Browsing by Subject "Salmonella"

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Analysis on the Metabolic Capabilities of five Salmonella Strains through Genome-Scale Metabolic Models
(2017-07) Ding, Tong
In every country of the world, foodborne diseases caused by Salmonella represent a severe problem to the food supply as well as the public health. The work presented here in this dissertation, looks to investigate food safety related to sustainable farming practices, genome evolution of pathogenic bacteria during host-interactions, and harness post-genomic data to use systems biology methods to elucidate differentiating metabolic capabilities and targets of control of numerous Salmonella serovars. The first chapter introduces detailed information about the background information about Salmonella as a foodborne pathogen. The second examines computational methods to determine if we can accurately predict genome evolution of pathogenic Escherichia coli and Salmonella during host interactions in niches in humans. The third chapter examines the food safety risks associated with the use of chicken manure for agricultural sustainable farming practices in Minnesota. Pathogenic bacteria including Salmonella are also a concern for sustainable farming in which organic fertilizers such as animal wastes are utilized. An analysis on microbiological hazards for such a sustainable farming system was presented in the third chapter. Finally, systems biology approaches were used in the study described in Chapter 4 to analyze strain to strain differences of metabolism of these pathogenic microorganisms. Throughout evolution bacteria have gained or lost certain metabolic properties to better compete with other microorganisms in the changing living condition found in environmental niches found in hosts. Therefore, to develop advanced strategies fighting against pathogenic bacteria, a solid understanding must be obtained on their capability to metabolize available nutrients within different hosts or environmental niches during infection. The genome-scale metabolic models (GEMs) constructed in silico allow us to conduct simulations mimicking real-life situation by interpreting complex bacterial metabolic systems to conduct predictions during bacteria-host/environment interactions. A publication reprinted in Chapter 2 presents work that we conducted to analyze the metabolism-related genes essential to various Salmonella and Escherichia coli species under simulated environments found in three niches where they cause disease. Chapter 4 discussed a study on analyzing five different Salmonella strains’ metabolic capabilities through a systems biology approach. The objective of the study was to gain a better understanding of differentiating metabolic capabilities among various Salmonella strains through efficient model construction and accurate prediction. Overall, the GEMs generated in this study can make good predictions when compared to experimental results, showing their great potentials in analyzing pathogenic bacteria and developing related pathogen control strategies, and the usefulness of this approach for the future examination of 100’s to 1,000s of genomes of Salmonella spp..
Association of Salmonella with Arachis hypgaea (peanut plants and seedpods)
(2016-01) Rossbach, Jonathan
Salmonella is the leading cause of bacterial foodborne illness in the United States and its presence in food results in the recall of many products every year. An increasing concern in the food industry is the contamination of peanuts with Salmonella. The goals of this research were to determine if S. Typhimurium was capable of internalizing in peanut seedpods and plants and identify factors involved in this uptake such as moisture, seedpod status, soil type and the influence of the peanut symbiont, Bradyrhizobium. Intact dry Virginia (DV) seedpods were exposed to S. Typhimurium suspensions and inoculated soil under different conditions. S. Typhimurium suspensions containing 7 Log CFU/mL were examined for different times, and temperatures. DV and green Virginia (GV) seedpods were also exposed to potting media and Hubbard series soil inoculated with 6.5 Log CFU/g. The initial moisture content of each soil was adjusted and the results were compared. Internalization was measured by swabbing and rinsing the interior surface of the seedpods following exposure. S. Typhimurium was quantified using differential tryptic soy agar (dTSA). Internalization of S. Typhimurium from soil into peanut plants was also examined. Soil was inoculated with 6 Log CFU/g. Sterile seeds were sowed and grown for 35 days after inoculation. Following surface sterilization, whole plants were divided into root, stem, and leaf samples. Each plant section was homogenized and quantified using dTSA. For experiments involving Bradyrhizobium, seeds were dip-inoculated in a suspension containing 8 Log CFU/mL of B. NC92 and then tested as described above. Intact DV seedpods were susceptible to S. Typhimurium internalization over all of the temperatures and times tested when immersed in cell suspensions. After exposing seedpods to the suspension for 22 h, S. Typhimurium was recovered internally at a level of 6.4 Log CFU/pod. Internalization also occurred rapidly in as little as 0.5 h. Significant differences were observed between the recovery of S. Typhimurium from the internal surface of DV and GV seedpods when exposed to soil. Overall differences were also identified in the ability of S. Typhimurium to infiltrate seedpods when delivered through potting media and Hubbard series soil at specific soil moisture contents. S. Typhimurium was capable of internalizing in peanut plant tissues and remained present at all testing times present. S. Typhimurium was recovered from stem samples (3.5 Log CFU/g) at greater levels than was observed for root (2.6 Log CFU/g) and leaf (1.7 Log CFU/g) samples. Overall results for stem, root, and leaf samples were recovered at lower levels when B. NC92 was inoculated on seeds before sowing. However, this difference was not significant for any time point, or plant section. Overall, this study’s results suggested the importance of water for S. Typhimurium to internalize within peanut seedpods. Moreover, the initial soil moisture content in relation to the water-holding capacity also impacted the ability of S. Typhimurium to internalize in seedpods. This work also observed that Salmonella was capable of internalization in peanut plants through inoculated soil. Moreover, the results indicated that the nodulating symbiont, B. NC92, did not significantly influence the internalization of S. Typhimurium when seeds are sowed in inoculated soil. This work provides some of the first evidence that peanut seedpods and plants are susceptible to Salmonella internalization, which may represent a potential route of entry of Salmonella into a processing facility from the field.
CD4 T cells in the protection and pathogenesis of persistent Salmonella infection
(2010-10) Johanns, Tanner Michael
CD4 T cells contribute a diverse and non-redundant role to host defense against infections by orchestrating the activation and quality of the innate and adaptive immune responses. The diversity of CD4 T cell function is accomplished by differentiating into a plethora of distinct effector and regulatory lineages that dictate the kinetics and extent of immune activation. However, due to the range and breadth of CD4 T cell function, the precise role and mechanism of these various effector and regulatory subsets in host immunity remains incompletely understood. As persistent infections represent a significant source of morbidity and mortality worldwide, and CD4 T cells play a critical role in protection against this class of pathogens, we sought to elucidate the relative contribution of effector and regulatory CD4 T cell subsets in the pathogenesis and protection of this class of pathogens. Using a murine model of persistent Salmonella infection, we demonstrate that CD4 T cells are required for protection during primary infection but dispensable for secondary immunity. Moreover, both host and pathogen factors limit the generation of a protective effector CD4 T cell response during primary disease including increased regulatory CD4 T cell suppressive function and Salmonella-associated virulence genes, respectively, that enables establishment and persistence of disease. Together, these findings provide novel insight into disease process of persistent Salmonella infection that will aid in the design of future therapeutic and prevention strategies.
Determining the antibacterial potential of a medium-chain fatty acid, caprylic acid, against multidrug-resistant Salmonella enterica enterica serovar Heidelberg in broiler chickens
(2020-04) Manjankattil Rajan, Shijina Raj
Salmonella is the leading bacterial cause of foodborne illness in the United States of America. Among the various foodborne infections, salmonellosis ranks first in terms of hospitalizations and death. More than 50% of foodborne salmonellosis is attributed to the consumption of contaminated poultry products. Salmonella colonizes the chicken cecum and gets excreted, leading to the contamination of the farm environment and poultry carcass during processing. Among the >2500 serovars of Salmonella causing human infections, 7% are associated with foodborne outbreaks through poultry. Salmonella enterica serovar Heidelberg (SH) has emerged through many foodborne outbreaks and found to be resistant to various commonly used and clinically relevant antibiotics. Multidrug-resistant (MDR) SH is one of the commonly isolated Salmonella from chicken carcasses and is associated with foodborne outbreaks through chicken products. Due to the federal initiatives to curb the use of antibiotic resistance development in animal agriculture, alternative antimicrobial strategies that control all Salmonella, including SH, is considered the urgent need of the poultry food industry. Since the pathogen is resistant to clinically important antibiotics, strategies are required to control them on farms and processing. Medium-chain fatty acids (MCFAs) could be an effective alternative to antibiotics approach as MCFAs have broad-spectrum antimicrobial activity. Among the MCFA’s, caprylic acid (CA) has been reported to have antibacterial potential against Salmonella. In this thesis, two questions were asked: Could a long-term supplementation of CA through feed control cecal colonization of MDR SH in broiler chickens (preharvest strategy), and could CA be effective against MDR SH on chicken drumsticks when applied in scalding water (processing aid). At first, we investigated the efficacy of CA in reducing MDR SH colonization in the cecum of 5-week old broiler chickens. Two independent studies were conducted. In each experiment, day-old Ross 708 broiler chicks were randomly allocated to four different groups (3 chicks/group; two studies). The four groups included in the study were: Negative control (NC), Positive Control (PC), Antibiotic group (AB), and CA group (CA). The birds in NC and PC were fed with a standard basal diet, whereas the broilers in the AB group received a standard diet containing 50g bacitracin methylene disalicylate (BMD)/ ton of feed for 5 weeks. The CA (1% w/w) was supplemented through the feed to the broilers in the CA group for 5 weeks. All birds except those in the NC group were challenged with 3.69 log10 CFU MDR SH (2014 Tennessee correctional facility outbreak strain) by crop gavage. Birds were euthanized 7-days after MDR SH inoculation by CO2 asphyxiation. Cecal samples were collected, and the cecal colonization of Salmonella was determined after plating the homogenates onto xylose lysine deoxycholate agar (XLD) plates. The bacterial counts were transformed to log values, and ANOVA was used for statistical analysis. The BMD supplementation resulted in 3.4 and 4.0 log10 CFU/g reduction of MDR SH in studies 1 and 2, respectively. The CA supplementation also resulted in a comparable reduction in cecal colonization of MDR SH as that of BMD. A reduction of 3.2 and 4.0 log10 CFU/g was observed in studies 1 and 2, respectively, in the CA group compared to birds in PC. Therefore, CA could be used as an effective control strategy against MDR SH colonization in 5-week old broilers. This results corroborate with other studies employing CA to control another major Salmonella serovar in broiler chickens, S. Enteritidis. In the second study, we determined the antimicrobial efficacy of CA against MDR SH on chicken drumsticks in simulated soft scalding conditions. Chicken drumsticks were spot inoculated with MDR SH [either lower (~3.0 log10 CFU/g) or higher (~5.0 log10 CFU/g) inoculum] and immersed in scalding water containing treatments for 2 min at 54oC (USDA-recommended time-temperature combination for soft scalding). The antimicrobial treatments included in the study were 0.5% CA, 1% CA, 0.05% peracetic acid (PAA), 0.5% CA + 0.05% PAA and 1.0% CA + 0.05% PAA. Samples inoculated with or without MDR SH and immersed in scalding water containing neither of the antimicrobial treatments served as the PC and NC groups, respectively. Immediately after scalding, the drumsticks were homogenized in phosphate-buffered saline (PBS), and surviving MDR SH populations were recovered on XLD agar plates (n=6). Similarly, MDR SH populations that survived in scalding water was also determined after surface plating (n=6). Additionally, the efficacy of the scalding treatments against MDR SH survival on drumsticks for a storage period of 48 h at 4oC was determined. Furthermore, the effect of these treatments on the surface color of the drumsticks was also evaluated. The antimicrobial treatments resulted in a significant reduction of MDR SH on drumsticks. For the lower inoculum, 0.5% CA, 1% CA, 0.05% PAA, 0.5% CA + 0.05% PAA and 1.0% CA + 0.05% PAA resulted in 0.7, 1.0, 2.5, 1.4 and 1.5 log10 CFU/g reduction of MDR SH on drumsticks (P<0.05). The same treatments resulted in 0.9, 1.3, 2.5, 2.2, and 2.6 log10 CFU/g reduction of MDR SH when the drumsticks were contaminated with the higher inoculum level (P<0.05). Moreover, the antimicrobial treatments completely inactivated MDR SH in scalding water to undetectable levels, whereas 2.0 to 4.0 log10 CFU/mL MDR SH survived in the PC group (P<0.05). Also, the scalding treatments were effective in inhibiting MDR SH on the drumsticks compared to the respective controls during a storage period of 48 h at 4oC (P<0.05), although the magnitude of reduction remained the same as observed during the scalding treatment. Additionally, none of the treatments affected the color of the drumsticks (P>0.05). The results indicated that CA could be used as an effective intervention strategy against MDR SH on chicken drumsticks at scalding to render safe meat production during subsequent stages of processing. The overall results from the MS studies indicated that CA could be used as an effective natural antimicrobial against MDR SH in the pre- and post-harvest stages in broiler production and could improve the microbiological safety of chicken meat.
The Development of a Plant-Growth Promoting Biocontrol Cocktail Using Microbiological Methods and Functional Genomics to Mitigate Salmonella enterica Typhimurium 4/74 Contamination in Alfalfa Sprouts
(2022-08) Vitt, Jacob
Salmonella enterica is a serious threat to public health that is responsible for the vast majority of reported Salmonella outbreaks annually. Despite interventions from private industry and governmental agencies, Salmonella’s main mode of transmission continues to be foodborne, thus eliminating Salmonella reservoirs in the food supply chain is critical to controlling this pathogen. Fresh sprouts present a unique food safety challenge due to minimal processing and ideal growth conditions created during sprouting. Currently, sprout producers have implemented interventions such as seed decontamination, regular pathogen testing of irrigation water and harvested sprouts, and post-harvest treatments. However, an intervention has yet to be introduced during the sprout germination process when conditions are the most optimal for Salmonella growth and proliferation. To address the current gap in pathogen reduction, great interest has been put towards the use of bacterial biocontrol agents to reduce Salmonella contamination during sprout germination.In this thesis, two research goals were established. The first goal was focussed on isolating/characterizing plant-associated bacteria from alfalfa sprouts and subsequently formulating a cocktail of plant-associated bacteria that would function as a biocontrol agent to reduce Salmonella contamination on alfalfa sprouts. This biocontrol cocktail was then assayed for its ability to positively impact alfalfa biomass production and reduce Salmonella cell density on alfalfa sprouts. A plant-associated bacterial cocktail was composed of Pantoea agglomerans, Priestia megaterium, Pseudomonas koreensis, and Pseudomonas putida KT-2440. With a complex community of plant-associated bacteria, the cocktail was found to increase alfalfa biomass production by 25% when compared to uninoculated seeds while not causing a detriment to the overall shelf-life of harvested alfalfa sprouts. Furthermore, the cocktail led to a 3.1 log reduction of Salmonella Typhimurium 4/74 on the sprout surface after six days of germination. The second goal was centered on utilizing a functional genomics approach through transposon insertion sequencing to determine Salmonella conditional gene essentiality to the colonization and infection of alfalfa sprouts. From identified conditionally essential metabolic genes, metabolites produced by alfalfa or metabolites inherent to the sprout germination environment can be targeted for consumption by biocontrol agents. Therefore, the results of the transposon insertion sequencing study could then be used to guide the process of formulating a biocontrol cocktail specifically tailored towards the consumption of metabolite targets to ultimately decrease Salmonella cell density on alfalfa sprouts. Due to time limitations, the results of the transposon insertion sequencing study were not available prior to the submission of this thesis for review, and the results were not included.
Development, validation, and application of molecular microbial source tracking methods to be used in the assessment of environmental waterways.
(2009-11) Sawdey, Richard Charles
The fecal loading of aquatic environments by various animal hosts is of concern for public and environmental health. The microbiological contamination of waterways is amongst the most commonly listed water quality impairment in the U. S. The federal Clean Water Act (CWA) requires individual states to provide the U. S. Environmental Protection Agency (USEPA) with an aggregate quality assessment of its waterways every biennium, in effort to identify waters that do not meet state and federal quality standards. Once waterways are deemed impaired, states must conduct a Total Maximum Daily Load (TMDL) assessment in order to mitigate the impairment and restore the water body to acceptable quality. Successful bacterial TMDL implementation strategies require the use of microbial source tracking (MST) technologies that accurately and efficiently characterize the host-specific source of bacterial loading of waterways, and the relative quantities of each bacterium. Here I report that suppression subtraction hybridization (SSH) was found useful to identify gene markers specific to E. coli derived from swine fecal sources. The ability of this marker gene set to identify 62.3% of E. coli isolated from swine hosts suggests it may be useful in determining their fecal contribution to impaired waterways. I also investigated the influence of cattle grazing operations on the microbiological impairment of a small stream system in Southeastern Minnesota. Impairment by fecal indicator bacteria (FIB) was assessed by using plate count analyses and a quantitative PCR (q-PCR) was developed to estimate the presence of a bovinespecific Bacteroides marker gene in the waterway. The q-PCR data were compared to E.coli plate count data, revealing a lack of correlation between the two methods. Several physical and environmental factors likely influenced the level of E. coli found in the stream, confirming the hypothesis that other information will be needed to supplement current efforts to monitor fecal indicator bacteria in order to determine accurate sourcespecific fecal impacts. Lastly, spatial and temporal variation in the population structure of a fecal pathogen (Salmonella) in association with an alternate host and habitats, including the green alga Cladophora found in stream and lake water, aquatic plants, beach sand, and sediments, was evaluated by use of horizontal fluorophore-enhanced rep-PCR (HFERP) DNA fingerprinting. It was revealed that Salmonella populations associated with Cladophora varied both spatially and temporally, suggesting potentially different input sources of Salmonella over space and time. In addition, differing environmental stressors may play a role in selecting particular Salmonella genotypes that are best suited for growth in these environments The use of rapid molecular-based assays to determine the presence and source-specific loading of fecal indicator bacteria and pathogens has the potential to improve the accuracy and efficacy of TMDL studies, and to expedite implementation strategies to remediate impaired waterways.
The effects of different feed additives on bird performance and the gastrointestinal microbiome of Salmonella-challenged broilers
(2018-01-16) Johnson, Timothy J; Evans, Nicholas; Karnezos, Peter; Sims, Michael; Youmans, Bonnie P; tjj@umn.edu; Johnson, Timothy J
A 42-day, 60-unit floor pen (10 pens per treatment, 25 birds per pen) Salmonella challenge study was conducted to determine the effects of supplementing broiler diets with virginiamycin (VM); medium chain fatty acids (MCFA); MCFA plus lactic acid (MCFA+LA) and a phytogenic blend (PB). Effects were assessed on bird performance and ileal, cecal, and litter microbiomes in birds challenged with Salmonella Typhimurium. Treatments were compared with a non-inoculated control group (NIC) and a Salmonella-challenged group without feed additives (IC). At days 14, 28, and 42 of age, all bird weights and intake were measured, 20 birds from each treatment were euthanized, and the ceca and ilea of euthanized birds were collected along with grab litter samples from each pen. Bacterial profiling was performed using 16S rRNA amplicon sequencing. Subsequent analyses were performed for measurements of alpha and beta bacterial community diversity, taxonomic classifications, and assessments of bacterial taxa that were shifted as a result of different treatments. At 42 days, body weights and mortality adjusted feed conversions for the UIC were significantly better (P<0.1) than the IC and VM while the MCFA, MCFA+LA and PB treatments were similar to the negative UIC. The Salmonella challenge itself had significant (P<0.01) effects on the bacterial microbiome of all sample types, with the greatest effects observed in the cecal microbiome of the bird. The VM treatment counteracted the effects of the Salmonella challenge on the overall bacterial communities of all sample types (P<0.05). While none of the antibiotic alternative treatments had significant effects on overall bacterial community structure consistent over time, specific bacterial taxa were impacted by several treatments. These included Candidatus Arthromitus (segmented filamentous bacteria), Peptostreptococcus, and Clostridium species. Unique signature taxonomic effects were identified for each treatment type, demonstrating attributes of each feed additive type in contributing to unique effects on the bird microbiota. Overall, this work identifies microbiome modulations conferred by different antibiotic alternatives under a Salmonella challenge.
Eliciting Th1 effector functions: A mechanism and role for innate amplification of the Th1 response during infection
(2014-06) O'Donnell, Hope
Innate and adaptive immunity have classically been considered as two distinct categories of cells and responses. Recently, however, an increasing appreciation for the dynamic interactions between these responses has developed. In this dissertation, this overlap is explored in the context of Th1 CD4 T cell production of IFN-γ (interferon-gamma) in response to innate stimuli. In particular, we first asked what triggers innate stimulation of Th1 cells, examining multiple ligands, infections and time points to show that this response occurred within broad contexts of intracellular infection. We then asked how T cells are able to recognize innate stimuli, focusing on whether the T cell intrinsic response relied upon direct LPS (lipopolysaccharide) recognition or indirect recognition of secondary signals. T cell intrinsic requirements were examined in mixed bone marrow chimeras that allowed T cells to be compared within the same environment. Upon demonstrating that the innate Th1 cell response required IL-18 (interleukin-18) receptor signaling, we next explored how T cell extrinsic PRRs (pattern recognition receptors) elicit effector functions through IL-18 secretion. Here, we showed a dual requirement for both TLR4 (toll-like receptor 4) and inflammasome pathways after LPS stimulation during Salmonella infection. The convergence of these pathways was required for increased IL-18 secretion, suggesting a dual level of control in production of such a proinflammatory cytokine. Finally, we asked whether the innate stimulation of Th1 cells is a required response pathway during clearance of Salmonella. Using Lck-cre x MyD88-loxP crossed mice, we demonstrated a deficiency in bacterial clearance in the absence of the signaling molecule MyD88 within T cells, which impairs the ability of Th1 cells to respond to IL-18, but not classical antigen stimulation. Together, this data suggests that the Th1 cell response utilizes a pathway of innate stimulation to amplify IFN-γ production under situations of severe inflammation, in which the classical adaptive response pathway may not be sufficient to mediate a strong and rapid response. Future work may explore additional infectious contexts, other CD4 T cell subsets, memory responses, and circumstances of immunopathology.
Evaluation and continuous improvement of foodborne disease surveillance.
(2012-08) Henke, Evan Elliot
The purpose of this thesis is to contribute to the evidence base of public health performance evaluation by applying innovative measurement and improvement methods to a foodborne disease outbreak detection and investigation process. This thesis is the results of investigation into the three following research questions: 1) can foodborne disease outbreak responder training and work experience be measured in an electronic survey, 2) what is the performance of the Minnesota Department of Health in detecting and investigating foodborne disease outbreaks, and 3) can quality improvement and evaluation tools be used to evaluate MDH process stability and capability? Three research studies were developed to address these research questions. In Chapter 1, the development and analysis of an online survey to ascertain foodborne disease outbreak responder training and work experience is described. In Chapter 2, a bacterial foodborne disease surveillance program in a state health department is described and its performance is evaluated. In Chapter 3, four processes that are conducted by a bacterial foodborne disease surveillance program are evaluated for stability and capability over three years using statistical process control charts. I conclude that methods for evaluating predictive factors of public health performance need to be improved, and that quantitative performance evaluation, within context, has great potential for improving evidenced-based public health preparedness, demonstrating changes in departmental performance, and enabling internal public health practice quality improvement.
Examining immune responses in a mouse model of Salmonella infection
(2010-09) Griffin, Amanda Jill
Salmonella infections are responsible for significant morbidity and mortality throughout the world. Although extensive research has elucidated the mechanisms of protective immunity following vaccination with live vaccine strains (LVS) of Salmonella, very little work has been done to examine immune responses during and following antibiotic treatment of virulent Salmonella infections. We have developed a murine model of naturally acquired immunity to Salmonella, where susceptible mice are orally infected with virulent S. typhimurium and treated with antibiotics for an extended period of time. These mice demonstrate weak protective immunity to rechallenge with virulent Salmonella, which is due to Th1 and antibody responses and can be augmented by the administration of a TLR5 agonist. We have also used antibiotic treatment to examine the development of Th1 responses to LVS Salmonella, which are vital for mediating protective immunity to this pathogen. We show that Th1 cells develop after sustained exposure to Salmonella antigens. Eradication of the bacteria by antibiotic intervention within one week of primary infection has profound effects on the ability of mice to survive rechallenge with virulent Salmonella. We also establish that full effector/memory function of Th1 cells, as determined by robust production of Th1 cytokines, requires two weeks of exposure to Salmonella antigens. Finally, we use short-term antibiotic treatment to establish a model of relapsing virulent Salmonella infection where mice appear to have cleared the bacteria soon after they begin treatment but suffer recurrent and fatal Salmonella infection upon withdrawal. We demonstrate that Salmonella harbored in CD11b+Gr1- resident monocytes in mouse mesenteric lymph nodes (MLNs) are the source of relapsing infection. In addition, the MLNs appear to act as a filter prohibiting the dissemination of Salmonella to systemic tissues. By using antibiotic treatment to examine immune responses to Salmonella, this thesis work may contribute to future research in the development of efficacious therapeutic and/or preventative typhoid vaccines. Moreover, these studies may stimulate future studies using these same tools in other infectious disease models.
The fate of Salmonella in ready to eat cereals.
(2010-04) Hedstrand, Eric Allan
This study was intended to provide insight into the ability of Salmonella to survive in ready-to-eat (RTE) cereal during storage, contaminated post processing. Sweetened toasted oat cereal (STOC) and toasted oat cereal (TOC) were used to elucidate the ability of Salmonella to remain viable for 3 months and the effect of sucrose on its survival. To date, this is the first study to report survival of Salmonella in ready to eat cereal during storage. Commercial cereal samples were inoculated with approx. 106 CFU/g of five different Salmonella strains belonging to four serovars (Agona, Typhimurium, Tennessee and Senftenberg) and re-dried within 24 h. Inoculated cereal was periodically sampled after drying on 1, 3, 5, 7, 14, 30, 60, and 90 days of storage at room temperature. The viable Salmonella count was determined using complex differential media and standard microbiological techniques. The count of most serovars increased during the cereal re-drying step in TOC, but not in STOC. During storage the Salmonella count remained greater than 107 CFU/g in TOC for the entire experimental period with the exception of serovar Senftenberg. The level of Salmonella in STOC declined during the first week of storage, but their final counts were more than 103 CFU/g. These results indicated that Salmonella was able to survive for at least ninety days in either type of cereal. The relevance of this research to the cereal industry is that it confirms the unique ability of this microorganism to survive conditions of very low water activity and stresses the importance of further processing to minimize the risk of transmission of this pathogen by cereal foods.
Isolation and Characterization of Bacteriophages for Bicontrol of Salmonella and Shiga Toxin-Producing Escherichia coli in Food Applications
(2021-08) Hansen, Eleanore
Salmonella poses a significant risk to public health, with tens of thousands of cases occurring each year. Food is the primary vehicle for Salmonella outbreaks, and several diverse foods are frequently attributed to outbreaks. Traditional methods of pathogen control in the food industry are often indiscriminate, killing microbes that may be beneficial alongside the pathogens. In addition, these methods can alter the organoleptic properties of foods and may not be usable for raw and ready-to-eat foods such as raw poultry or fresh produce. Use of chemical antimicrobials is also growing out of favor in some settings as concerns rise over antimicrobial resistance in foodborne pathogens. Interest is growing in using phage cocktails as an alternative method to combat Salmonella and other foodborne pathogens.Bacteriophages, or phages, are viruses that infect bacteria. They are highly host specific, safe to consume, relatively inexpensive, and do not alter the organoleptic properties of food, making them ideal as a biocontrol agent in a variety of food applications. Using several phages combined in a cocktail can increase their success in killing pathogens and lower the chance of resistance to the phages developing. Phages are the most abundant biological entity on the planet, and most remain undiscovered. A few commercial phage cocktails exist that may be used in the food industry, but identifying novel cocktails of unique phages increases the diversity of the tools available to handle troublesome pathogens that arise. In this study, phages were isolated from local Minnesota wastewater samples. The newly isolated phages were tested for their ability to lyse and kill several serotypes of Salmonella and a few serotypes of Shiga-toxin producing E. coli. Six promising phages were picked for a putative novel cocktail. This putative cocktail was assessed for its ability to reduce Salmonella levels in a raw chicken breast model. The cocktail shows promise as a tool to manage both Salmonella and Shiga-toxin producing E. coli in food and food processing environments.
Produce Safety in the United States: Epidemiological Trends and Risk Management Utilizing a Novel Screening Method for Shiga-Toxin Producing E. coli and Salmonella in Irrigation Water
(2019-12) Wu, Yan
Despite significantly improved technologies in food science and public health and tremendous efforts being put by governments to ensure food safety, foodborne outbreaks are still abundant worldwide. Produce products have been frequently implicated in foodborne illness outbreaks in recent years due to changes in consumer demands, consumption habits and production practices. A better understanding on epidemiology changes of produce outbreaks is needed to evaluate current risks associated with produce supply chain and to understand safety regulations regarding produce safety. In addition, it is evident that water used in produce production plays an important role in potentially introducing microbial contaminations. Therefore, its risk management is crucial for safety assurance of the produce supply chain. The goal of this thesis research is to analyze the epidemiological trends of produce outbreaks and to improve the risk management of microbial quality of irrigation water. It summarizes the changing epidemiology of produce outbreaks in the United States from 1998-2007, establishes the baseline to further evaluate the potential impact from the recently implemented Food Safety Modernization Act (FSMA). The study also describes the development, optimization, and evaluation of a novel selective medium for sensitive enrichment and screening of Shiga-toxin producing E. coli and Salmonella in irrigation water. The developed enrichment-indicator system meets the increasing demand of method for multi-pathogen enrichment and detection in a single assay format allowing cost effective detection of STEC and Salmonella within 24 hours.
Protective B cell responses during Salmonella infection
(2013-07) Nanton, Minelva Romelia
In this thesis, we aim to further define the protective role of B cells during Salmonella infection. Understanding how B cells contribute to protective immunity is essential for designing more efficacious next generation vaccines against Typhoid and new vaccines against Paratyphoid and Non-Typhoid Salmonella strains. In the second chapter we test the hypothesis that systemic IgG production is an essential mechanism by which B cells protect against virulent infection. Surprisingly, we found that unlike B cells deficient mice, mice that lack the ability to secrete IgG or all antibodies are still largely protected from virulent Salmonella following resolution of infection with a vaccine strain. We further show that B cells, in the absence of antibody, are required for establishing robust memory Th1 responses. That B cells are important for protective immunity during secondary infection, suggests that we should better understand how this protection develops during a primary infection. In the third chapter, we aimed to define the kinetics and characteristics of the primary endogenous ovalbumin-specific B cell response during live vaccination with attenuated Salmonella expressing ovalbumin and OVA-specific B cell tetramers. We show that unlike immunization with OVA and adjuvant alone, there was a delay in the OVA-specific B cell responses until the infection had cleared. Additionally, we observed a delay in germinal center formation in these OVA-specific B cells, also in contrast to immunization with OVA and adjuvant alone. Upon co-administration of OVA with Salmonella infection, there was a decrease in the expansion and germinal center formation of OVA-specific B cells compared to mice that received OVA alone. We further discovered that, unlike culling of T cells during Salmonella infection, this inhibition of the B cell might not only be linked to expression of Salmonella Pathogenicity Island II proteins, but to effectors that modify the intraphagosomal load of bacteria.
Restaurants And Salmonella: Using Surveillance Data To Improve Policy Development For The Enhancement Of Food Safety
(2020-03) Firestone, Melanie
As the landscape of the diet in the United States changes in response to shifts in consumer preferences and migrating populations, foodborne illnesses remain an important public health challenge. Public health surveillance – the ongoing, systematic collection and analysis of data to prevent and control disease and injury – is the foundation of a prevention-focused food system. As technological advancements simultaneously enhance and disrupt our current surveillance efforts, there is an increasing need to adapt investigation and prevention activities. There is an opportunity to identify novel methods that use existing data to improve surveillance activities. Salmonella – a bacterial foodborne pathogen – is estimated to cause more than one million illnesses per year and is the leading cause of foodborne illness hospitalization. Restaurants are a frequent setting for outbreak and sporadic (non-outbreak) cases of Salmonella infection. Since Salmonella can take advantage of the major pathways for foodborne illness transmission in a restaurant, understanding and controlling its transmission in restaurants is a useful prevention strategy. This dissertation aims to identify opportunities to improve surveillance activities to drive declines in the incidence of foodborne illness using restaurants and Salmonella as a model. The application of novel methods to outbreak investigations and special studies are used to demonstrate an expanded role for surveillance in enhancing food safety. An outbreak of Salmonella infections associated with a chain of quick-service restaurants (Chapter 2) highlights the importance of using all available information in a restaurant-associated outbreak investigation in order to make informed conclusions. An evaluation of restaurant inspection disclosure methods in New York City (Chapter 3) demonstrates the use of surveillance data to evaluate the public health impact of a public disclosure program for restaurant inspections and creates a framework for evaluating future programs. A survey of Minnesota State Fairgoers (Chapter 4) assesses the consumer interest and preferred formats for public disclosure of restaurant inspection results. A second Salmonella outbreak (Chapter 5) explores routine restaurant inspection data to better understand transmission dynamics in a restaurant-associated outbreak, highlighting the value of these data as hazard surveillance. Collectively, the findings from this dissertation demonstrate an important role for linking routine food establishment inspection data with conventional illness surveillance data to improve our food safety systems. These findings will be useful for policymakers, public health officials, and restaurant operators for surveillance-driven prevention of foodborne illness and create a framework for future work in this area.
Risk-Based Evaluation Of The Public Health Impact Of Food Safety Interventions For The Control Of Salmonella Spp. In The Chicken Meat Production Chain
(2017-05) Gonzalez, Rolando
The aim of this work was to develop a risk-based decision analysis framework of farm to table food safety interventions for the control of Salmonella spp. in the chicken meat production chain, using chicken breasts and ground chicken as the model food systems. This framework should assist chicken producers, processors and policy makers when evaluating and selecting the most cost-effective and feasible pre-harvest and post-harvest interventions to control Salmonella spp. The approach included defining the risk factors for Salmonella spp. contamination in the chicken meat production chain, identifying existing and proposed pre- and post-harvest interventions for controlling Salmonella spp., prioritizing pre- and post-harvest interventions based on the reduction of the overall public health risk, developing a quantitative risk assessment to predict the number of Salmonella cases in the US population per year and the impact of individual and combined intervention strategies in reducing the Salmonella public health burden, and finally, applying cost-benefit analysis to identify the most cost-effective measures. The results suggest that the use of peroxyacetic acid as a single intervention applied at post-chill is the most cost-effective intervention to both control Salmonella spp. and meet regulatory performance standards in chicken meat production. It also became evident that there is a need to update the body of published literature to better understand the impact of all stages of the chicken meat production chain, from pre- and post-harvest through consumer handling and cooking, particularly on levels of Salmonella spp.
Salmonella enterica Typhimurium as a tumor-targeting immunotherapy vector
(2015-08) Drees, Jeremy
Interest in cancer immunotherapy has grown in recent years due to its potential for significant and durable therapeutic responses. Immune checkpoint blockade has emerged as an immunotherapy as a single agent but has even greater appeal when it is used in combination with other immunostimulatory approaches. However, the dosing of checkpoint blockade and its combinatorial use with other immunotherapies has been limited by systemic immune-related adverse side effects. One way to overcome these adverse effects is to deliver the therapeutic agents specifically to the tumor microenvironment. Salmonella enterica Typhimurium (S. Typhimurium) has been studied for cancer therapy due to its genetic manipulability and tumor-targeting propensity, and in this thesis, the potential of S. Typhimurium as a tumor-targeting immunotherapy vector was investigated. Functional antagonistic single chain antibodies (scFvs) against the immune checkpoints CTLA-4 and PD-L1 were isolated from an immunized chicken library and engineered for secretion from S. Typhimurium. The inherent anti-tumor properties and tumor-targeting capability of S. Typhimurium were then tested in transplanted primary and metastatic tumor models as well as a genetically engineered autochthonous BALB-neuT breast cancer model. In each of these models, S. Typhimurium demonstrated native anti-tumor efficacy; however the bacteria did not adequately colonize the autochthonous tumors of the BALB-neuT model. Disruption of tumor vasculature by treating BALB-neuT mice with a vasculature disrupting agent (VDA) improved the colonization of autochthonous tumors over 1000-fold to levels similar to those observed for transplanted tumors. Subsequent comparison of the tumor targeting capability and efficacy of S. Typhimurium engineered to secrete the antagonistic ?PD-L1 (scFv) versus a control strain showed that secretion of the scFv may further improve the colonization of autochthonous tumors, leading to a greater reduction in tumor burden of treated mice. These findings provide a proof of principle for the expression and delivery of functional immunotherapeutic single chain antibodies using S. Typhimurium, demonstrate S. Typhimurium's native tumoricidal activity independent of tumor-targeting, illustrate the importance of clinically representative tumor models when studying bacterial cancer therapy, and demonstrate the potential of VDA treatment to improve bacterial tumor-targeting. Collectively, this work illustrates S. Typhimurium's promise as a tumor-targeting immunotherapy vector.
Salmonella in Restaurants: Evaluating the Relationship between Certified Food Managers, 3rd Party Auditors, Risk Factor Violations and Sporadic Salmonella Cases
(2018-10) Appling, Xarviera
Every year in the United States, an estimated 48 million Americans are affected by foodborne illness, 1.2 million from Salmonella. Food establishments are identified as a frequent setting for outbreaks and sporadic cases of Salmonella. Routine health inspections are conducted to prevent foodborne illness by ensuring that proper food handling and safe food preparation procedures are followed. Certified food manager (CFM) certifications are required to ensure proper food safety practices are followed to decrease risk factors associated with foodborne illnesses. Routine inspections are conducted by regulatory agencies. We evaluated the relationships between CFMs, third-party inspectors, risk factor violations cited by health inspectors during routine health inspections, and sporadic Salmonella case exposures in restaurants. Routine inspection and sporadic Salmonella case data from the Cities of Bloomington and Richfield, Minnesota were analyzed. Fewer risk factor violations were found out of compliance during routine inspections when the food establishment had a CFM, the CFM was the person-in-charge (PIC), and used a third-party inspector to audit food safety practices. CFMs were found to improve safety practices specifically related to Salmonella transmission within a food establishment. Lack of compliance with food contact surfaces and handwash facilities being stocked were associated with serving a sporadic case of Salmonella. The results emphasize the importance of food establishments following food safety procedures to reduce risk factors being cited out of compliance on routine inspection reports. By reducing risk factor violations in the establishment, food establishments can potentially reduce patrons’ risk of foodborne Salmonella exposure. Sporadic exposures to Salmonella appear to be useful indicators of food safety performance.
Salmonella’s Desiccation Survival and Thermal Tolerance: Genetic, Physiological, and Metabolic Factors
(2017-07) Maserati, Alice
Salmonella can survive for long periods under extreme desiccation and low water activity conditions (aw < 0.6) while becoming tolerant to heat. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular processes involved in this adaptation mechanism and its potential for cross-protection. This dissertation consists of three distinct studies focused on elucidating this mechanism. The objective of the first study was to identify the genes involved in Salmonella’s resistance to desiccation. A global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low aw (aw 0.11) and cells equilibrated to high aw (aw 1.0) determined that 719 genes (16% of the total number of genes in the genome) were differentially expressed between the two conditions. The genes that were up-regulated at aw 0.11 (290) were mostly involved in metabolic pathways, DNA replication/repair, regulation of transcription and translation, and virulence. Based on the transcriptomic analysis, we created deletion mutants for two virulence genes, sseD and sopD, and tested their ability to survive desiccation and low aw on glass beads. The two mutants exhibited significant cell viability reductions after desiccation compared to the wild-type and additional decrease after exposure to aw 0.11 for 7 days. Under scanning electron microscopy, the mutants displayed a different cell morphology and extracellular matrix production when compared to the wild-type under the same conditions. The findings of this study suggested that sopD and sseD are required for Salmonella’s survival during desiccation. The objective of the second study was to determine the effect of food and inert matrices, nutrient availability, and growth conditions on desiccation survival and thermal tolerance of S. enterica serovar Typhimurium. Salmonella was grown in LBglc and M9 media, in the presence or absence of EDTA and dipyridyl. Cultures were inoculated on toasted oat cereal (TOC) or glass beads, dried, and equilibrated for a week at aw 0.11 and 1.0, before being thermally treated at 75, 85, 90, and 95oC. For all growth conditions and temperatures tested, cells exposed to aw 0.11 had inactivation rates (δ-values) at least 10-fold longer than cells equilibrated at aw 1.0. Our results showed that growth in the presence of EDTA or Dipyridyl did not have any effect on Salmonella’s thermal tolerance at either aw on TOC. In control conditions, recovery after drying and thermal tolerance was higher on TOC than on glass beads, suggesting that the food matrix was protective for desiccation and thermal treatment. Growth in M9 resulted in lower survival to drying and exposure to low aw on glass beads, compared to LBglc. On the contrary, thermal tolerance increased in cells grown in M9 compared to LBglc at both aw. Cells grown in LBglc and M9 displayed differences in the production of extracellular matrix, in particular during equilibration to aw 0.11 and after thermal treatment at both aw. Additionally, when Salmonella was grown on glass beads in LBglc as biofilm, the thermal tolerance was greater than free cells dried on beads. Our observations suggest that the presence of nutrients during growth and before exposure to desiccation and thermal treatment influenced Salmonella’s ability to survive desiccation and develop thermal tolerance. The objective of the third study was to identify proteins involved in Salmonella’s resistance to desiccation and thermal treatment using iTRAQ. Proteins were extracted from S. enterica servorar Typhimurium cells dried, equilibrated at high aw (1.0) and low aw (0.11), and thermally treated at 75°C. Our analysis determined that 734 proteins were differentially expressed among samples, and of these 175 proteins were the most significant in determining differences in the proteomic profiles among treatments. Based on their proteomic expression profiles, the samples were clustered in two main groups by PCA analysis, “dry” samples and “wet” samples, while we did not observe significant differences between the thermally treated samples and the non-heated samples, at both aw. Protein profiles indicated shifts in cell metabolism in both samples, as well as a strict regulation of DNA repair, replication, transcription, and translation. “Dry” samples had higher levels of 50S and 30S ribosomal proteins, indicating that ribosomal proteins might be important for extra-ribosomal regulation of cellular response even when the synthesis of proteins is slowed down. Stress response proteins were more frequently present in “wet” samples compared to “dry” samples, including SspA, GorA, and Dps, suggesting that “wet” cells were activating stress systems in response to rehydration. In conclusion, our study indicated that pre-adaptation to dry conditions was linked to increased thermal tolerance, while reversion from a dry state into a wet state implied a significant change in protein expression that is linked with reduced thermal tolerance.
Systematic review protocol of the efficacy of prebiotics and probiotics in reducing the colonization and shedding of Campylobacter and Salmonella in broilers and turkeys
(2020) Valeris-Chacin, Robert; Nault, Andre J.; Hwang, Haejin; Weber, Bonnie; Bueno, Irene; Johnson, Timothy J.; Pieters, Maria; Singer, Randall S.
Use of prebiotics and probiotics as an alternative to antibiotics is increasing in poultry 16 production. However, there is still uncertainty about their efficacy in controlling the spread of 17 human pathogens in poultry. This paper describes the protocol for a systematic review assessing18 the efficacy of prebiotics and probiotics in reducing the colonization and shedding of 19 Campylobacter and Salmonella in broiler chickens and turkeys. The objective of this protocol is 20 to document the methodology that will be used for the systematic review a priori.

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