For years the issue of food waste has been recognized and quantified; however, food safety issues often go unrecognized as a source of food waste. One objective of this research is to estimate quantities and monetary value of fruits and vegetables implicated in food safety recalls, and thus wasted. Using publicly available data we identified all recalls involving vegetable or fruit commodities contaminated with Listeria monocytogenes, pathogenic E. coli, or Salmonella during 2015-2018. When quantities were provided, monetary value of recalled product was calculated using USDA ERS 2016 average retail prices. Although data limitations only allowed analysis of 17% of the recalls that met the criteria of this study, we estimated an annual loss of 38 million pounds and $61 million in revenue. Overall this shows that food safety issues can result in food waste, therefore mitigation strategies are needed. There are many ways that produce can become contaminated, however contaminated soils are a potential source of produce contamination and treatments to mitigate this risk while maintaining soil health is lacking. Current biofumigation methods that use glucosinolate hydrolysis products in mustard seed meal to control plant pathogens could also be effective against foodborne pathogens in soil. The purpose of this research is to determine the fate of E. coli O157:H7, Salmonella, and L. monocytogenes in soil treated with Brassica spp seed meal and plant material. Seed meals were successful in reducing pathogen concentrations in soil, significant reductions (p < 0.05) of E. coli O157:H7, L. monocytogenes, and Salmonella were observed in soil over 72 hours with the addition of 1.0 and 1.5 g of mustard seed meal. Increasing the seed meal concentration did not significantly (p > 0.05) increase the observed log reduction for L. monocytogenes or Salmonella, reductions ranged from 5.6 – 5.9 log CFU/g. However, for E. coli O157:H7 seed meal concentration was significant (p < 0.05). A 5.7 log CFU/g decrease was observed when 1.5 g of seed meal was used which was larger than 3.5 log CFU/g reduction observed with 1.0 g. Findings suggest that biofumigation with mustard seed meal could potentially be used to reduce E. coli O157:H7, L. monocytogenes, and Salmonella in contaminated soil. However, the use of plant material was not as successful as the use of the processed seed meals. In soil or in the absence of soil Brassica spp. plant material at 10% 15%, and 75% significantly increased E. coli O157:H7, L. monocytogenes, and Salmonella concentrations (p < 0.05). The results of these studies support literature indicating Brassica spp. processed plant products, like seed meals or extracts may be a more effective strategy in reducing human pathogen concentrations in contaminated agricultural soils. While the process of Biofumigation using Brassica spp. cover crops has been successful in eliminating plant pests from agricultural soils, due to its low isothiocyanate release efficiency and reactivity in soil organic matter, it may not be sufficient as a soil decontamination method against human pathogens.

This study focused on evaluating the antimicrobial activity of chlorine dioxide (ClO2) and different plant-based antimicrobials (carvacrol, thymol, and eugenol as bioactive compounds and muscadine extract- ME and blueberry extract- BBE as plant extracts) against selected foodborne pathogens under in vitro conditions and on produce (spinach and blueberries). In addition, bacterial microbiota associated with blueberries and blueberry farm environments from three different regions: Cundinamarca, Colombia; Mississippi, United States; and Guadalajara, Mexico, was determined. Under in vitro conditions, carvacrol and thymol were more effective (lower MICs and MBCs) than eugenol against Salmonella spp., Escherichia coli O157:H7 (MIC=MBC=0.2 mg/ml), and Listeria monocytogenes (MIC=MBC=0.4 mg/ml). Both plant extracts had the same MIC and MBC for Salmonella spp. while BBE had stronger bactericidal effect on Escherichia coli O157:H7 (MBC=150 mg/ml) and ME on L. monocytogenes (MBC=100 mg/ml). ClO2 had stronger bacteriostatic effect on L. monocytogenes (MIC=1 ppm) than on the Gram-negative bacteria (MIC=3 ppm). For the produce study, 300 mg/ml ME exerted the highest (P ≤ 0.05) E. coli O157:H7 reduction (4.5 log CFU/g at day 1) on spinach, and 3 ppm ClO2 + 300 mg/ml ME had the highest (P ≤ 0.05) L. monocytogenes reduction on both (4.5-5.6 log CFU/g). There was a similar (P>0.05) E. coli O157:H7 reduction on blueberries, regardless of antimicrobial treatment. For the microbiota study, Proteobacteria was the most abundant phylum in blueberries, soil, and water, with the exception of fruits from Mexico. Blueberries grown on the different regions shared two predominant genera: Heliorestis (10.5-47.4%) and Thiomonas (5.0-9.1%). Nonetheless, alpha and beta diversity revealed that blueberry microbiota structures were distinctive. PCoA plots revealed that within regions the microbial composition distribution was different (P ≤0.05) among fruits, soil, and water. Based on the results, ME combined with ClO2, could represent an antimicrobial alternative against foodborne pathogens for the produce industry. Furthermore, the study of the microbiota provided a good understanding on the bacterial community profile in blueberries and the blueberry farming environment across regions.

A bifidobacteria mix (nine strains) was evaluated for its effect on the growth and survival of Escherichia coli O157:H7 (ATCC 43890) in 11% NFDM and MRS broth under the optimum growth conditions for E. coli O157:H7 growth (37¡ãC, aerobic). Preliminary experiments were conducted to obtain the growth curves of the 9 strains of bifidobacteria and E. coli O157:H7 and confirm the inhibitory effect of acidity on E. coli O157:H7 (pH of media adjusted to 3.8). Acidapted E. coli O157:H7 showed no difference in resistance toward bifidobacteria (P>0.05) when compared to the non-acid adapted one. Escherichia coli O157:H7 did not survive in the supernatant of the bifodbacteria mix collected after incubation (37¡ãC) with aerobic shaking (8 h). However, the pathogen was able to grow after the pH of the supernatant was adjusted to 6.50 (pH of fresh MRS broth). Results suggest that a high content of bifidobacteria has a strong inhibitory effect on E. coli O157:H7, in part due to the low pH. However, products from bifidobacteria may also exert inhibitory effects.

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<p>Though pathogenic strains represent a small portion of the total variety of existing <em>Escherichia coli </em>strains, they contribute extensively to human morbidity and mortality. Disease outbreaks caused by enterohaemorrhagic <em>E. coli</em> of the serotype O157:H7 and the “Big Six” serotypes (i.e., O26, O45, O103, O111, O121 and O145) have driven the development of assays for pathogen detection. From culture-based assays requiring several days for confirmation of target organisms, to quantitative PCR (qPCR) tests that provide pathogen identification in several hours’ time, the sensitivity, specificity and speed of bacterial diagnostics have seen improvements that increased the efficacy of assays used to detect pathogens at clinically relevant levels. One relatively unexplored field of diagnostics is the use of conserved signature insertion/deletions (CSIs) as stable genetic markers for pathogen detection. This thesis presents two qPCR assays that target an <em>E. coli</em> O157:H7-specific insertion in a CSI. In a more preliminary study, an EvaGreen-based qPCR assay was developed that had a detection limit of 16 <em>E. coli</em> O157:H7 genome equivalents. An improved format of the O157:H7-specific CSI assay, using TaqMan probes, was later established. TaqMan probes are sequence-specific, while DNA-intercalating EvaGreen dye is sequence-independent. Though the TaqMan probe-based assay had a higher detection limit of 100 genome equivalents, the assay maintained detection sensitivity in presence of genetically similar (<em>E. coli</em> K-12) and dissimilar (fish sperm) DNA in excess amounts (1000-fold and 800-fold excess of target DNA, respectively), demonstrating its potential for pathogen detection in environmental samples where the presence of background flora may influence detection. These assays thus represent an exploration into the use of CSIs as diagnostic tools. This thesis also provides a guide for future developments of pathogen detection using CSIs, such as those that may be present in toxigenic species of Cyanobacteria and human pathogens, including <em>Vibrio</em> and <em>Campylobacter</em>.</p> / Master of Science (MSc)

Fresh produce is increasingly associated with foodborne outbreaks. In order to develop effective intervention and measures to reduce microbial risks, it is essential to attain a better understand the mechanisms of attachment of foodborne pathogens to fruits and vegetables. Using lettuce as a model, the attachment of Escherichia coli O157:H7 to produce surfaces was studied. Strains expressing various extracellular proteins (curli, O157-antigen, and intimin) known to influence attachment of E. coli to intestinal cells were evaluated for their physicochemical properties and ability to adhere to cut edge and whole leaf lettuce. Escherichia coli O157:H7 strains included: 0018, 43894 and 43895 (curli producing and non-producing); 86-24 (WT), F-12 (O157-antigen negative), pRFBE (O-antigen replaced on plasmid); and 86-24, 86-24Ã eae10 (intimin negative). The eleven strains were surveyed for their hydrophobicity and cell charge using hydrophobic interaction chromatography (HIC) and electrostatic interaction chromatography (ESIC) techniques. Iceberg lettuce squares (2 x 2 cm) were inoculated with E. coli O157:H7 strains separately (7.0 log CFU/square) and dried in a laminar flow hood. Lettuce was sampled before (unrinsed) and after being rinsed twice with sterile de-ionized water (rinsed). Strips (2 mm wide) of each cut edge of the lettuce were aseptically removed. Cut-edge and whole-leaf samples were homogenized and spiral plated onto Luria-Bertani agar, supplemented with nalidixic acid (50ppm), to assess levels of bacteria remaining on the lettuce leaf after rinsing. The rinse steps were not effective in significantly removing bacteria from lettuce (p>0.05). Curli-producing and non-producing strains preferentially attached to cut edge versus the whole leaf portions of lettuce (p<0.05); however the 86-24 strains showed no preference for attachment. With the exception of 0018 curli-producing and non-producing strains, presence/absence of extracellular proteins surveyed did not influence attachment of E. coli O157:H7 to either cut edge or whole leaf lettuce. There was significantly greater attachment of the curli-producing 0018 strain over the curli non-producing 0018 strain to cut and whole lettuce surfaces (p<0.05). Production of curli and O-polysaccharide significantly increased (p<0.05) the cell's overall hydrophobicity of the cell; however this did not affect attachment (p<0.05). The overall cell charge of all strains was negative; however, charge did not affect attachment of E. coli O157:H7 to lettuce. The presence of extracellular appendages (curli, O157-antigen, intimin) as well as hydrophobicity and cell charge properties had no affect on attachment of the cell to lettuce. / Ph. D.

A Quantitative Microbial Risk Assessment (QMRA) has been undertaken to utilize research on Shiga-toxin Escherichia coli (STEC) contamination in beef for the benefit of public health. The QMRA operates as a 2nd order Monte Carlo simulation to create stochastic mathematical models that incorporate all of the key components of STEC contamination from farm to fork. The resulting model is able to identify knowledge gaps, public health risks, and simulate theoretical changes in the beef system. However, high variability in processing plant intervention literature has prompted a meta-analysis to determine informed estimates of intervention effectiveness for QMRA parameterization. Meta-analysis derived least-squares means bacterial log reductions for acetic acid, lactic acid, steam vacuum, and water wash interventions on carcass surfaces (n=249) were 1.44 [95% CI: 0.73 – 2.15], 2.07 [1.48 – 2.65], 3.09 [2.46 – 3.73], and 1.90 [1.33 – 2.47] log CFU/cm2, respectively. Least-squares means log reductions for acetic acid, lactic acid, sodium hydroxide, and water wash on hide surfaces (n=47) were 2.21 [1.36 – 3.05], 3.02 [2.16 – 3.88], 3.66 [2.60 – 4.72], and 0.08 [-0.94 – 1.11] log CFU/cm2, respectively. Meta-regressions showed that temperature, duration of application, microbial starting concentration, extra water washes, inoculation type, organism type, sample method, surface type, and antimicrobial concentrations were all significant predictors of intervention effectiveness. Finally, after observing authors use substituted values for samples found below a detection limit in primary plant intervention literature, simulations were run to assess the impact of substitution on a random-effects meta-analysis. Simulation results show that substitution practices artificially decrease effectiveness estimates and increase heterogeneity. / Master of Science

The antibacterial efficacy of hydrogen peroxide treatments in four fruit juices was determined. Preservative free apple cider, white grape, and purple grape juice were inoculated with ~ 6.4 log CFU/ml of a five strain, acid adapted, nalidixic acid resistant E. coli O157:H7 cocktail. Orange juice was inoculated with a comparable Salmonella spp. cocktail. In the first study, 0.017% and 0.012% H₂O₂ was added in combination with 0.1% and 0.3% of the dominant organic acid (OA) to 4°C and 25°C juices, with samples taken each day for 21 days. H₂O₂ was a significant factor in all juices (p < 0.05) except white grape (lack of data), and both 0.017% H₂O₂ treatments reduced counts in apple cider, orange juice, and white grape to undetectable numbers within 48 hrs as cultured on tryptone soy agar + 0.05% nalidixic acid (TSAN). Treatments in purple grape juice were less effective overall, and more dependent on OA concentration (p < 0.001) than H₂O₂. There were instances where bacterial survival in apple cider, purple grape, and orange juice continued for 21 days after treatment, and sometimes outlasted the control. These occurrences were dependent on temperature (25°C) and H₂O₂ (0.012%), but not on OA. However, OA concentration was a significant factor (p < 0.05) overall in apple cider and purple grape juice, but not in orange juice. In the second study, 0.015% and 0.03% H₂O₂ was added to 10, 25, and 40°C apple cider and orange juice inoculated with 6.4 log CFU/ml E. coli O157:H7 and Salmonella spp. respectively. Only 0.03% H₂O₂ was effective in reducing counts to undetectable numbers in both juices. However, both temperature and H₂O₂ were significant factors (p < 0.0001) in bacterial destruction, with 0.03% H₂O₂ at 40°C giving undetectable numbers at ≤ 3 and ≤ 6 hours in orange juice and apple cider respectively. It has been demonstrated that at ~ ≥ 0.017%, H₂O₂ can provide a 5 log reduction of these pathogens in fruit juice. Increasing temperature and organic acid concentration can improve its rate of effectiveness in certain juices. However, sensory concerns may negate its use in some products. / Master of Science