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The first aim of this study (Chapter III), the effects of chlorate and nitroethane on foodborne pathogens and rumen fermentation were evaluated. The experimental chlorate product, reduced (P < 0.001) fecal, but not ruminal (P > 0.05) E. coli concentrations by 1000- and 10-fold by 24 and 48 h after chlorate feeding when compared to pre-treatment concentrations (> 5.7 log10 colony forming units/g). Nitroethane treatment decreased (P < 0.01) ruminal (8.46, 7.91 and 4.74 ± 0.78 μmol/mL h-1) and fecal (3.90, 1.36 and 1.38 ± 0.50 μmol/g h-1) methane-producing activity for treatments 0, 80 and 160 mg nitroethane/kg body weight per day, respectively. Whole animal methane emissions, expressed as L/d or as a proportion of gross energy intake (%GEI) were unaffected by nitroethane treatment (P > 0.05). The second aim of this study (Chapter IV) was conducted to examine the effects of nitroethane and monensin on ruminal fermentation and nitro-metabolizing bacterial populations in vitro. The addition of nitroethane decreased methane production (μmol/mL) by at least 90%. The most probable number (MPN) of nitro-metabolizing bacterial populations was increased (P < 0.01) with the addition of nitroethane by at least 3 log10 cells/mL compared with monensin, monensin plus nitroethane or the control group. The final aim of this study (Chapter V) evaluated the effect of two sources of tannins, chestnut (CT) and mimosa (MT) on foodborne pathogens when applied as a hide-intervention and as a feed additive to feedlot cattle. Tannin spray application showed no effect of treatment or application-time (P > 0.05) on E. coli/total coliforms and total aerobes. Chestnut tannin decreased bacterial load of ruminal E. coli and total coliform by at least 0.4 log10 CFU/mL. However, fecal E. coli concentrations were increased with mimosa by 0.3 log10 CFU/g. Also, fecal total coliforms increased with the addition of chestnut or mimosa by at least 0.3 log10 CFU/g. Fecal Campylobacter concentrations (log10 CFU/g) increased with the addition of chestnut and mimosa by at least 0.4 log10 CFU/g.

The increase in production farming, also known as concentrated animal feeding operations (CAFOs), garners more investigations on the implications to public health regarding the disposal of the wastes of food production animals. In addition to the vast amount of animal manure produced, human biosolids is another waste residual that must be managed. The research focus was the sustainability of foodborne pathogens in waste products and the variables that manipulate these environments such as moisture, temperature, organic matter and time. The first study was designed to analyze spatial differences in microbial populations in broiler litter by investigating the relationship of intra-house location, age of flock, bedding moisture, and seasonality. Antibiogram profiles of selected isolates were explored to determine if antibiotic resistant bacteria are common in these environments and if multiple class resistance is present. These findings provided insight into new targets that may reduce zoonotic bacteria that are problematic from a food safety prospective as well as nuisance bacteria that threaten broiler health. The second study was designed to establish current decay rates of viral and bacterial pathogens when seeded in various waste residuals and the effects soil type and application method have on those rates. Decay rates were established by standard culture and molecular methods, such as qPCR. A comparison of both derived inactivation rates were analyzed to determine if these methods were significantly different. Both cultural and molecular methods have limitation and advantages, and the argument that both are useful and needed is asserted. The decay rates associated with each method were used to simulate a one-time exposure to a land application site to assess the microbial risk of Salmonella using a Quantitative Microbial Risk Assessment model.

Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have caught the attention of federal agencies and researchers due to their great economic impact when illnesses occur. To reduce the presence of these pathogens, different approaches have been used. However, since the global consumer’s demand for natural ingredients is steadily increasing, the investigation of the effectiveness of potential natural antimicrobials is necessary. In this study, the in vitro antimicrobial activity of Hibiscus sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was investigated. Furthermore, H. sabdariffa L and ε-polylysine were evaluated to reduce populations of Salmonella and E. coli O157:H7 in ground beef. The minimum inhibitory concentration (MIC) of H. sabdariffa L extracts against Salmonella and E. coli O157:H7 was 6,489 μg/mL and for L. monocytogenes, 5,309 μg/mL. The minimum bactericidal concentration (MBC) of H. sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was 19,467, 58,400 and 29,200 μg/mL, respectively. The exposure to 58,400 μg/mL of H. sabdariffa extract at 25 oC for 12 h resulted in reductions of more than 6.0 log CFU/mL for any of the 3 pathogens tested. Ground beef inoculated with S. Agona (GFP) and E. coli O157:H7 (RFP) was subjected to 5 decontamination treatments. Three of the treatments were using H. sabdariffa L and the remaining ε-polylysine. S. Agona (GFP) was reduced in 1.1 log cycles using 10% of ground H. sabdariffa L and E. coli O157:H7 (RFP) was reduced 0.9 log cycles using 400 ppm of ε-polylysine. If these natural antimicrobials are combined with current antimicrobial technologies to form a hurdle effect, higher pathogen reductions could be achieved. Reductions in the presence of pathogens in food may lead into reductions in the incidence of foodborne diseases.

Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have caught the attention of federal agencies and researchers due to their great economic impact when illnesses occur. To reduce the presence of these pathogens, different approaches have been used. However, since the global consumer’s demand for natural ingredients is steadily increasing, the investigation of the effectiveness of potential natural antimicrobials is necessary. In this study, the in vitro antimicrobial activity of Hibiscus sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was investigated. Furthermore, H. sabdariffa L and ε-polylysine were evaluated to reduce populations of Salmonella and E. coli O157:H7 in ground beef. The minimum inhibitory concentration (MIC) of H. sabdariffa L extracts against Salmonella and E. coli O157:H7 was 6,489 μg/mL and for L. monocytogenes, 5,309 μg/mL. The minimum bactericidal concentration (MBC) of H. sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was 19,467, 58,400 and 29,200 μg/mL, respectively. The exposure to 58,400 μg/mL of H. sabdariffa extract at 25 oC for 12 h resulted in reductions of more than 6.0 log CFU/mL for any of the 3 pathogens tested. Ground beef inoculated with S. Agona (GFP) and E. coli O157:H7 (RFP) was subjected to 5 decontamination treatments. Three of the treatments were using H. sabdariffa L and the remaining ε-polylysine. S. Agona (GFP) was reduced in 1.1 log cycles using 10% of ground H. sabdariffa L and E. coli O157:H7 (RFP) was reduced 0.9 log cycles using 400 ppm of ε-polylysine. If these natural antimicrobials are combined with current antimicrobial technologies to form a hurdle effect, higher pathogen reductions could be achieved. Reductions in the presence of pathogens in food may lead into reductions in the incidence of foodborne diseases.

Factors that affect liquid chemical intervention methods of controlling pathogens on fresh-cut produce were investigated. The relationship between produce tissue structure (intercellular space, cell size, and cell distribution) and the sanitizing effectiveness of liquid chemical treatment was studied. Experiments determined if sanitizer contact with bacteria could be improved through the use of surfactants and different application methods (drop application method, negative pressure differential, and sonication). To test these factors, a model sanitizer, H2O2, and a model microorganism: Salmonella Typhimurium, along with various fresh-cut produce (apple, pear, carrot, and potato) were tested. Microscopic analysis revealed a very complicated pore structure consisting of irregular capillaries. S. Typhimurium was found to survive in all produce tested, and washing did not significantly reduced inoculated bacteria regardless of the bacterial incubation time or produce type. The results showed that a 3% H2O2 solution reduced S. Typhimurium in produce and the solution’s efficiency varied in the following descending order: potato>apple>carrot>pear. In seven min treatments, bacteria were reduced by 2.5 CFU/ml in potato, 2.3 CFU/ml in apple, 1.5 CFU/ml in carrot, and 0.7 CFU/ml in pear. There was no direct evidence on how intercellular space, its percentage or cellular distribution and shape affected efficiency, but some possibilites were discussed. The rate and extent of liquid penetration, and how varying pore diameter in each cell or air space prevent complete chemical treatment penetration were also analyzed. It was determined that bacterial density has a slight effect in bacterial reduction but this depends on type of produce inoculated. The use of surfactants did not improve bacterial reduction in either washing or chemical treatments, and neither did the use of drop application method or temperature differential. On the other hand, applying the chemical treatment with a surfactant while using a sonicator did improve the treatment’s efficiency. This thesis provides a number of factors to be considered when designing a chemical treatment and a guideline for further research in areas such as rate and extent of liquid chemical treatment penetration into fresh-cut produce.

Hard cider is an alcoholic drink made from fermented crushed fruit, typically apples. The popularity of this fermented alcoholic beverage has been on the rise within the last decade. Historically, hard cider has been deemed safe due to the presence of ethanol and the low pH. Although there is lack of scientific evidence to prove that hard cider will and can be safe from foodborne pathogens. Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes are three predominate foodborne bacterial pathogens of concern in the food and beverage industry. Escherichia coli O157:H7 in particular has been associated with fresh produce and more specifically apples, and apple products such as apple juice. The purpose of this study was to determine the bactericidal effects of pH, ethanol, and malic acid on Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes to evaluate the safety parameters for safe hard cider production and storage. The fate of foodborne pathogens in cider was determined during hard cider fermentation, in a cider model system, and in commercial cider. Escherichia coli O157:H7, Salmonella spp, and Listeria monocytogenes did not survive a 5-day fermentation period resulting in a > 7 log CFU/mL reduction of each pathogen with no significant change in pH. The final ABV of the cider at the end of the 5-day fermentation was 4.4%. In the cider model system, the lower the pH and higher the ABV the quicker die off was observed, at pH 2.8, 3.0, 3.2, and 3.4 with 7, 8, and 9% ethanol concentration there was a 6.6 log reduction in E. coli O157:H7 population after 1 day. By the 7-day incubation period, no pathogens were detected at all pH and ABV combinations except for at pH 3.6 and 3.8 with 4% ethanol having ≤0.6 log CFU/mL of the population surviving. Similar E. coli O157:H7 inactivation patterns were observed in the model system and in the commercial ciders. The six commercial ciders observed had varying pH, ABV (%), and malic acid concentrations but successfully resulted in a > 6 log CFU/mL reduction in population of E. coli O157:H7 within 4 days of incubation. The ciders with the highest ABV’s, 8.7 and 9.6% observed a > 6 log reduction by 1 day. It was observed that at some point in time pH plays a bigger role in the presence of less ethanol, but it is clear that ethanol and pH work synergistically to kill of pathogens present in cider fermentation, a cider model, and commercial cider.

Doctor of Philosophy / Department of Food Science / Randall K. Phebus / Intervention techniques to control foodborne pathogens, and rapid identification of pathogens in food are of vital importance to ensure food safety. Therefore, the first objective of this research was to study the efficacy of radio frequency dielectric heating (RFDH) against C. sakazakii and Salmonella spp. in nonfat dry milk (NDM) at 75, 80, 85, or 90°C. Using thermal-death-time (TDT) disks, D-values of C. sakazakii in high heat (HH)- and low heat (LH)-NDM were 24.86 and 23.0 min at 75°C, 13.75 and 7.52 min at 80°C, 8.0 and 6.03 min at 85°C, and 5.57 and 5.37 min at 90°C, respectively. D-values of Salmonella spp. in HH- and LH-NDM were 23.02 and 24.94 min at 75°C, 10.45 and 12.54 min at 80°C, 8.63 and 8.68 min at 85°C, and 5.82 and 4.55 min at 90°C, respectively. The predicted (TDT) and observed (RFDH) destruction of C. sakazakii and Salmonella spp. were in agreement, indicating that the organisms' behavior was similar regardless of the heating system (conventional vs. RFDH). However, RFDH can be used as a faster and more uniform heating method for NDM to achieve the target temperatures. The second objective of this research was to study if hyperspectral imaging can be used for the rapid identification and differentiation of various foodborne pathogens. Four strains of C. sakazakii, 5 strains of Salmonella spp., 8 strains of E. coli, and 1 strain each of L. monocytogenes and S. aureus were used in the study. Principal component analysis and kNN (k-nearest neighbor) were used to develop classification models, which were then validated using a cross-validation technique. Classification accuracy of various strains within genera including C. sakazakii, Salmonella spp. and E. coli, respectively was 100%; except within C. sakazakii, strain BAA-894, and within E. coli, strains O26, O45 and O121 had 66.67% accuracy. When all strains were studied together (irrespective of their genera) for the classification, only C. sakazakii P1, E. coli O104, O111 and O145, S. Montevideo, and L. monocytogenes had 100% classification accuracy; whereas, E. coli O45 and S. Tennessee were not classified (classification accuracy of 0%).

Experimental studies into the use of cold atmospheric plasmas for inactivating foodborne pathogens are presented in this thesis. Eliminating the possibility that treatment delivered by a plasma to a population or assemblage of micro-organisms is unevenly distributed is an essential pre-requisite to attempting to interpret inactivation kinetics with a view to elucidating mechanisms of inactivation. A filtration method of depositing cells evenly on the surface of a membrane without cell stacking was developed and used throughout the work described here. Two atmospheric plasma systems were evaluated and each brought about microbial inactivation in a distinct way. A pulsed radio frequency plasma jet operated at 3.47 MHz caused gross morphological changes to L. innocua whereas a low frequency air mesh plasma system operated at a frequency of 24 kHz led to the inactivation of these bacteria without inducing observable structural changes. Changing the operating parameters of the plasma jet system had a significant effect on the composition of the reactive plasma species generated as revealed by changes to the mode of inactivation of bacteria. In addition to inactivating bacteria, the pulsed plasma jet was shown to be highly effective in degrading and removing amyloid aggregates from the surface of mica coupons. Amyloids have widely been used as a non-infectious model for prions, and the results obtained here show potential for the application of gas plasma technology for removing prions from abiotic surfaces in medical and other applications. It has widely been assumed that bacterial envelopes are the principal sites at which reactive plasma species bring about damage to cells. However, changing the composition of the bacterial membranes of E. coli and Listeria innocua by cultivating them at widely different temperatures to induce changes proved not to result in enhanced inactivation. Flow cytometry was also used to provide additional insights into possible mechanisms of inactivation. The following fluorescent dyes were used either singly or in combination; SYTO 13, DiBAC4(3), cFDA and PI. The results obtained with the dyes DiBAC4(3) and PI showed that Gram positive bacteria became depolarised prior to the bacterial membrane becoming compromised, possibly suggesting that the inactivating plasma species are affecting membrane proteins responsible for maintaining the bacterial charge. Differences between the fluorescent dye staining of Gram negative and Gram positive species were obtained using SYTO13 and PI demonstrating that the different membrane structures affect their interaction with the plasma. In additional studies, the air mesh plasma was used to treat multi-drug resistant strains of Methicillin resistant Staphylococcus aureus (MRSA) in an attempt to reverse antibiotic resistance. MRSA PM 64 was shown to reverse its antibiotic resistance to Oxacillin, Kanamycin and Trimethoprim. Culturing the bacteria in a nutrient limited media led to increased resistance towards plasma treatment and maintenance of their high levels of antibiotic resistance.

Water quality, and therefore human health, may be significantly affected by the presence of pathogenic enteric microorganisms derived for improper disposal of wastewater to aquatic environments. Detection of waterborne viruses is complex due to the difficulties in concentrating the sample and then in detecting the virus by cell culture or molecular techniques. Methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years. The U.S. Environmental Protection Agency requires the use of 1MDS electropositive filters for concentrating enteric viruses from water; however, these filters are expensive for routine viral monitoring. The NanoCeram® filter, an electropositive cartridge filter, has been proposed as a new alternative for large volumes of water. The objective of the study was: to evaluate the effectiveness of NanoCeram® filters for the concentration of poliovirus-1 from wastewater samples and compare to 1MDS cartridge filters. This study suggested that NanoCeram® filters are a viable alternative to the use of 1MDS filters for viral monitoring in surface waters and wastewaters. L. monocytogenes outbreaks with Latin-style soft cheese have been well-documented; however, more information to characterize the human health risk associated with the consumption of queso fresco (QF) using unpasteurized milk is needed. The objectives of the study were: i) to evaluate the prevalence of Listeria, Escherichia coli, Salmonella and fecal coliforms in QF obtained from markets in the northwestern state of Sinaloa, Mexico, and ii) to address the human health impact associated with the consumption of QF contaminated with L. monocytogenes using quantitative microbial risk assessment (QMRA). The study suggested that QF produced in Culiacan, Sinaloa, Mexico have microbial loads above the maximum values recommended by the Official Mexican Regulations; and QMRA can be used to interpret microbial contamination data for impacts on public health.