Renee Raiden Boyer

Changes in spinach phylloepiphytic bacteria communities following minimal processing and refrigerated storage described using pyrosequencing of 16S rRNA amplicons

Aims:  To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures.

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.

Enhancement of Plant Essential Oils' Aqueous Solubility and Stability Using Alpha and Beta Cyclodextrin

Sodium benzoate has been shown to produce benzene in combination with ascorbic acid. This has led to research for safe alternatives from plant essential oils and parabens that have shown some antimicrobial activity, but many of these compounds exhibit poor solubility in aqueous solutions. Cyclodextrins can increase the solubility of many compounds. This work aimed to investigate the solubility of 23 plant essential oils and 4 parabens in water and an apple juice medium. Four of these compounds were chosen for their low aqueous solubility to determine if complexing the compound with α- and β-cyclodextrin would increase solubility. Three of the complexes were dissolved in an acidified aqueous solution and then studied in glass and polyethylene terephthalate (PET) to determine if storage material would affect the stability. Solubility of the 27 compounds in distilled water ranged from 1.6 mg/L to 2460.6 mg/L and the solubility of 18 of the compounds decreased from 2.5 to 84.7% in apple juice medium (pH = 3.4, 12-13 °Brix). Complexation with cyclodextrin dramatically increased the solubility of the compounds, up to 10-fold. Packaging material had no effect on concentration of compounds present over 7 days. Cyclodextrins were able to increase solubility of these compounds to more suitable concentrations, and may lead to viable natural alternatives to sodium benzoate.

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.

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.

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 cells 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 cells 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.

Effect of α-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.

Use of UV light for the inactivation of Listeria monocytogenes and lactic acid bacteria species in recirculated chill brines.

Ready-to-eat meat products have been implicated in several foodborne listeriosis outbreaks. Microbial contamination of these products can occur after thermal processing when products are chilled in salt brines. The objective of this study was to evaluate UV radiation on the inactivation of Listeria monocytogenes and lactic acid bacteria in a model brine chiller system. Two concentrations of brine (7.9% [wt/wt] or 13.2% [wt/wt]) were inoculated with a approximately 6.0 log CFU/ml cocktail of L. monocytogenes or lactic acid bacteria and passed through a UV treatment system for 60 min. Three replications of each bacteria-and-brine combination were performed and resulted in at least a 4.5-log reduction in microbial numbers in all treated brines after exposure to UV light. Bacterial populations were significantly reduced after 5 min of exposure to UV light in the model brine chiller compared with the control, which received no UV light exposure (P < 0.05). The maximum rate of inactivation for both microorganisms in treated brines occurred between minutes 1 and 15 of UV exposure. Results indicate that in-line treatment of chill brines with UV light reduces the number of L. monocytogenes and lactic acid bacteria.

Survival of Listeria monocytogenes, Listeria innocua, and lactic acid bacteria in chill brines.

Listeria monocytogenes is the pathogen of concern in ready-to-eat (RTE) meat products. Salt brines are used to chill processed meats. L. monocytogenes and lactic acid bacteria (LAB) can grow under saline conditions, and may compete with each other for nutrients. The objective of this study was to determine the effect of lactic acid bacteria (Enterococcus faecalis, Carnobacterium gallinarum, and Lactobacillus plantarum) on the survival of L. monocytogenes and Listeria innocua in brines stored under low temperatures for 10 d. Sterile tap water (STW) and 2 brine solutions (7.9% and 13.2% NaCl) were inoculated with 1 of 5 cocktails (L. monocytogenes, L. innocua, LAB, L. monocytogenes+ LAB, or L. innocua+ LAB) at initial concentrations of 7 log CFU/mL. Brines were stored for 10 d at 4 or 12 degrees C. Three replications of each brine concentration/cocktail/temperature combination were completed. No significant reductions of L. monocytogenes occurred in 7.9%[w/v] or 13.2%[w/v] brines when LAB were present; however, there were significant reductions after 10 d of L. monocytogenes in the STW solution when LAB were present (1.43 log CFU/mL at 4 degrees C and 3.02 log CFU/mL at 12 degrees C). L. innocua was significantly less resilient to environmental stresses of the brines than L. monocytogenes, both with and without LAB present (P< or = 0.05). These strains of lactic acid bacteria are not effective at reducing L. monocytogenes in brines at low temperatures. Furthermore, use of L. innocua as a model for L. monocytogenes is not appropriate under these environmental conditions.

Tracking cross-contamination transfer dynamics at a mock retail deli market using GloGerm.

Ready-to-eat (RTE) deli meats are considered a food at high risk for causing foodborne illness. Deli meats are listed as the highest risk RTE food vehicle for Listeria monocytogenes. Cross-contamination in the retail deli market may contribute to spread of pathogens to deli meats. Understanding potential cross-contamination pathways is essential for reducing the risk of contaminating various products. The objective of this study was to track cross-contamination pathways through a mock retail deli market using an abiotic surrogate, GloGerm, to visually represent how pathogens may spread through the deli environment via direct contact with food surfaces. Six contamination origination sites (slicer blade, meat chub, floor drain, preparation table, employees glove, and employees hands) were evaluated separately. Each site was inoculated with 20 ml of GloGerm, and a series of standard deli operations were completed (approximately 10 min of work). Photographs were then taken under UV illumination to visualize spread of GloGerm throughout the deli. A sensory panel evaluated the levels of contamination on the resulting contaminated surfaces. Five of the six contamination origination sites were associated with transfer of GloGerm to the deli case door handle, slicer blade, meat chub, preparation table, and the employees gloves. Additional locations became contaminated (i.e., deli case shelf, prep table sink, and glove box), but this contamination was not consistent across all trials. Contamination did not spread from the floor drain to any food contact surfaces. The findings of this study reinforce the need for consistent equipment cleaning and food safety practices among deli workers to minimize cross-contamination.