Fate of Listeria monocytogenes on Broccoli and Cauliflower at Different Storage Temperatures.

ABSTRACT: Understanding a food's ability to support the growth and/or survival of a pathogen throughout the supply chain is essential to minimizing large-scale contamination events. The purpose of this study was to examine the behavior (growth and/or survival) of Listeria monocytogenes on broccoli and cauliflower florets stored at different postharvest temperatures utilized along the supply chain. Broccoli and cauliflower samples were inoculated with L. monocytogenes at approximately 3 log CFU/g and stored at 23 ± 2, 12 ± 2, 4 ± 2, and -18 ± 2°C. Samples were evaluated for L. monocytogenes levels after 0, 0.167 (4 h), 1, 2, 3, and 4 days at 23 ± 2°C; 0, 0.167, 1, 2, 3, 4, 7, 10, and 14 days at 12 ± 2°C; 0, 0.167, 1, 2, 3, 4, 7, 10, 14, 21, and 28 days at 4 ± 2°C; and 0, 1, 7, 28, 56, 84, 112, 140, and 168 days at -18 ± 2°C. L. monocytogenes populations were determined by plating samples onto tryptic soy agar and modified Oxford agar supplemented with nalidixic acid. Broccoli and cauliflower supported the growth of L. monocytogenes at 23, 12, and 4°C, and higher growth rates were observed at higher temperatures. Populations of L. monocytogenes on broccoli and cauliflower samples significantly increased within 1 day at 23°C (by 1.6 and 2.0 log CFU/g, respectively) (P ≤ 0.05). At 12°C, populations of L. monocytogenes on broccoli and cauliflower samples significantly increased over 14 days by 1.4 and 1.9 log CFU/g, respectively (P ≤ 0.05). No significant difference over time was observed in L. monocytogenes populations on broccoli and cauliflower samples held under refrigeration until populations began to grow by day 10 in both commodities (P > 0.05). Under frozen storage (-18°C), populations of L. monocytogenes survived on broccoli and cauliflower at least up to 168 days. Storage of broccoli and cauliflower at lower temperatures can minimize L. monocytogenes growth potential; growth rates were lower at 4°C than at 12 and 23°C.

Correlation of Salmonella enterica and Listeria monocytogenes in Irrigation Water to Environmental Factors, Fecal Indicators, and Bacterial Communities.

Outbreaks of foodborne illnesses linked to fresh fruits and vegetables have been key drivers behind a wide breadth of research aiming to fill data gaps in our understanding of the total ecology of agricultural water sources such as ponds and wells and the relationship of this ecology to foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. Both S. enterica and L. monocytogenes can persist in irrigation water and have been linked to produce contamination events. Data describing the abundance of these organisms in specific agricultural water sources are valuable to guide water treatment measures. Here, we profiled the culture independent water microbiota of four farm ponds and wells correlated with microbiological recovery of S. enterica (prevalence: pond, 19.4%; well, 3.3%), L. monocytogenes (pond, 27.1%; well, 4.2%) and fecal indicator testing. Correlation between abiotic factors, including water parameters (temperature, pH, conductivity, dissolved oxygen percentage, oxidation reduction potential, and turbidity) and weather (temperature and rainfall), and foodborne pathogens were also evaluated. Although abiotic factors did not correlate with recovery of S. enterica or L. monocytogenes (p > 0.05), fecal indicators were positively correlated with incidence of S. enterica in well water. Bacterial taxa such as Sphingomonadaceae and Hymenobacter were positively correlated with the prevalence and population of S. enterica, and recovery of L. monocytogenes was positively correlated with the abundance of Rhizobacter and Comamonadaceae (p < 0.03). These data will support evolving mitigation strategies to reduce the risk of produce contamination by foodborne pathogens through irrigation.

Consumer Response to Mechanically Tenderized Beef (MTB) and MTB Labels: An Exploratory Focus Group Study.

In May 2016, labeling of certain nonintact mechanically tenderized beef (MTB) products was mandated in the United States. MTB products should be handled differently by the consumer because pathogens can be transferred from the exterior to the interior of the meat during the tenderization process. Without labeling, it is difficult to visually distinguish between some intact beef and MTB products, which is a concern because MTB products require higher internal cooking temperatures for safety. An exploratory study was conducted to understand consumer understanding of MTB products and consumer responses to the new label. Thirteen focus groups were convened in rural and urban settings across Virginia and North Carolina between December 2015 and May 2016. Sessions were audiorecorded, transcribed verbatim, and analyzed through constant-comparison thematic analysis. Although MTB products were commonly bought, prepared, and consumed, consumer awareness of MTB products and the MTB process was limited. Generally, the label confused participants, and they did not understand the message. Specifically, terminology such as "blade tenderized" and "mechanically tenderized" were preferred over the term "needle tenderized" on labels. Once explained, many individuals wanted more information and better messaging. Through a multiprong approach, other messaging methods (e.g., in stores, through technology, and with certifications) were highly valued by consumers and may result in increased message clarity. Ultimately, the intrinsic and extrinsic properties of the beef rather than the MTB product continued to be the primary guide for purchasing and preparation. This study is the first to be conducted regarding American perceptions of MTB products. An understanding of consumer awareness of MTB products and labels is needed to develop targeted risk messaging communication tools.

Gamma Irradiation Influences the Survival and Regrowth of Antibiotic-Resistant Bacteria and Antibiotic-Resistance Genes on Romaine Lettuce.

Contamination of romaine lettuce with human pathogens, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs) occurs during production. Post-harvest interventions are emplaced to mitigate pathogens, but could also mitigate ARB and ARGs on vegetables. The objective of this research was to determine changes to lettuce phyllosphere microbiota, inoculated ARB, and the resistome (profile of ARGs) following washing with a sanitizer, gamma irradiation, and cold storage. To simulate potential sources of pre-harvest contamination, romaine lettuce leaves were inoculated with compost slurry containing antibiotic-resistant strains of pathogenic (Escherichia coli O157:H7) and representative of spoilage bacteria (Pseudomonas aeruginosa). Various combinations of washing with sodium hypochlorite (50 ppm free chlorine), packaging under modified atmosphere (98% nitrogen), irradiating (1.0 kGy) and storing at 4°C for 1 day versus 14 days were compared. Effects of post-harvest treatments on the resistome were profiled by shotgun metagenomic sequencing. Bacterial 16S rRNA gene amplicon sequencing was performed to determine changes to the phyllosphere microbiota. Survival and regrowth of inoculated ARB were evaluated by enumeration on selective media. Washing lettuce in water containing sanitizer was associated with reduced abundance of ARG classes that confer resistance to glycopeptides, ß-lactams, phenicols, and sulfonamides (Wilcoxon, p < 0.05). Washing followed by irradiation resulted in a different resistome chiefly due to reductions in multidrug, triclosan, polymyxin, ß-lactam, and quinolone ARG classes (Wilcoxon, p < 0.05). Irradiation followed by storage at 4°C for 14 days led to distinct changes to the ß-diversity of the host bacteria of ARGs compared to 1 day after treatment (ANOSIM, R = 0.331; p = 0.003). Storage of washed and irradiated lettuce at 4°C for 14 days increased the relative abundance of Pseudomonadaceae and Carnobacteriaceae (Wilcoxon, p < 0.05), two groups whose presence correlated with detection of 10 ARG classes on the lettuce phyllosphere (p < 0.05). Irradiation resulted in a significant reduction (∼3.5 log CFU/g) of inoculated strains of E. coli O157:H7 and P. aeruginosa (ANOVA, p < 0.05). Results indicate that washing, irradiation and storage of modified atmosphere packaged lettuce at 4°C are effective strategies to reduce antibiotic-resistant E. coli O157:H7 and P. aeruginosa and relative abundance of various ARG classes.

Effect of post-harvest interventions on surficial carrot bacterial community dynamics, pathogen survival, and antibiotic resistance.

Strategies to mitigate antibiotic-resistant bacteria (ARB), including human pathogens, on raw vegetables are needed to reduce incidence of resistant infections. The objective of this research was to determine the effectiveness of standard post-harvest interventions, sanitizer washing and cold storage, to reduce ARB, including antibiotic resistant strains of the human pathogen E. coli O15:H7 and a common spoilage bacterium Pseudomonas, on raw carrots. To provide a background inoculum representing potential pre-harvest carryover of ARB, carrots were dip-inoculated in dairy cow manure compost slurry and further inoculated with known ARB. Inoculated carrots were washed with one of three treatments: sodium hypochlorite (50 ppm free chlorine), peroxyacetic acid (40 ppm peroxyacetic acid; 11.2% hydrogen peroxide), tap water (no sanitizer), or no washing (control). Washed carrots were air dried, packaged and stored at 10 °C for 7d or 2 °C for up to 60 d. Enumeration was performed using total heterotrophic plate counts (HPC), HPCs on antibiotic-containing media ("ARBs"), E. coli O157:H7, and Pseudomonas sp. immediately after washing (0 d) and after 7 d of storage. In addition to the cultured bacteria, changes to the surficial carrot microbiota were profiled by sequencing bacterial 16S rRNA gene amplicons to determine the effect of sanitizer wash, storage temperature, and time of storage (0, 1, 7, 14 and 60 d). Storage temperature, addition of a sanitizer during wash, and duration of storage significantly affected the bacterial microbiota (Wilcoxon, p < 0.05). Inclusion of either sanitizer in the wash water significantly reduced the log CFU/g of E. coli O157:H7 and Pseudomonas sp., as well as HPCs enumerated on cefotaxime- (10 µg/ml), sulfamethoxazole- (100 µg/ml), or tetracycline (3 µg/ml) supplemented media compared to the unwashed control (ANOVA, p < 0.05). However, no significant reductions to bacteria resistant to vancomycin or clindamycin occurred after washing and storage. Members of the Proteobactetria, Firmicutes, Actinobacteria, Planctomycetes, and Acidobacteria comprised the bacterial carrot microbiota. The diversity of the carrot microbiota was significantly affected by the temperature of storage and by extended storage (60 d), when spoilage began to occur. There were no significant differences to the relative abundance of bacterial groups associated with the type of sanitizer used for washing. Results of this study indicate that inclusion of a sanitizer in wash water, followed by storage at 2 °C, might be an effective strategy to prevent re-growth of pathogenic E. coli O157:H7 and reduce levels of bacteria resistant to certain antibiotics on carrots.

Cetylpyridinium chloride direct spray treatments reduce Salmonella on cantaloupe rough surfaces.

Cetylpyridinium chloride (CPC) solutions (0, 0.5, or 1.0%) were applied to cantaloupe ("Athena" and "Hale's Best Jumbo" cultivars) rind plugs, either before or after inoculation with a broth culture of Salmonella Michigan (109 CFU/mL) and held at 37°C for 1 or 24 hr. Rind plugs were diluted, shaken, and sonicated, and solutions were enumerated. Texture quality and color were evaluated over 14 days storage at 4°C after 0 and 1% CPC spray applications. A 0.5 or 1.0% (vol/vol) application of CPC after Salmonella reduced the pathogen levels between 2.34 log CFU/mL and 5.16 log CFU/mL in comparison to the control (p < .01). No differences were observed in the firmness and color of 1% CPC treated cantaloupes. Salmonella concentrations on cantaloupes, treated with 1.0% CPC, were lower after 1 hr storage as compared to 24 hr. And, Salmonella on "Athena" surfaces were more susceptible to CPC spray treatments than on "Hale's Best Jumbo." PRACTICAL APPLICATIONS: Cetylpyridinium chloride (CPC) is the active ingredient of some antiseptic oral mouth rinses, and has a broad antimicrobial spectrum with a rapid bactericidal effect on gram-positive pathogens. The spray application of CPC solutions to cantaloupe may reduce the level of Salmonella surface contamination during production from irrigation water and manure fertilizers and, during food processing by contaminated equipment and food handlers. Since the surfaces of cantaloupes are highly rough or irregular, bacteria can easily attach to these surfaces and become difficult to remove. Appropriate postharvest washing and sanitizing procedures are needed that can help control Salmonella and other pathogens on melons, especially on cantaloupes with nested surfaces. A direct surface spray application of CPC may be an alternative antimicrobial postharvest treatment to reduce pathogen contamination of cantaloupe melons, while providing an alternative to chlorine-based solutions.

Survival of Tomato Outbreak Associated Salmonella Serotypes in Soil and Water and the Role of Biofilms in Abiotic Surface Attachment.

Salmonella serotypes linked to tomato-associated outbreaks were evaluated for survival in soil and water over a 40-day period. Salmonella enterica serotypes Anatum, Baildon, Braenderup, Montevideo, Newport, and Javiana were inoculated separately into sterile soil and water, followed by plating onto TSAYE and XLT4 at 10-day intervals. Biofilm production by Salmonella serotypes was measured on both quartz particles (soil surrogate) and glass coverslips, and was evaluated using a crystal violet dye assay. Salmonella populations in soil and water over 40 days indicated no significant differences between Salmonella serotypes tested (p > 0.05). Over a 40-day period, there was a 1.84 ± 0.22 log CFU/g and 1.56 ± 0.54 CFU/mL decrease in populations of Salmonella in soil and water, respectively. Enumeration indicated that Salmonella population fluctuated in water but decreased linearly in soil. All serotypes tested produced the "red dry and rough" morphotype on Congo Red agar. Biofilm produced by all the Salmonella serotypes tested was significantly different on quartz particles than on glass coverslips (p < 0.0001), indicating that material and surface characteristics could affect biofilm development. The ability of Salmonella serotypes to persist in soil or water and attach to abiotic surfaces through biofilm formation affirms that contact surfaces, soil, water, and sediment should be considered as possible sources of cross-contamination in the farm environment.

Incidence of Listeria spp. in Ready-to-Eat Food Processing Plant Environments Regulated by the U.S. Food Safety and Inspection Service and the U.S. Food and Drug Administration.

A multiyear survey of 31 ready-to-eat (RTE) food processing plants in the United States was conducted to determine the incidence of Listeria spp. in various RTE production environments. Samples were collected from 22 RTE plants regulated by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS) and from 9 RTE food plants regulated by the U.S. Department of Health and Human Services' Food and Drug Administration (FDA). Only nonfood contact surfaces in the RTE manufacturing areas with exposed RTE product were sampled. Each sample was individually analyzed for the presence of Listeria spp. by using a PCR-based rapid assay. In total, 4,829 samples were collected from various locations, including freezers, equipment framework, floors, walls, wall-floor junctures, drains, floor mats, doors, and cleaning tools. Nine (29%) of the facilities had zero samples positive for Listeria spp. in the production environment, whereas 22 (71%) had one or more samples positive for Listeria spp. The total incidence of Listeria spp. in all RTE food plants was 4.5%. The positive rate in plants regulated by the FSIS ranged from 0 to 9.7%, whereas the positive rate in plants regulated by the FDA ranged from 1.2 to 36%.

Delmopinol hydrochloride reduces Salmonella on cantaloupe surfaces.

Since the surfaces of cantaloupes are highly rough or irregular, bacteria can easily attach and become difficult to remove. Appropriate postharvest washing and sanitizing procedures can help control Salmonella and other pathogens on cantaloupe or other melons during postharvest operations. Delmopinol hydrochloride (delmopinol) is a cationic surfactant that is effective for treating and preventing gingivitis and periodontitis. The application of delmopinol to two cantaloupe cultivars was evaluated for reducing the level of inoculated Salmonella. Athena and Hale's Best Jumbo (HBJ) cantaloupe rind plugs (2.5 cm. dia.) were inoculated with nalidixic acid-resistant Salmonella Michigan (approx. 1.0 × 109 CFU/ml). After 15 min, rind plugs were sprayed with 10 ml of a delmopinol spray solution (0% or 1.0% vol/vol) and held at 35°C for 1 hr or 24 hr. Rind plugs were diluted with Butterfield's phosphate buffer, shaken and sonicated, and solutions were enumerated on 50 ppm nalidixic acid-tryptic soy agar. The texture quality and color of additional cantaloupes were evaluated, after 1% delmopinol spray treatment, over 14-day storage at 4°C. A 1.0% application of delmopinol after 1 hr reduced Salmonella concentration by ~3.1 log CFU/ml for both "HBJ" skin rind plugs and "Athena" stem scar rind plugs in comparison to the control (p < .05). No differences were observed in the texture and color (L*, a*, b* values) of 1% delmopinol-treated cantaloupes as compared to control. Storage of cantaloupes treated with 1.0% delmopinol solution for 1 hr had a greater effect on reducing concentration of Salmonella compared to 24-hr treatment. A surface spray application of 1% delmopinol on cantaloupes could be an alternative antimicrobial postharvest treatment that could make surface bacteria more susceptible to sanitizers or physical removal.

Evaluation of peanut skin and grape seed extracts to inhibit growth of foodborne pathogens.

Peanut skin extract (PSE) and grape seed extract (GSE) are derived from waste products in the wine and peanut industries, respectively. Both have high concentrations of polyphenols, known to possess antioxidant and antimicrobial properties. PSE primarily contains "A-type" procyanidins, while GSE primarily contains "B-type" procyanidins. These differ structurally, but are both isomers of epicatechin dimers. The objective of this study was to evaluate the antimicrobial effects of PSE containing A-type procyanidins and GSE containing B-type procyanidins against select foodborne pathogens (Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium). The minimum inhibitory concentration (MIC) of the two extracts on L. monocytogenes, E. coli O157:H7, and S. Typhimurium was determined using the pour plate method. GSE had a significantly lower MIC (p ≤ .05) than PSE for L. monocytogenes (GSE = 60.6 ppm, PSE > 68.2 ppm) and S. Typhimurium (GSE = 45.7 ppm, PSE = 60.6 ppm), but no difference in inhibition of E. coli O157:H7. Since GSE contributed to greater inhibition, GSE extract was fractionated into monomer-rich (consisting primarily of catechins, epicatechins, and epicatechin gallates) and oligomer-rich (consisting of dimers, trimers, tetramers, up to decamers) components. Growth curves of all three pathogens in the presence of full extract, monomer and oligomer fractions were compared separately. None of the extracts inhibited S. Typhimurium growth. Generally, the extract containing greater oligomer components inhibited growth of L. monocytogenes and E. coli O157:H7 when compared to the control. Results indicate that an extract with type B procyanidins higher in oligomers may have greater antimicrobial properties.

Airborne soil particulates as vehicles for Salmonella contamination of tomatoes.

The presence of dust is ubiquitous in the produce growing environment and its deposition on edible crops could occur. The potential of wind-distributed soil particulate to serve as a vehicle for S. Newport transfer to tomato blossoms and consequently, to fruits, was explored. Blossoms were challenged with previously autoclaved soil containing S. Newport (9.39log CFU/g) by brushing and airborne transfer. One hundred percent of blossoms brushed with S. Newport-contaminated soil tested positive for presence of the pathogen one week after contact (P<0.0001). Compressed air was used to simulate wind currents and direct soil particulates towards blossoms. Airborne soil particulates resulted in contamination of 29% of the blossoms with S. Newport one week after contact. Biophotonic imaging of blossoms post-contact with bioluminescent S. Newport-contaminated airborne soil particulates revealed transfer of the pathogen on petal, stamen and pedicel structures. Both fruits and calyxes that developed from blossoms contaminated with airborne soil particulates were positive for presence of S. Newport in both fruit (66.6%) and calyx (77.7%). Presence of S. Newport in surface-sterilized fruit and calyx tissue tested indicated internalization of the pathogen. These results show that airborne soil particulates could serve as a vehicle for Salmonella. Hence, Salmonella contaminated dust and soil particulate dispersion could contribute to pathogen contamination of fruit, indicating an omnipresent yet relatively unexplored contamination route.

Survey of food safety practices on small to medium-sized farms and in farmers markets.

As produce consumption has increased, so have foodborne disease outbreaks associated with fresh produce. Little research has addressed food safety practices used on small to medium-sized farms selling locally or in farmers markets. This study evaluated current food safety practices used by farmers on small to medium-sized farms and managers of farmers markets in Georgia, Virginia, and South Carolina based on responses to surveys. Surveys were developed, pretested, and revised before implementation with target audiences and were implemented via mail and the Web to maximize participation, with reminders sent to nonrespondents. Data were collected from 226 farmers and 45 market managers. Frequencies and percentages were calculated for all response variables. Responses from farmers indicated that more than 56% of them use manures. Of those who use manures, 34% use raw or mixtures of raw and composted manure, and over 26% wait fewer than 90 days between application of raw manure and harvest. Over 27% use water sources that have not been tested for safety for irrigation, and 16% use such water sources for washing produce. Over 43% do not sanitize surfaces that touch produce at the farm. Only 33% of farmers always clean transport containers between uses. Responses from market managers indicated that over 42% have no food safety standards in place for the market. Only 2 to 11% ask farmers specific questions about conditions on the farm that could affect product safety. Less than 25% of managers sanitize market surfaces. Only 11% always clean market containers between uses. Over 75% of markets offer no sanitation training to workers or vendors. While farmers and market managers are using many good practices, the results indicate that some practices being used may put consumers at risk of foodborne illness. Consequently, there is a need for training for both farmers and market managers.

Application of proanthocyanidins from peanut skins as a natural yeast inhibitory agent.

UNLABELLED: Proanthocyanidins were extracted from peanut skins and investigated for their antimicrobial activity against Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus in traditional growth media (Sabouraud Dextrose and Maltose broth) and a simulated apple juice beverage. Peanut skins extracts (PSE) were prepared through a multisolvent extraction procedure. The PSE extended the lag phase growth of the 3 yeasts studied at a concentration of 1 mg/mL and at 10 mg/mL yeast growth was totally inhibited for 120 h. PSE was fractionated by normal phase high performance liquid chromatography and the active components/fractions were determined. Compounds present in the fractions were identified by liquid chromatography-mass spectrometry to determine the compounds responsible for inhibition. Fractions consisting mostly of A-type proanthocyanidin dimers, trimers, and tetramers showed the highest percent inhibition toward the yeasts tested in this study. Both optical density (OD) and standard enumeration plating methods were performed in this study. The OD method led to an overestimation of the inhibitory effects of PSE, the 2 methods agreed in respect to treatment effects but not the severity of the inhibition. PRACTICAL APPLICATION: There is a growing consumer demand for "fresh like" products containing reduced amounts of chemical preservatives without compromising food safety and quality. Therefore, the goal of this study was to determine if an extract of peanut skins containing flavonoid rich compounds could function as a natural antimicrobial in a model beverage system. Proteins were removed through the process of producing the peanut skin extract, thus it is unlikely to contain peanut allergens. The antimicrobial compounds mentioned in this study were successfully integrated into a model beverage system, and were found to have antimicrobial effect. However, the incorporation of these compounds would likely lead to negative sensory attributes at the concentration needed to achieve an appreciable antimicrobial effect alone.

Characterization of interactions between Escherichia coli O157:H7 with epiphytic bacteria in vitro and on spinach leaf surfaces.

This study characterized the types of interactions between Escherichia coli O157:H7 and spinach phylloepiphytic bacteria and identified those that influence persistence of E. coli O157:H7 on edible plants. A total of 1512 phylloepiphytic bacterial isolates were screened for their ability to inhibit or to enhance the growth of E. coli O157:H7 in vitro and on spinach leaf surfaces. Fifteen different genera, the majority belonging to Firmicutes and Enterobacteriaceae, reduced growth rates of E. coli O157:H7 in vitro by either nutrient competition or acid production. Reduced numbers of E. coli O157:H7 were recovered from detached spinach leaves that were co-inoculated with epiphytic isolates belonging to five genera. A 1.8 log reduction in E. coli O157:H7 was achieved when co-inoculated with Erwinina perscinia and 20% cellobiose, a carbon source used by the phylloepiphytes but not E. coli O157:H7. The reduction on leaves was significantly less than reduction measured in vitro. Phylloepiphytic bacteria belonging to eight different genera, increased numbers of E. coli O157:H7 when co-cultured in vitro on spent medium and when co-cultured on detached spinach leaves. The results, showing reduction of E. coli O157:H7 numbers by natural epiphytic bacteria, support the hypothesis that native plant microbiota can be used for bio-control of foodborne pathogens, however, other epiphytes may promote the persistence of enteric pathogens on the phyllosphere.

High hydrostatic pressure processing reduces Salmonella enterica serovars in diced and whole tomatoes.

Fresh and fresh-cut tomatoes have been associated with numerous outbreaks of salmonellosis in recent years. One effective post harvest treatment to reduce Salmonella enterica in tomatoes may be high pressure processing (HPP). The objectives of the study were to determine the potential for HPP to reduce S. enterica serovars Newport, Javiana, Braenderup and Anatum in tryptic soy broth (TSB) and to determine the effect of HPP to reduce the most pressure resistant of the four serovars from fresh diced and whole tomatoes. To evaluate pressure resistance, TSB containing 8 log CFU/ml of one of the four serovars was packaged in sterile stomacher bags and subjected to one of three different pressures (350, 450 or 550MPa) for 120s. The most pressure resistant S. enterica serovar evaluated was Braenderup. Subjecting the broth culture to 350, 450 and 550MPa resulted in a 4.53, 5.74 and 7.09 log reduction in S. Braenderup, respectively. Diced tomatoes (150g) and whole red round tomatoes (approximately 150g) were inoculated with 0.1ml of 9.1 log CFU/ml S. Braenderup, and subjected to the same pressure treatments (350, 450 or 550MPa). Significant reductions of S. Braenderup concentrations in diced tomatoes (P<0.05) were seen after processing at 350 (0.46CFU/g), 450 (1.44 log CFU/g), and 550MPa (3.67 log CFU/g). In whole tomatoes, significant reductions (P<0.05) were also seen at 350 (1.41 log CFU/g), 450 (2.25 log CFU/g) and 550MPa (3.35 log CFU/g). HPP may be an effective post harvest strategy to reduce low levels of S. enterica contamination in whole and diced tomatoes.

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.

Alterations of the phylloepiphytic bacterial community associated with interactions of Escherichia coli O157:H7 during storage of packaged spinach at refrigeration temperatures.

This study investigated the effects of packaging and storage temperature on the spinach phylloepiphytic bacterial community and fate of Escherichia coli O157:H7. Freshly harvested spinach was rinsed and/or disinfected, packaged and stored under typical retail conditions (4 degrees C) or under temperature abuse conditions (10 degrees C) for a period of 15 days. The final population size of culturable epiphytic bacteria after 15 days of storage was not affected by the temperature of storage or the presence of E. coli O157:H7. However, analysis of the bacterial community using denaturing gradient gel electrophoresis of 16s rDNA revealed changes with time of storage and the presence of E. coli O157:H7. Excision and sequencing of prominent DGGE bands identified that the majority of sequences belonged to the phyla Actinobacteria, Bacteroidetes, Firmicutes and Alphaprotebacteria. After 10 days of storage at 4 degrees C or 10 degrees C the population became more dominated by psychrotrophic bacteria. Removal of the epiphytic bacteria resulted in significant increases in numbers of E coli O157:H7 at 10 degrees C and was associated with decreased expression of E. coli O157:H7 virulence (stxA, curli, eaeA) and stress response (rpoS, sodB) genes. In conclusion, storage temperature and time of storage of packaged spinach affected the diversity of the epiphytic spinach microbiota which influenced the growth, establishment, physiology and potentially virulence of E. coli O157:H7.

Effect of alpha-cyclodextrin-cinnamic acid inclusion complexes on populations of Escherichia coli O157:H7 and Salmonella enterica in fruit juices.

Cinnamic acid (CA), a naturally occurring organic acid found in fruits and spices, has antimicrobial activity against spoilage and pathogenic bacteria, but low aqueous solubility limits its use. The purpose of this study was to determine the effectiveness of solubility-enhancing alpha-cyclodextrin-CA inclusion complexes against Escherichia coli O157:H7 and Salmonella enterica serovars suspended in apple cider or orange juice at two different incubation temperatures (4 and 26 degrees Celsius). Two concentrations (400 and 1,000 mg/liter) of alpha-cyclodextrin-CA inclusion complex were aseptically added to apple cider inoculated with E. coli O157:H7 (7 log CFU/ml) and orange juice inoculated with a cocktail of six Salmonella enterica serovars (7 log CFU/ml). Samples were extracted at 0 min, at 2 min, and at 24-h intervals for 7 days, serially diluted in 0.1 % peptone, spread plated in duplicate onto tryptic soy agar, and incubated at 35 degrees Celsius for 24 h. Populations of E. coli O157:H7 in apple cider were significantly reduced (P < or = 0.05) during the 7-day sampling period in all solutions regardless of temperature. Compared with the controls, populations were significantly reduced by the addition of 400 and 1,000 mg/liter inclusion complex, but reductions were not significantly different (P > or = 0.05) between the two treatment groups (400 and 1,000 mg/liter). Salmonella was significantly reduced in all solutions regardless of temperature. There were significant differences between the control and each inclusion complex concentration at 4 and 26 degrees Celsius. Coupled with additional processing steps, alpha-cyclodextrin-CA inclusion complexes may provide an alternative to traditional heat processes.

Internalization of Salmonella enterica serovar Montevideo into greenhouse tomato plants through contaminated irrigation water or seed stock.

Tomatoes have been linked to outbreaks of salmonellosis, demonstrating the need to identify sources of contamination. Objectives of this study included determining the ability for Salmonella enterica serovar Montevideo to be internalized into tomatoes from contaminated irrigation water and seed stock, and establishing whether Salmonella Montevideo can survive in fertilizer solutions. Six treatment groups (five plants per group) were irrigated with 350 ml of 7 log CFU/ml of Salmonella Montevideo every 14 days for 70 days, each group receiving an increased number of contaminated water events progressively: group 1 received one contaminated watering at day 0, and group 6 received a total of six contaminated waterings. Group 7 was a control, and group 8 was grown from seeds soaked in 8 log CFU/ml of Salmonella Montevideo for 24 h. All plants were watered daily with uncontaminated water. Three replications were completed. Fruit from every plant, and roots, stems, and leaves of one plant per treatment were sampled. All tomatoes were negative for Salmonella Montevideo; five root samples tested positive. For fertilizer studies, a commercially available fertilizer, two custom mixed and 1.0% dilutions of each (total of six solutions), and sterile water were inoculated with 8 log CFU/ml of Salmonella Montevideo and stored at 25 degrees C. Solutions were sampled at 24, 48, and 72 h. There were no differences (P > or = 0.05) between survival of Salmonella Montevideo in diluted fertilizers and the control. Results indicate Salmonella Montevideo is unable to contaminate tomato fruit via irrigation water and seed stock but can survive in fertilizer solutions.

Influence of curli expression by Escherichia coli 0157:H7 on the cell's overall hydrophobicity, charge, and ability to attach to lettuce.

Curli fibers are produced by some Escherichia coli cells in response to environmental stimuli. These extracellular proteins enhance the cell's ability to form biofilms on various abiotic surfaces. E. coli 0157:H7 cells readily attach to a variety of fruit and vegetable surfaces. It is not known whether the expression of curli influences the cell's ability to attach to produce surfaces. In this study, the effect of curli expression on the cell's overall hydrophobicity, charge, and ability to attach to cut and whole iceberg lettuce surfaces was examined. All strains, regardless of curli expression, attached preferentially to the cut edges of lettuce (P < 0.05). The curli-producing cells of E. coli 0157:H7 strain E0018 attached in significantly greater numbers to both cut and whole lettuce pieces than did the non-curli-producing E0018 cells (P < 0.05); however, no significant attachment differences were observed between the curli-producing and non-curli-producing cells of E. coli 0157:H7 strains 43894 and 43895. All curli-producing E. coli 0157:H7 strains were significantly more hydrophobic (P < 0.01); however, no association between the cells' hydrophobic characteristics and lettuce attachment was observed. Overall surface charge of the cells did not differ among strains or curli phenotypes. Results indicate that overall hydrophobicity and cell charge in E. coli 0157:H7 strains do not influence attachment to iceberg lettuce surfaces. The presence of curli may not have any influence on attachment of E. coli 0157:H7 cells to produce items. Additional factorsmay influence the attachment of E. coli 0157:H7 to plant surfaces and should be further examined.