we aimed to establish a baseline level of normal brain AChE activity in wild birds. AChE activity was measured in the brains of 87dead wild birds (26 species). The level of AChE activity ranged from 6.40 to 15.9 µmol/min/g of brain tissue in normal wild birds. However, the brain tissue AChE activity level in wild birds exposed to organophosphate (OP) pesticide was 48.0%-96.3% of that in the normal birds. These results may serve as reference values to facilitate routine diagnosis and monitoring of OP-poisoned wild birds.

High-content analysis of in vitro hepatocyte injury induced by various hepatotoxicants.

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.

Fate of Enterobacter sakazakii attached to or in biofilms on stainless steel upon exposure to various temperatures or relative humidities.

Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22 degrees C decreased significantly (P < or = 0.05) at 4, 25, and 37 degrees C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25 degrees C and subsequent survival of cells at 25 degrees C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.

Heat Tolerances of Salmonella, Cronobacter sakazakii, and Pediococcus acidilactici Inoculated into Galactooligosaccharide.

Food-grade galactooligosaccharide (GOS) with low water activity (aw of ca. 0.7) is used as an ingredient in various foods. We evaluated heat tolerances of Salmonella, Cronobacter sakazakii, and Pediococcus acidilactici at temperatures (70 to 85°C) used during the saturation process of GOS by comparing decimal reduction time (D-values) and thermal resistance constants (z-values). To determine the D- and z-values, GOS containing Salmonella (5.1 to 5.8 log CFU/g) or C. sakazakii (5.3 to 5.9 log CFU/g) was heat treated at 70, 77.5, or 85°C for up to 40, 25, or 15 s, respectively, and GOS containing P. acidilactici (6.1 to 6.5 log CFU/g) was heat treated at 70, 77.5, or 85°C for up to 150, 75, or 40 s, respectively. The D-values were calculated using a linear model for heating time versus microbial population for each bacterium. When the D-values for Salmonella, C. sakazakii, and P. acidilactici in GOS were compared, the thermal resistance of all bacteria decreased as the temperature increased. Among the three bacteria, P. acidilactici had higher D-values than did Salmonella and C. sakazakii. The z-values of Salmonella, C. sakazakii, and P. acidilactici were 30.10, 33.18, and 13.04°C, respectively. Overall order of thermal resistance was P. acidilactici > Salmonella ≈ C. sakazakii. These results will be useful for selecting appropriate heat treatment conditions for the decontamination of pathogenic microorganisms during GOS manufacturing.

Sanitizing radish seeds by simultaneous treatments with gaseous chlorine dioxide, high relative humidity, and mild heat.

Sanitizing radish seeds intended for edible sprout production was achieved by applying simultaneous treatments with gaseous chlorine dioxide (ClO2), high relative humidity (RH, 100%), and mild heat (55°C). Gaseous ClO2 was produced from aqueous ClO2 (0.66ml) by mixing sulfuric acid (5% w/v) with sodium chlorite (10 mg/mL) in a sealed container (1.8L). Greater amounts of gaseous ClO2 were measured at 23% RH (144ppm after 6h) than at 100% RH (66ppm after 6h); however, the lethal activity of gaseous ClO2 against naturally occurring mesophilic aerobic bacteria (MAB) on radish seeds was significantly enhanced at 100% RH. For example, when exposed to gaseous ClO2 at 23% RH, the number of MAB on radish seeds decreased from 3.7logCFU/g to 2.6logCFU/g after 6h. However, when exposed to gaseous ClO2 at 100% RH for 6h, the MAB population decreased to 0.7logCFU/g after 6h. Gaseous ClO2 was produced in higher amounts at 55°C than at 25°C, but decreased more rapidly over time at 55°C than at 25°C. The lethal activity of gaseous ClO2 against MAB on radish seeds was greater at 55°C than at 25°C. When radish seeds were treated with gaseous ClO2 (peak concentration: 195ppm) at 100% RH and 55°C, MAB were reduced to populations below the detectable level (<-0.7logCFU/g) within 2h without decreasing the seed germination rate (97.7%). The lethality of combined treatments against artificially inoculated Escherichia coli O157:H7 was also evaluated. When exposed to gaseous ClO2 at 100% RH and 55°C for 6h, the initial number of E. coli O157:H7 (3.5logCFU/g) on radish seeds decreased to below the detection limit (0.7logCFU/g) by direct plating but it was not eliminated from seeds. The germination rate of radish seeds was not significantly (P>0.05) decreased after treatment for 6h. The information reported here will be useful when developing decontamination strategies for producing microbiologically safe radish seed sprouts.

Efficacy of gaseous chlorine dioxide in inactivating Bacillus cereus spores attached to and in a biofilm on stainless steel.

We evaluated the lethal activity of gaseous chlorine dioxide (ClO2) against Bacillus cereus spores attached to and in biofilm formed on a stainless steel surface. Aqueous ClO2 was prepared by mixing sulfuric acid (5% w/v) with sodium chlorite (10mg/mL), and gaseous ClO2 was produced by vaporization of aqueous ClO2 in an air-tight container. The concentration of gaseous ClO2 in the air within the container increased rapidly at first but gradually decreased over time. The lethality of gaseous ClO2 against B. cereus spores attached to stainless steel coupons (SSCs) and in biofilm formed by the pathogen on SSCs was determined. The B. cereus spores attached to SSCs (5.3±0.1logCFU/coupon) were completely inactivated within 1h at 25°C when treated with gaseous ClO2 (peak concentration: 115.3±5.0 parts per million [ppm]). The total number of vegetative cells and spores in biofilm formed by B. cereus on SSCs was 5.9±0.3logCFU/coupon; the spore count was 5.3±0.1logCFU/coupon. The vegetative cells and spores in biofilm were completely inactivated within 6h (peak concentration: 115.3±5.0ppm). Results show that B. cereus spores in biofilms are more resistant to gaseous ClO2 than are attached spores not in biofilms. Gaseous ClO2 was, nevertheless, very effective in killing B. cereus spores in biofilm on the surface of stainless steel. Results show promise for application of gaseous ClO2 to enhance the microbiological safety of foods that may come in contact with stainless steel and possibly other hard surfaces on which B. cereus biofilms have formed.

Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying.

We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P ≤ 0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO2) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood>plastic>glass>stainless steel. The synergistic lethal effects of sequential ClO2 and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO2 (200 µg/ml, 10 min), rinsed with water, and subsequently dried at 43% relative humidity and 22 °C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4 CFU/coupon within 6 h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0 log CFU/coupon, respectively, after 12 h of drying. This indicates that ClO2 treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on food-contact surfaces in food processing, distribution, and preparation environments.

Combined effects of plant extracts in inhibiting the growth of Bacillus cereus in reconstituted infant rice cereal.

A study was done to determine the potential use of plant extracts to inhibit the growth of Bacillus cereus in reconstituted infant rice cereal. A total of 2116 extracts were screened for inhibitory activity against B. cereus using an agar well diffusion assay. The minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) of 14 promising extracts in tryptic soy broth (TSB) were determined. Dryopteris erythrosora (autumn fern) root extract showed the lowest MIC (0.0156 mg/ml), followed by Siegesbeckia glabrescens (Siegesbeckia herb) leaf (0.0313 mg/ml), Morus alba (white mulberry) cortex (0.0313 mg/ml), Carex pumila (sand sedge) root (0.0625 mg/ml), and Citrus paradisi (grapefruit) seed (0.0625 mg/ml) extracts. The order of MLCs of extracts was D. erythrosora root (0.0156 mg/ml)

Inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms.

We investigated the potential use of biofilm formed by a competitive-exclusion (CE) microorganism to inactivate Escherichia coli O157:H7 on a stainless steel surface. Five microorganisms showing inhibitory activities against E. coli O157:H7 were isolated from vegetable seeds and sprouts. The microorganism with the greatest antimicrobial activity was identified as Paenibacillus polymyxa (strain T5). In tryptic soy broth (TSB), strain T5 reached a higher population at 25 °C than at 12 or 37 °C without losing inhibitory activity against E. coli O157:H7. When P. polymyxa (6 log CFU/mL) was co-cultured with E. coli O157:H7 (2, 3, 4, or 5 log CFU/mL) in TSB at 25 °C, the number of E. coli O157:H7 decreased significantly within 24h. P. polymyxa formed a biofilm on stainless steel coupons (SSCs) in TSB at 25 °C within 24h, and cells in biofilms, compared to attached cells without biofilm formation, showed significantly increased resistance to a dry environment (43% relative humidity [RH]). With the exception of an inoculum of 4 log CFU/coupon at 100% RH, upon exposure to biofilm formed by P. polymyxa on SSCs, populations of E. coli O157:H7 (2, 4, or 6 log CFU/coupon) were significantly reduced within 48 h. Most notably, when E. coli O157:H7 at 2 log CFU/coupon was applied to SSCs on which P. polymyxa biofilm had formed, it was inactivated within 1h, regardless of RH. These results will be useful when developing strategies using biofilms produced by competitive exclusion microorganisms to inactivate foodborne pathogens in food processing environments.

Development of a random genomic DNA microarray for the detection and identification of Listeria monocytogenes in milk.

We developed a DNA microarray that contains random genomic DNA fragments of Listeria monocytogenes, validated its diagnostic abilities using cells grown in laboratory media and milk, and established enrichment conditions for detection of a low population of L. monocytogenes in milk. Genomic DNA of L. monocytogenes strain ATCC 19111 was fractionated by agarose gel electrophoresis after being cleaved using several different pairs of restriction enzymes. Sixty DNA fragments of different sizes were randomly selected and spotted onto an amine-coated glass slide. To validate diagnostic ability, probes on the DNA microarray were hybridized with genomic DNA extracted from L. monocytogenes, other Listeria spp., and foodborne pathogenic bacteria belonging to other genera grown in laboratory media. The DNA microarray showed 98-100% positive hybridization signals for the 16 strains of L. monocytogenes tested, 7-85% positive signals for 9 strains of other Listeria spp., and 0-32% positive signals for 13 strains of other types of foodborne pathogens. In milk, the detection limit of the DNA microarray was approximately 8 log CFU/mL. When milk contained L. monocytogenes (3-4 log CFU/mL) with other types of bacteria (Bacillus spp., B. cereus, Salmonella Montevideo, Peudomonas aeruginosa, and Yersinia enterocolitica; ca. 3 log CFU/mL each), L. monocytogenes enriched in UVM modified Listeria enrichment broth at 37°C for 24h was successfully detected by the DNA microarray. Results indicate that the DNA microarray can detect L. monocytogenes and distinguish it from other Listeria spp. and other foodborne pathogens in laboratory media and milk. This platform will be useful when developing a DNA microarray to rapidly and simultaneously detect and identify various foodborne pathogens in foods.

Development of an antimicrobial sachet containing encapsulated allyl isothiocyanate to inactivate Escherichia coli O157:H7 on spinach leaves.

A sachet releasing allyl isothiocyanate (AIT) vapor was developed and its effectiveness as an antimicrobial packaging system for fresh spinach was evaluated. AIT was encapsulated in calcium alginate beads (AIT beads) and the release of AIT as affected by temperature and relative humidity (RH) was determined. The release rate of AIT from beads increased with increased temperature, but was not significantly affected by RH. A low-density polyethylene (LDPE) sachet containing AIT beads (AIT sachet) was prepared and the rate of release of AIT as affected by film thickness and temperature was studied. The release of AIT from sachets increased significantly with decreased LDPE thickness and increased temperature. Finally, antimicrobial effects of the AIT sachet against Escherichia coli O157:H7 and molds and yeasts (MY) on fresh spinach leaves were determined. E. coli O157:H7 and MY had similar sensitivities to AIT vapor. The number of E. coli O157:H7 on spinach leaves (5.6logCFU/leaf) decreased by 1.6-2.6logCFU/leaf at 4°C and 2.1-5.7logCFU/leaf at 25°C within 5days. The reduction of E. coli O157:H7 was significantly greater at 85% RH than at 43% RH. Reductions on control leaves (no AIT sachet) did not exceed 1.2logCFU/leaf under any of the test conditions. Results of the study will be useful when developing antimicrobial packaging systems to increase the microbiological safety and extend the shelf life of spinach and possibly other fresh produces.