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Title
THE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME
Creator
Quinn, Melissa
Date
2020
Collection
Electronic Theses & Dissertations
Description
ABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression....
Show moreABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression. METHODS. A cross-fostering, protein-restricted nutritive model (8% protein) was used to induce undernutrition during gestation (GUN) or lactation (PUN). At 21 days of age (PN21), all mice were weaned to a control diet (CON; 20% protein), isolating undernutrition to specific windows of early life. Fecal samples were collected weekly PN18-PN80 to determine longitudinal programming effects of growth restriction on the gut microbiome (CON N=5, GUN N=6, PUN N=6) and metabolome. Fecal sample DNA was extracted for amplification of the bacterial 16S rRNA genes using PCR, and then the amplicons were sequenced with the Illumina pipeline and analyzed using the Qiita bioinformatics software. Cecum samples were also collected at PN21 (CON N=4, GUN N=6, PUN N=5) and PN80 (CON N=5, GUN N=6, PUN N=6) for microbiome analysis. Liver samples were collected at PN21(CON N=12, GUN N=6, PUN N=7) and PN80 (CON N=13, GUN N=9, PUN N=11) and analyzed along with the cecum for metabolomics via tandem mass spectrometry (LC-MS/MS) and analyzed with the Global Natural Products Social Molecular Networking (GNPS) bioinformatics software. IGF-1 expression in the liver and gastrocnemius (CON N=15, GUN N=12, PUN N=13) was analyzed via a Total Protein NIR western blot to establish a connection between the gut microbiome, tissue metabolome and organ growth. RESULTS. The Beta-Diversity of the fecal microbiome was significantly separated by treatment group using Weighted UniFrac measures (PERMANOVA p=0.0001). Differences in the microbiome were not evident through analysis at the Phylum level (Firmicutes/Bacteroidetes ratio) but were instead driven by longitudinal alterations in the abundance of specific genera and species in PUN. Linear mixed model (LMM) analysis revealed PUN having significantly higher abundance of specific bacteria compared to GUN and CON across the lifespan including: Bacteroides uniformis, B. acidifaciens, B. ovatus, Bifidobacterium sp. and Clostridium sacchrogumia. Rikenellaceae was the only microbe that was significantly lower in abundance in the PUN group over time compared to GUN and CON. Additionally, the PUN metabolome was significantly altered compared to GUN and CON, primarily characterized by reduced: essential amino acids (EAAs: methionine, phenylalanine and tyrosine), riboflavin (B2), primary bile acids, and decreased Dehydroepiandrosterone (DHEA); and increased acylcarnitines and fecal peptides. NIR Western blot analysis revealed significantly lower IGF-1 expression in the liver at PN21 in GUN (p=0.0012) and PUN (p<0.001) as well as overall lower expression in the muscle in PUN (p=0.037) and GUN (p=0.007) compared to CON. CONCLUSION: The gut microbiome and metabolome are altered by early life growth restriction at PN21 and through adulthood. Elevated sugar-fermenting bacteria in the PUN group represent gut microbiome immaturity and delayed development. Temporary metabolic alterations of early life growth restriction are seen in decreased primary bile acids and increased synthesis of liver acylcarnitines, both of which are indicative as adaptations of the pups being calorie-restricted as a result of the low-protein fed dam. More permanent outcomes of growth restriction were evident by increased peptide excretion over the lifespan, significantly decreased methionine and riboflavin – which prevented protein synthesis to occur during early life development, and overall decreased muscle IGF-1 expression and DHEA levels in the PUN mice. Many of the metabolic pathways permanently altered by growth restriction are seen in the liver, making this organ an important site for future research on the development of treatment modalities that can limit growth restriction induced chronic disease.
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Title
Effects of dietary fiber sources on the gastro-intestinal microbiota, fermentation metabolites, and listeria monocytogenes in vivo and in vitro
Creator
Walker, Ryan (Ryan Adam)
Date
2019
Collection
Electronic Theses & Dissertations
Description
Dietary fiber consumption influences the gastro-intestinal microbiota, gastro-intestinal function, and health. This research investigated the effects of dietary fiber sources on bacterial composition, short-chain fatty acid (SCFA) production, gastro-intestinal barrier function, and Listeria monocytogenes challenge in mice and human colon cells. L. monocytogenes causes life-threatening illness in humans and animals. Some dietary fibers protect animals against illness in models of infection,...
Show moreDietary fiber consumption influences the gastro-intestinal microbiota, gastro-intestinal function, and health. This research investigated the effects of dietary fiber sources on bacterial composition, short-chain fatty acid (SCFA) production, gastro-intestinal barrier function, and Listeria monocytogenes challenge in mice and human colon cells. L. monocytogenes causes life-threatening illness in humans and animals. Some dietary fibers protect animals against illness in models of infection, while others enhance infection. Therefore, it is unclear if consuming certain dietary fiber sources confers protection against foodborne illnesses. The objectives of this research were: 1) describe how dietary fiber sources affect bacterial composition in vivo and in vitro; 2) quantify SCFAs produced by bacterial fermentation; 3) determine if fiber sources differentially affect L. monocytogenes infection in vivo; and 4) determine if bacterial metabolites promote or inhibit L. monocytogenes infection by affecting in vitro epithelial barrier function.In this project, dietary fiber sources differentially affected gastro-intestinal bacterial composition in vivo and in vitro, L. monocytogenes infection in mice, and enhanced barrier integrity in vitro. The overall results of this project demonstrate that dietary fiber sources differentially influence certain gastro-intestinal bacterial populations, measures of diversity, and that resulting compositional changes are important in the pathogenicity of L. monocytogenes. Additionally, fermentation end products enhance gastro-intestinal barrier function in this in vitro model, which may be an important factor in the prevention of enteric infection in humans. In summary, both the gastro-intestinal microbiota and its metabolites are important factors for maintaining gastro-intestinal health.
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Title
Quantitative detection of rotavirus and enterovirus in raw sewage using reverse transcription droplet digital PCR
Creator
Kiulia, Nicholas Mukaria
Date
2017
Collection
Electronic Theses & Dissertations
Description
"The goal of this study was to survey the concentrations of rotaviruses (RVs) and enteroviruses (EVs) in raw sewage using reverse transcription droplet digital PCR (RT-ddPCR) and to compare the use of ViroCap filters, polyethylene glycol/sodium chloride (PEG/NaCl) precipitation and mixed cellulose ester (HA) filter methods for virus recovery." -- Abstract.
Title
Environmental regulation of cyclic di-GMP turnover in vibrio cholerae
Creator
Koestler, Benjamin J.
Date
2014
Collection
Electronic Theses & Dissertations
Description
3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine...
Show more3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine bacterium and human pathogen Vibrio cholerae utilizes c-di-GMP signaling to interpret and respond to environmental cues. The central hypothesis that underpins my research is that V. cholerae senses environmental signals with DGCs and PDEs to modulate c-di-GMP concentrations in different environments. As c-di-GMP is a widely conserved second messenger utilized by many different bacteria, the mechanisms by which V. cholerae utilizes c-di-GMP in different environments can be applied to other bacterial systems to further comprehend how they behave in and adapt to various surroundings. To examine the influence of environmental factors on c-di-GMP synthesis and hydrolysis, I have developed a novel method named The Ex vivo Lysate c-di-GMP Assay (TELCA) that systematically measures total DGC and PDE cellular activity. I have shown that V. cholerae grown in different environments exhibits distinct intracellular levels of c-di-GMP, and using TELCA have determined that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. These findings highlight that modulation of both total DGC and PDE activity alters the intracellular concentration c-di-GMP.While searching for specific environmental cues that regulate c-di-GMP, I have found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. This bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. These findings lead me to propose that V. cholerae senses distinct microenvironments within the small intestine, using bile and bicarbonate as chemical cues, and responds by modulating intracellular c-di-GMP.In addition to its function as a bacterial second messenger, c-di-GMP has potent immunostimulatory properties in eukaryotes; these properties make c-di-GMP a prime candidate for use as a vaccine adjuvant. Here, I present a novel method of delivering c-di-GMP into eukaryotic cells using adenovirus. I have demonstrated that c-di-GMP can be synthesized in vivo by delivering DGC DNA into the cell, and that this c-di-GMP increases the secretion of numerous cytokines and chemokines. This novel adenovirus c-di-GMP delivery system offers a more efficient and cost-effective method to administer c-di-GMP as an adjuvant to stimulate innate immunity.
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Title
Modeling the movement of water, bacteria and nutrients across heterogeneous landscapes in the Great Lakes region using a process-based hydrologic model
Creator
Niu, Jie
Date
2013
Collection
Electronic Theses & Dissertations
Description
The development and application of process-based hydrologic models (PBHMs) continues to be a topic of significant interest to the hydrologic community. Although numerous studies have applied PBHMs at small scales ranging from plot and field scales to small-watershed scales, the application of PBHMs to understand large-scale hydrology remains a topic that is relatively unexplored. Understanding controls on large-scale hydrology is key to climate change assessments and effective water resources...
Show moreThe development and application of process-based hydrologic models (PBHMs) continues to be a topic of significant interest to the hydrologic community. Although numerous studies have applied PBHMs at small scales ranging from plot and field scales to small-watershed scales, the application of PBHMs to understand large-scale hydrology remains a topic that is relatively unexplored. Understanding controls on large-scale hydrology is key to climate change assessments and effective water resources management; therefore, to quantify the nature and magnitude of fluxes in regional Great Lakes watersheds, we use a new distributed hydrologic model (PAWS+CLM). Here we describe the application of the model to several large watersheds in the State of Michigan including the Grand River, Saginaw Bay, Kalamazoo and Red Cedar River watersheds and evaluate model performance by comparing model results with different types of data including point measurements of streamflows, groundwater heads, soil moisture, soil temperature as well as remotely-sensed datasets for evapotranspiration (ET) and land water thickness equivalent (GRACE). We then report a budget analysis of major hydrologic fluxes and compute annual-average fluxes due to infiltration, ET, surface runoff, sublimation, recharge, and groundwater contributions to streams etc. as percentage of precipitation and use this information to understand the inter-annual variability of these fluxes and to quantify storage in these large watersheds. After testing the model for its ability to describe hydrologic fluxes and states, we describe the development of solute transport models at the watershed scale by using a mechanistic, reactive transport modeling framework in which the advection, dispersion and reaction steps are solved using an operator-splitting strategy. The solute transport models are tested extensively using available analytical solutions for different hydrologic domains and then applied to describe transport with surface - subsurface interactions and to describe the fate and transport of fecal indicator bacteria such as Escherichia coli in the Red Cedar River watershed in Michigan. Following the successful application of the bacterial fate and transport model, we describe detailed reactive transport modules for predicting the levels of nutrients (N and P). The models are tested using available field observations for the Kalamazoo River watershed in Michigan. The watershed-scale fate and transport modules are expected to aid management by quantifying the impacts of upstream watershed influences on water quality in downstream receiving water bodies such as lakes and oceans. Together with the flow modules they represent a comprehensive suite of process-based models to describe the terrestrial hydrologic cycle coupled with vegetation/land surface and biogeochemical processes.
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Title
Identifying the underlying mechanisms of Marek's disease vaccine synergy
Creator
Umthong, Supawadee
Date
2019
Collection
Electronic Theses & Dissertations
Description
Marek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more...
Show moreMarek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more virulent MDV strains have repeatedly emerged in vaccinated flocks. Thus, there is a need to understand how MD vaccines work in order to design future vaccines that are more protective, especially against more virulent MDVs. One promising insight for vaccine development is based upon protective synergism, a phenomenon where two vaccines when combined provide greater protection compared to either original vaccine when administered alone as a monovalent vaccine. The mechanism that underlines the synergistic effect between SB-1 (a Gallid herpesvirus 3, aka, serotype 2 strain) and HVT (herpesvirus of turkey, aka, Meleagrid herpesvirus 1 or serotype 3), two of the most widely used MD vaccines, has never been investigated, and thus, provides a highly relevant and useful model to explore. To investigate the mechanisms of protective synergy of SB-1 and HVT, we used three approaches. First, we investigated how monovalent SB-1 or HVT replicates when they were alone in the host or together as a bivalent vaccine. We observed that the replication patterns of SB-1 and HVT were different with respect to time after administration into the bird and the organs that they were found to replicate in regardless if the other vaccine were present. Based on the observation that HVT replicated primarily early in the bursa, we found that this organ was necessary for protection using both HVT and bivalent HVT + SB-1 vaccines. Second, we measured the effects of CD8 T cells in monovalent SB-1, HVT, and bivalent SB-1+HVT vaccine treatment. Specifically, we reduced CD8 T cells to see their effect of CD8 T cells on MD incidence and vaccinal protection by injecting the chickens with a monoclonal antibody directed against chicken CD8 T cells. In this study, we found that CD8 T cells were necessary for protection induced by vaccines. Third, we identified the cytokine profiles induced by SB-1, HVT, and the bivalent vaccine to see if cytokine synergy could be one of the mechanisms to explain protective synergy. We found that SB-1 induced an innate anti-viral response typified by IFN-α, IFN-β, IL-1β, T-cell proliferation cytokine IL-21, and Th2 cytokine IL-5, while HVT suppressed TGF-β3 and TGF-β4. The early stimulation of IL-1β and IL-21 (IFN-γ-promoting cytokines) at 4 days post vaccination (DPV) by SB-1 combined with the suppression of TGF-β (IFN-γ- suppressing cytokine) at 1 day post challenge (DPC) by HVT could result in the strong induction of IFN-γ found in the bivalent vaccine at 10 DPC. The induction of IFN-γ supports the synergistic effect of cytokines by a cooperative action mechanism where multiple cytokines work together to enhance the signal. Based on these findings, we propose a model to explain bivalent SB-1 and HVT vaccine synergy, which combines the replication of vaccines, T cell response to vaccinations, and cytokine synergy between SB-1 and HVT vaccine. Our proposed mechanism provides insights on how to generate rationally designed MD vaccines.
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Title
The impact of lactobacillus and bacteriophage on group b streptococcus and the placental membranes
Creator
Shiroda, Megan
Date
2019
Collection
Electronic Theses & Dissertations
Description
The human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in...
Show moreThe human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in this site, but few studies have been conducted to characterize their interactions in vitro or to understand their impact on the placental membranes. Further, our knowledge of the viral component of the microbiome in human health remains incomplete.In this dissertation, Lactobacillus, a well-studied probiotic in other body sites, was evaluated for its effect in the placental membranes. As these membranes were previously considered sterile, we sought to assess the ability of Lactobacillus to colonize these cells and evaluate its impact on them. A cell line model of the outermost layer of these membranes, the decidual cells, was used to establish that Lactobacillus can associate and impact a known cell signaling pathway, the Mitogen Activated Protein Kinase (MAPK) pathway, which is associated with inflammation and host cell death. Total protein level of p38, an important upstream protein in this pathway, was found to be significantly lower with Lactobacillus than in mock infection. These data suggest that Lactobacillus could maintain a commensal interaction in the placental membranes as described in other body sites. Lactobacillus is also known to inhibit pathogen invasion. Group B Streptococcus (GBS) can ascend from the vaginal tract to infect placental membranes, triggering premature birth or neonatal infection. Four Lactobacillus strains representing three species were characterized to assess their impact on two GBS strains (colonizing and invasive). We found live Lactobacillus does not affect GBS growth or biofilm production. L. gasseri increased association of both strains of GBS to the decidual cells but did not result in increased invasion of the cells. Instead, co-culture with Lactobacillus reduced host cell death. Secreted products of Lactobacillus drastically reduced growth in 35 GBS strains that broadly represent GBS diversity and could prevent biofilm formation; this inhibition was strain dependent. Unfortunately, increased GBS-induced host cell death with Lactobacillus supernatants was observed. Collectively, these data suggest that both live Lactobacillus and its supernatant could impact GBS interactions with the placental membranes.Bacteriophage are one of the most abundant members of the microbiome but their impact on opportunistic pathogens such as GBS remains unknown. As GBS can be isolated from gastrointestinal tract, we hypothesized fecal phage communities would inhibit the growth of GBS in vitro. Approximately 6% of the tested communities inhibited the growth of 35 GBS strains. To better understand differences in GBS strain inhibition, we examined capsule, sequence and clinical types of the strains. As no significant differences were found with these traits, we next examined Clustered Regularly Interspaced Palindromic Repeats (CRISPR), which serve as an adaptive immune system against invading foreign DNA by the acquisition of spacer sequences. GBS strains with fewer than nine spacer sequences were less likely to be lysed by a phage community than strains with more than sixteen spacers. We further hypothesized that presence of GBS in the corresponding bacterial component of each phage community would correlate to its ability to inhibit GBS growth. While this correlation did not exist across all GBS strains tested, sensitive strains of GBS were significantly more likely to be inhibited by phage communities with a lower abundance of GBS. Collectively, these data suggest that the phage component of the intestinal microbiome could impact GBS colonization. To further characterize these interactions, an individual bacteriophage should be isolated from these communities.
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Title
WILDFIRE IMPACTS ON SOIL CARBON POOLS AND MICROBIAL COMMUNITIES IN MIXED-CONIFER FORESTS OF CALIFORNIA
Creator
Adkins, Jaron
Date
2021
Collection
Electronic Theses & Dissertations
Description
Forest ecosystems are important reservoirs for long term carbon (C) storage. Forests of the western United States account for 20-40% of total U.S. carbon C sequestration, and nearly half of the total C in these forests is stored in soil. However, many forests in the western U.S are experiencing wildfire conditions that diverge from historical fire regimes. Prior to Euro-American settlement, California’s mixed-conifer forests typically experienced frequent surface fires of low to moderate burn...
Show moreForest ecosystems are important reservoirs for long term carbon (C) storage. Forests of the western United States account for 20-40% of total U.S. carbon C sequestration, and nearly half of the total C in these forests is stored in soil. However, many forests in the western U.S are experiencing wildfire conditions that diverge from historical fire regimes. Prior to Euro-American settlement, California’s mixed-conifer forests typically experienced frequent surface fires of low to moderate burn severity, but, due to the combined effects of altered forest structure and climate change, now experience fires that are larger and more severe than historical conditions. Fires have numerous direct and indirect effects on the soil biological, chemical, and physical characteristics that influence the soil C cycle. Understanding how altered soil characteristics influence the cycling and persistence of soil C, and how they vary with severity, is important for managing forests for C storage and for predicting fire-climate feedbacks. My dissertation work incorporates observational and manipulative experiments to understand the direct and indirect effects of burn severity on soil C cycling and microbial communities over the short to intermediate term, with a particular focus on the distribution of soil C between active and slow cycling pools. Soil C can be conceptualized as discrete pools of variable persistence in soil. The active C pool is quickly decomposed, contributing to the return of CO2 to the atmosphere, whereas the non-active C pool is more stable and contributes to long term C storage. I leveraged a burn severity gradient resulting from a wildfire in a California mixed-conifer forest to determine the structure and kinetics of these C pools at an intermediate time point in post-fire recovery (i.e. three years). I found that the size of the non-active C pool was smaller in burned areas than unburned areas, and the kinetic rate of the non-active C pool was negatively related to burn severity. I also characterized the soil microbial communities across this severity gradient and identified the environmental characteristics responsible for differences. I found that fungal-to-bacterial ratio and oligotroph-to-copiotroph bacteria ratio decreased with burn severity, and these effects were driven by differences in live and dead tree basal area, soil nutrients, and pH. Leveraging another burn severity gradient, I then determined whether differences in microbial communities and soil C pools were related one-year post-fire in a mixed-conifer forest. I again found lower non-active C pool kinetic rates, and higher abundances of copiotrophic bacteria in burned compared to unburned areas. Differences in soil C pool kinetics were related to tree basal area, soil nutrients, and bacterial communities. I determined the short-term impacts of fire on soil C pools and cycling using lab experiments in which I manipulated soil heating intensity and pyrogenic organic matter (PyOM) additions. I found that high intensity soil heating can decrease microbial biomass C (MBC) accumulation, whereas PyOM had minimal effects on MBC in the short-term. Finally, I found that the size of the active C pool increased with soil heating intensity, while the kinetic rate of the non-active C pool decreased; PyOM primarily increased the size of the non-active C pool. Taken as a whole, my research suggests that fire induces short-term soil C losses by increasing the size of the active C pool, but, over the intermediate-term, residual soil C is more persistent. Fire severity is predicted to increase globally throughout the 21st century, and my research contributes to understanding how forest C storage will be affected by disrupted wildfire regimes.
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Title
Genetic diversity of clinical and bovine non-o157 shiga toxin-producing escherichia coli (stec
Creator
Blankenship, Heather Marie
Date
2019
Collection
Electronic Theses & Dissertations
Description
Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised...
Show moreShiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised questions about the genetic diversity of this pathogen population. Six serogroups, O26, O45, O103, O111, O121, and O145, have been denoted as "big six" non-O157 STEC serogroups since they are frequently associated with clinical outcomes.In this dissertation, 895 non-O157 STEC isolates recovered from patients in Michigan between 2001-2018 were analyzed using whole genome sequencing (WGS) to identify virulence gene profiles and apply new typing methods to better discriminate closely related strains. The recovery of a wide range of serogroups from cases presenting with symptoms ranging from mild diarrhea to hemorrhagic colitis, indicates that genetic diversity and variation may have an impact on disease outcomes. The number and richness of serogroups identified over the past 18 years has been steadily increasing and serogroup alone lacks the discriminatory capabilities to classify related isolates. Indeed, strains representing the same sequence types (ST) were often found to be unrelated by serogroup. Notably, some serogroups, STs, virulence gene profiles and alleles were associated with clinical outcomes and patient demographics. Contrast to national surveillance, cases between 11 and 29 years of age had the highest frequency of STEC infections in Michigan.Additionally, a subset of 44 non-O157 STEC recovered from Michigan patients between 2000 and 2006 were examined more comprehensively while making comparisons to 114 clinical STEC isolates from Connecticut to examine the impact of geographic location on risk factors for non-O157 STEC infections. Lastly, a subset of STEC isolates associated with outbreaks in Michigan were examined to identify the impact of WGS on identification of strain relatedness for surveillance compared to pulsed-field gel electrophoresis.While most of the work outlined in this dissertation focused on characterizing clinical non-O157 STEC isolates, a comparative analysis of cattle isolates was also performed since cattle are an important reservoir of STEC. Indeed, numerous outbreaks and illnesses have been traced back to contaminated cattle-based food products or fecal contamination of water and crops. The ability of STEC to persist in the cattle reservoir and farm environment may give rise to more pathogenic strains due to the accumulation of horizontally acquired genes. 66 STEC isolates recovered from a beef herd over four samplings were examined to identify the genetic diversity within the cattle population and longitudinal persistence. The ability of a strain to form a strong biofilm was associated with the ability to persist and be recovered at multiple sampling phases from the same animal. Further, to better understand the genetic diversity of STEC recovered from the cattle reservoir, an additional 12 STEC isolates from three bovine herds (n = 78) and 241 clinical O157 STEC isolates (n = 1,135) were included to identify shared profiles. The similarity in serogroups and virulence gene profiles warrant a continued surveillance of the cattle environment to better understand crossover events and the ability of strains to evolve into new virulent STEC lineages. The work described in this dissertation helped to elucidate the genetic characteristics important for clinical outcomes and identified targets for future surveillance to better understand lineages that may be important for disease.
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Title
Characterizing single-cell behavior of flagellated pathogenic bacteria in mucus and visco-elastic environments
Creator
Nguyen, Nhu Thi Quynh
Date
2020
Collection
Electronic Theses & Dissertations
Description
Flagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of...
Show moreFlagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of Vibrio cholerae and Salmonella enterica in different visco-elastic environments. First, I tested if V. cholerae and S. enterica were still motile in unprocessed pig intestinal mucus. Second, I studied factors that supported their motility in mucus. The first factor I investigated was the effect of pH on V. cholerae motility. I also studied the role of curvature in V. cholerae motility within mucus and some other visco-elastic environments, including liquid and agar. Last, I began investigation of the role of flagellar number in S. enterica motility. In these studies, I focused on analyzing the effective diffusion of bacteria and factors promoting the diffusion in each environment. My findings demonstrate that V. cholerae and S. enterica are able to swim in mucus, and that the torque, and the curvature of V. cholerae, and the flagellar number of S. enterica, play a significant role in bacteria motility in mucus and liquid. Moreover, my findings help elucidate the significance of motility in pathogenesis.
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Title
Characterization of map3773c, ferric uptake regulator protein, in iron metabolism of mycobacterium avium subsp. paratuberculosis
Creator
Miyagaki Shoyama, Fernanda
Date
2020
Collection
Electronic Theses & Dissertations
Description
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a...
Show moreMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a major hurdle to diagnosis (Bannantine et al., 2002). Currently, no current vaccine can protect animals against JD or prevent shedding of MAP (Garcia and Shalloo, 2015). These challenges establish a need for a better understanding of the MAP physiology during infection. Unlike other mycobacteria, MAP has a unique requirement for supplementation of an iron-binding siderophore (mycobactin J) for optimal growth in laboratory media. A whole genome sequence of MAP K10 revealed a truncation of the mbtA gene that was speculated to have led to its mycobactin dependency (Li et al., 2005). Zhu et al (2008) showed that MAP is able to transcribe all mycobactin synthesis genes in an intra-macrophage environment. Furthermore, several genes responsible for iron acquisition in infected tissues, including genes responsible for mycobactin biosynthesis have been shown to be upregulated in naturally infected tissues (Janagama et al., 2010). Its known that iron plays an important role in vital biological processes; however high intracellular concentration of free iron can lead to toxicity to the bacteria. As such, bacteria activate expression of specific group of genes that are controlled by a metal-sensing regulatory transcription factor. In 2009, Janagama and others identified and characterized the iron dependent regulator (IdeR) in MAP. In addition, in a MAP-specific genomic island, MAP carries three putative ferric uptake regulator (Fur) boxes, an iron regulated transcriptional control motif, (Stratmann et al., 2004). To date, nothing is known about the role of Fur in MAP. To elucidate the mechanisms of iron homeostasis in MAP, we investigated LSP15 using a transposon mutant MAP3776c. We demonstrated a phenotype for LSP15 genes in a culture model of cell entry and survival and suggest a function in epithelial cell pathogenesis, however further functional analysis with complementation by a MAP3776c::Tn strain would be necessary to confirm these findings. Additionally, full characterization of a Fur-like protein (MAP3773c) was performed. Using PRODORIC for computational analysis, 23 different pathways that were likely regulated by MAP3773c were identified. These findings were confirmed using a chromatin immunoprecipitation assay followed by high-throughput sequencing (ChIP-seq), that revealed 58 regions where Fur binds under iron replete and deplete conditions. From those, three were directed related to iron regulation MAP3638c (hemophore-like protein), MAP3736c (Fur box) and MAP3776c (ABC transporter). Using the Fur box consensus sequence, we confirmed binding specificity and Mn2+ availability by a chemiluminescent electrophoresis mobility shift assay (EMSA). A transcriptional profile of the parent MAP K10, deletion mutant of MAP3773c and the complemented strains was developed under iron replete and depleted conditions. However, under the current experimental conditions, we are unable to conclude if the lack of transcriptional responses in our study was indicative of a lack of FUR activity.
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Title
Establishment of novel Wolbachia symbiosis and characterization of sex determination genes in dengue mosquito vectors
Creator
Liang, Xiao
Date
2020
Collection
Electronic Theses & Dissertations
Description
As one of the deadliest animals on earth, mosquitos transmit numerous diseases to humans, including dengue, Zika and malaria, which account for over one million human deaths every year. Due to insufficiency of traditional vector control strategies, significant efforts have recently been made to develop novel genetic approaches to either directly suppress mosquito populations or reduce mosquito’s ability to transmit pathogens to humans. One of them is based on the maternally transmitted...
Show moreAs one of the deadliest animals on earth, mosquitos transmit numerous diseases to humans, including dengue, Zika and malaria, which account for over one million human deaths every year. Due to insufficiency of traditional vector control strategies, significant efforts have recently been made to develop novel genetic approaches to either directly suppress mosquito populations or reduce mosquito’s ability to transmit pathogens to humans. One of them is based on the maternally transmitted intracellular symbiotic bacterium Wolbachia. Estimated to infect more than 60% of arthropods in nature, Wolbachia can spread through host populations by means of a reproduction-interfering referred to as cytoplasmic incompatibility (CI). By altering the host’s physiological environment, including immune priming or metabolic perturbation, Wolbachia can also confer antiviral resistance in mosquito vectors. Successful field trials have been conducted to release Wolbachia-infected mosquito males to induce incompatible matings for population suppression or spread Wolbachia into mosquito populations to reduce or block dengue transmission by population replacement. Both population suppression and replacement require for establishment of an artificial Wolbachia symbiosis in mosquito to make it incompatible with target populations. In order to develop a Wolbachia-based strategy for dengue/Zika control in Singapore and Mexico, I have established the transinfected line WB2. By comparing with another transinfected line WB1 which developed 15 years ago, I have demonstrated that wAlbB maintains a stable symbiosis with Ae. aegypti. Further assays show that Wolbachia induces strong resistance to dengue, Zika and Chikungunya viruses in WB2. WB2 line has now been released for field trials in both Mexico and Singapore. In order to improve Wolbachia-based mosquito control, transinfected mosquitoes must be optimized to display maximum pathogen blocking, the desired CI pattern, and the lowest possible fitness cost. Achieving such optimization, however, requires a better understanding of the interactions between the host and various Wolbabachia strains. Thus, we transferred the Wolbachia wMel strain into Ae. albopictus, resulting in a transinfected line, HM (wAlbAwAlbBwMel), no CI was induced when the triply infected males were crossed with the wild-type GUA females or with another triply infected HC females carrying wPip, wAlbA, and wAlbB, but removal of wAlbA from the HM line resulted in the expression of CI after crosses with lines infected by either one, two, or three strains of Wolbachia. These results show that introducing a novel strain of Wolbachia into a Wolbachia-infected host may result in complicated interactions between Wolbachia and the host and between the various Wolbachia strains, with competition likely to occur between strains in the same supergroup. In order to manage the potential risk of failure in population suppression in Singapore, I developed another Ae. aegypti carrying wMal. The transinfected line showed 100% maternal transmission. To facilitate developing a perfect sex separation approach for Wolbachia-based population suppression, I established the CRISPR/Cas9 approach to characterize the function of sex determination pathway genes in Ae. aegypti. By individually knocking out doublesex (dxl) and transformer-2 (tra-2), two essential genes in mosquito sex determination pathway, we show that dxl is not essential gene for female development while knockout of tra-2 results in male-biased sex ratio and absence of female mosquito with homozygous tra-2. These results indicate that the tra-2 is a potential sex determination target that can be explored to develop the female-specific lethality for mosquito sex separation.
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Title
Crop microbiomes and the search for effective biocontrol of Fusarium graminearum on wheat
Creator
MacCready, Kristi Gdanetz
Date
2018
Collection
Electronic Theses & Dissertations
Description
"Manipulation of naturally occurring microbial communities to reduce plant diseases or increase crop yields requires a thorough understanding of interactions within the phytobiome, in particular, how microbial communities change as plants age, across plant species and organs, and under different land management regimes. Plants were sampled from a wheat-maize-soybean crop rotation site that implements four different land management strategies (conventional, no-till, reduced inputs, and organic...
Show more"Manipulation of naturally occurring microbial communities to reduce plant diseases or increase crop yields requires a thorough understanding of interactions within the phytobiome, in particular, how microbial communities change as plants age, across plant species and organs, and under different land management regimes. Plants were sampled from a wheat-maize-soybean crop rotation site that implements four different land management strategies (conventional, no-till, reduced inputs, and organic). The fungal and bacterial communities of leaves, stems, and roots of wheat, maize, and soybean throughout the growing season were analyzed using fungal internal transcribed spacer and bacterial 16S rRNA gene amplicon sequencing. Analysis of sequence-based fungal communities has some limitations due to the unreliable phylogenetic resolution of DNA sequence alignments. To improve this deficiency, a tool that improved phylogenetic resolution was developed. This tool increases the number of operational taxonomic units which are identified at genus and species levels. Endophytes were isolated from the wheat plants used for microbial community analysis and tested for antagonistic activity toward the wheat pathogen Fusarium graminearum during wheat seedling and head infection. Endophytes on crops can be developed to manage disease, and endophyte-based biocontrols could solve current limitations in F. graminearum disease control. Additionally, functional analysis of F. graminearum secondary metabolite genes provides insight into the function of their gene products for this fungal pathogen. Microbial community structure is affected by various genetic factors of the host plant, environmental factors, and interactions with other organisms. Understanding community responses to these factors is necessary for targeted manipulation of communities to reduce plant disease."--Pages ii-iii.
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Title
Postmortem microbiome computational methods and applications
Creator
Kaszubinski, Sierra Frances
Date
2020
Collection
Electronic Theses & Dissertations
Description
Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample...
Show moreMicrobial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that pipeline significantly affected the microbial communities. Increasing minimum library size and sample size increased the number of low abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice significantly affect the resulting microbial communities, which is important for forensic applications. One such forensic application is the potential postmortem reflection of manner of death (MOD) and cause of death (COD). Microbial community metrics have linked the postmortem microbiome with antemortem health status. To further explore this association, we demonstrated that postmortem microbiomes could differentiate beta-dispersion among M/COD, especially for cardiovascular disease and drug-related deaths. Beta-dispersion associated with M/COD has potential forensic utility to aid certifiers of death by providing additional evidence for death determination. Additional supplemental files including tables of raw data and additional statistical tests are available in supplemental files online, denoted in the text as table 'S'.
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Title
Evaluation of a household contact disinfection device for inactivation of bacteriophage MS2 and murine norovirus
Creator
Leak, Emaly
Date
2013
Collection
Electronic Theses & Dissertations
Description
Obtaining safe drinking water is problematic in many developing nations around the world. The HaloPure disinfection canister designed by HaloSource was created to provide household water treatment for middle-class families in India. The Waterbird device was tested for its effectiveness in reducing viruses. The main objective of this study was to determine the effectiveness of the Waterbird device in removing or inactivating bacteriophage MS2 and murine norovirus as surrogates for human...
Show moreObtaining safe drinking water is problematic in many developing nations around the world. The HaloPure disinfection canister designed by HaloSource was created to provide household water treatment for middle-class families in India. The Waterbird device was tested for its effectiveness in reducing viruses. The main objective of this study was to determine the effectiveness of the Waterbird device in removing or inactivating bacteriophage MS2 and murine norovirus as surrogates for human pathogens. Secondary objectives were to determine the potential impacts, if any, that added organic contaminants (in the form of raw sewage) and pH adjustment have on the effectiveness of the device. The Waterbird device was tested by adding MS2 and murine norovirus stock to five liters of well water (with or without sewage); the pH of the water was adjusted to 7.5 or 9 before treatment. The murine norovirus samples were all reduced to the detection limit, achieving a minimum of 2.0 to 4.0 log10 reductions after treatment, but the performance of the device could not be accurately examined using these results. For the MS2, the Waterbird device inactivated or removed an average of 5.4 log10. The MS2 log reduction was affected by pH (p=0.006) and sampling times (p<0.001). Overall, the Waterbird device met the U.S. EPA's guidelines for water purifiers (which requires at least 4 log10 removal of viruses).
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Title
Improving pathogen-reduction validation methods for pistachio processing
Creator
Casulli, Kaitlyn Eileen
Date
2016
Collection
Electronic Theses & Dissertations
Description
The 2015 FDA Preventive Controls for Human Foods Rule requires firms to validate pathogen-reduction steps. Some thermal processes, such as pistachio roasting, are not yet well-characterized with respect to the impact of product and process variables on Salmonella lethality. The objective was to quantify and model the effects of product and process factors on Salmonella lethality for in-shell pistachios. In-shell pistachios were inoculated with Salmonella Enteritidis PT30 (~8.5 log CFU/g), and...
Show moreThe 2015 FDA Preventive Controls for Human Foods Rule requires firms to validate pathogen-reduction steps. Some thermal processes, such as pistachio roasting, are not yet well-characterized with respect to the impact of product and process variables on Salmonella lethality. The objective was to quantify and model the effects of product and process factors on Salmonella lethality for in-shell pistachios. In-shell pistachios were inoculated with Salmonella Enteritidis PT30 (~8.5 log CFU/g), and thermally treated at various levels of temperature, process humidity, and product moisture, under dry and presoaked conditions. Salmonella survivors, moisture content, and aw were quantified at six time points during each treatment, targeting a cumulative lethality of ~3-5 log. Increasing product temperature or process dew point increased Salmonella inactivation rates (P < 0.05). For dry and presoaked treatments, analyzed separately, initial product water activity did not affect inactivation rates (P > 0.05). However, when comparing dry against presoaked treatments, inactivation rates were greater (P < 0.05) for the presoaked pistachios. Models were developed to quantify Salmonella inactivation in pistachios as a function of product temperature, moisture, and process humidity. These models successfully described the effect of these variables on Salmonella inactivation and are the first published attempts to model pathogen reduction in low-moisture products as a function of multiple dynamic variables. Based on these findings, the impact of product and process factors on Salmonella inactivation in pistachios must be considered when designing and validating industrial thermal processes for pathogen reduction.
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Title
Spread of Escherichia coli O157:H7 during flume washing and drying of fresh-cut romaine lettuce
Creator
Wang, Siyi (Of Michigan State University)
Date
2016
Collection
Electronic Theses & Dissertations
Description
ABSTRACTSPREAD OF ESCHERICHIA COLI O157:H7 DURING FLUME WASHING AND DRYING OF FRESH-CUT ROMAINE LETTUCE By Siyi WangThe microbiological safety of leafy greens remains a concern as evidenced from recent outbreaks. This study assessed the spread of E. coli O157:H7 during washing and drying of fresh-cut romaine lettuce. Radicchio was spot-inoculated at 10-1, 101 and 103 CFU/leaf and mixed with uninoculated romaine lettuce to obtain 5 kg batches with inoculated vs uninoculated ratios of 0.5:100,...
Show moreABSTRACTSPREAD OF ESCHERICHIA COLI O157:H7 DURING FLUME WASHING AND DRYING OF FRESH-CUT ROMAINE LETTUCE By Siyi WangThe microbiological safety of leafy greens remains a concern as evidenced from recent outbreaks. This study assessed the spread of E. coli O157:H7 during washing and drying of fresh-cut romaine lettuce. Radicchio was spot-inoculated at 10-1, 101 and 103 CFU/leaf and mixed with uninoculated romaine lettuce to obtain 5 kg batches with inoculated vs uninoculated ratios of 0.5:100, 1:100, 5:100 and 10:100. After 90 s of sanitizer-free flume washing followed by shaker table and centrifugal dryer, the radicchio was removed and the lettuce was divided into 225 g samples to test presence/absence of E. coli O157:H7 using a GeneQuence assay. Based on triplicate trials, lower inoculation levels led to decreased E. coli O157:H7 transfer to romaine lettuce (P < 0.05). All lettuce samples yielded E. coli O157:H7 when radicchio was inoculated at 103 CFU/leaf. At 101 CFU/leaf, the percentage of positive samples decreased from 96.8% to 93.7%, 81.0% and 63.5% while at 10-1 CFU/leaf, 22.2%, 6.3%, 4.8% and 6.3% were positive at 10:100, 5:100, 1:100 and 0.5:100 ratios. Within each inoculation level, there were no significant differences (P>0.05) among four product ratios. These findings are critical to predict the extent of cross-contamination under realistic conditions and will provide important data for improving exposure assessment in risk assessments for leafy greens.
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Title
Transfer and inactivation of Salmonella during post-harvest processing of tomatoes
Creator
Wang, Haiqiang
Date
2015
Collection
Electronic Theses & Dissertations
Description
Salmonella outbreaks have been historically linked to tomatoes, with cross-contamination during post-harvest processing having become a major public health concern. In response, a series of studies were developed to assess the extent to which dump tanks, conveyors, slicers, and dicers can spread Salmonella and other microorganisms. An evaluation of the microbiological quality of tomatoes and dump tank water was conducted during three visits to a local tomato packinghouse. At the beginning of...
Show moreSalmonella outbreaks have been historically linked to tomatoes, with cross-contamination during post-harvest processing having become a major public health concern. In response, a series of studies were developed to assess the extent to which dump tanks, conveyors, slicers, and dicers can spread Salmonella and other microorganisms. An evaluation of the microbiological quality of tomatoes and dump tank water was conducted during three visits to a local tomato packinghouse. At the beginning of whole-day processing and after 3 h of operation, bacteria and yeast/mold populations decreased < 2 logs on tomatoes, with these microbial counts greatly impacted by changes in organic load and sanitizer concentration during washing. When the spread of Salmonella was assessed during washing of tomatoes with various sanitizers and subsequent conveying in a pilot-scale packing line, ~ 90% of the original Salmonella inoculum transferred to sanitizer-free water. Acidified chlorine yielded the greatest Salmonella reduction on tomatoes (3.1 log CFU/g). After processing with sanitizers, Salmonella populations decreased to non-detectable levels (< 0.2 log CFU/100 cm2) on the equipment surfaces. Thereafter, Salmonella transfer was assessed during conveying of tomatoes with plastic, foam, or brush rollers. Overall, cross-contamination was greatest using foam, followed by plastic and brush rollers (P < 0.05). After 5 inoculated tomatoes were roller conveyed, 24 and 76% of all uninoculated subsequently conveyed tomatoes were cross-contaminated with Salmonella of 10 - 100 and 1 - 10 CFU/tomato, respectively, compared to 8% of 25 tomatoes using brush rollers.The next two studies focused on Salmonella transfer during slicing and dicing of tomatoes. For tomato slicing, one red round tomato was inoculated with Salmonella Typhimurium LT2 (~ 5 log CFU/g) and sliced using a manual or electric slicer, followed by 20 uninoculated tomatoes, all of which yielded quantifiable numbers of Salmonella after slicing. The quantitative data was fitted to a two-parameter exponential model (Y = A  e(B  X)). While significantly higher (P ≤ 0.05) percentages of Salmonella were transferred to wet (12.2%) as opposed to dry tomatoes (1.1%), with the variety of tomato also impacting transfer, post-contamination hold time, processing temperature and tomato slice thickness did not significantly impact the overall percentage of cells transferred. When one 0.9 kg batch of inoculated Roma tomatoes (~5 log CFU/g) was mechanically diced, followed by ten batches of uninoculated tomatoes, all uninoculated tomatoes yielded Salmonella, with populations exponentially decreasing from 3.3 to 1.1 log CFU/g. Flume tank washing in sanitizer-free water or water containing 80 ppm peroxyacetic acid, 80 ppm mixed peracid, or 80 ppm total chlorine decreased the Salmonella populations on diced tomatoes 1.3 ± 0.2, 2.3 ± 0.3, 2.4 ± 0.4, and 2.4 ± 0.1 log CFU/g, respectively. Spray sanitation on conveyor belts proved to be an effective way to enhance safety of diced tomatoes, with electrolyzed water being especially attractive due to its relatively low cost and ease of preparation.Finally, the impact of temperature, pH, and wash water organic load on Salmonella morphology and early-biofilm formation was assessed on different surfaces encountered in tomato packing houses. Both pH and temperature significantly affected the surface hydrophobicity of Salmonella. Early-biofilm formation on tomatoes was significantly affected by both time (P = 0.0004) and temperature (P < 0.0001). After 6 d, early-biofilms consistently developed on stainless steel and HDPE surface, with the former being more evenly distributed.
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Title
EPIDEMIOLOGY OF ANTIBIOTIC RESISTANT SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) AND NON-TYPHOIDAL SALMONELLA (NTS) IN MICHIGAN
Creator
Mukherjee, Sanjana
Date
2018
Collection
Electronic Theses & Dissertations
Description
The enteric pathogens, Shiga toxin-producing E. coli (STEC) and non-typhoidal Salmonella (NTS), are leading causes of foodborne infections in the US, resulting in 265,000 and 1.2 million illnesses every year, respectively. The emergence of antibiotic resistance in these pathogens has been documented and is of great concern due to negative patient health outcomes and the possibility of transfer of resistance genes to other clinically relevant pathogens. However, there is a scarcity in...
Show moreThe enteric pathogens, Shiga toxin-producing E. coli (STEC) and non-typhoidal Salmonella (NTS), are leading causes of foodborne infections in the US, resulting in 265,000 and 1.2 million illnesses every year, respectively. The emergence of antibiotic resistance in these pathogens has been documented and is of great concern due to negative patient health outcomes and the possibility of transfer of resistance genes to other clinically relevant pathogens. However, there is a scarcity in information about frequencies of antibiotic resistant and factors associated with resistant STEC and NTS infections in Michigan. It is necessary to have a complete understanding about the of emerging antibiotic resistance and factors driving the rise of resistance in STEC and NTS to help develop effective control strategies. In this dissertation, 980 STEC isolates collected from patients in Michigan between 2001 and 2014 were examined for resistance to clinically relevant antibiotics. The examination of STEC strains for resistance, revealed high frequencies of resistance to ampicillin and trimethoprim-sulfamethoxazole, with significant increases in antibiotic resistance rates observed over this 14-year period. Multivariate logistic regression analysis identified non-O157 serotypes to be independently associated with antibiotic resistance. The recent increase in incidence of non-O157 serotypes observed in the US, coupled with the high frequencies of antibiotic resistance observed in this study, suggest the emergence of antibiotic resistant non-O157s as important human pathogens. Additionally, antibiotic resistant STEC isolates from patients in recent years (2010-2014) were more likely to cause hospitalizations than pansusceptible STEC isolates, suggesting that resistant STEC infections may result in adverse patient outcomes. Using whole genome sequencing, we also identified chromosomal mutations and 33 horizontally acquired genes present in the genomes of non-O157 STEC, likely conferring resistance. Importantly, by creating a co-occurrence network of these genes, we identified the co-occurrence of certain resistance genes, which are possibly present on the same mobile genetic element, thus resulting in multi-drug resistance. In addition to examining resistance in STEC, a total of 198 clinical NTS isolates collected between 2011 and 2014 were also examined for antibiotic resistance in this dissertation. Resistance to tetracycline, trimethoprim-sulfamethoxazole and ampicillin were commonly observed. Concerningly, high frequencies of multidrug resistant NTS were also observed with significant increases in their prevalence observed between 2011 and 2014. These high multidrug resistant rates have important implications on patient care as the efficacy of multiple antibiotics is reduced. Antibiotic resistant NTS isolates were also found to result in significantly longer mean hospital stays compared to pansusceptible NTS. Serovar specific differences in frequencies of antibiotic resistance were observed; S. Enteritidis were observed to have lower resistance frequencies than other serovars. Lastly, to better understand the role that cattle reservoirs play in harbouring antibiotic resistant STEC strains, we examined 121 STEC isolates collected in 2012 from six cattle farms in Michigan for antibiotic resistance. While high resistance frequencies to tetracycline and trimethoprim-sulfamethoxazole were observed in certain herds, no resistance to ampicillin was observed, unlike what was observed in STEC isolates collected from patients. While different populations of resistant STEC may be circulating in the clinical and agricultural environments, continuous monitoring of resistance in the cattle reservoir is warranted to determine if animal reservoirs can serve as potential sources of resistant infections in humans.
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Title
Combination of physical and chemical interventions for reduction of loosely and tightly associated bacteria on broiler carcass skin
Creator
Singh, Pranjal
Date
2016
Collection
Electronic Theses & Dissertations
Description
The study was conducted to develop processing interventions to reduce pathogen load on broiler carcasses and to understand patterns of bacterial association to broiler skin with the following purposes: 1) assess the effect of hot water spray (HWS, 71oC, 1 min) on broiler carcasses for reduction of loosely, intermediately, and tightly associated bacteria, 2) quantify loosely and tightly associated bacteria on broiler skin using different sampling protocols, 3) assess the effect of trisodium...
Show moreThe study was conducted to develop processing interventions to reduce pathogen load on broiler carcasses and to understand patterns of bacterial association to broiler skin with the following purposes: 1) assess the effect of hot water spray (HWS, 71oC, 1 min) on broiler carcasses for reduction of loosely, intermediately, and tightly associated bacteria, 2) quantify loosely and tightly associated bacteria on broiler skin using different sampling protocols, 3) assess the effect of trisodium phosphate dip, hot water dip, and their combinations with/without brushing on reduction of loosely- and tightly-associated bacteria on broiler carcasses and related structural changes in the skin, and 4) assess the characteristics of Salmonella association and penetration to broiler skin with and without stomaching. In study 1, broiler skin was quantitatively assessed for loosely, intermediately, and tightly associated bacteria by rinsing, stomaching, and grinding the same skin. Hot water spraying (71oC, 1 min) of broiler carcasses followed by water immersion chilling reduced mesophilic aerobic bacteria (MAB) populations by 2.07, 1.84, and 2.04 log CFU/g and Salmonella prevalence by 65, 65, and 73% at loose, intermediate and tight association levels, respectively. Hot water spraying and chilling reduced loosely associated Campylobacter prevalence by 45% but no reduction was observed at intermediate and tight association levels. Broiler carcasses suffered from an undesirable partially cooked appearance after the hot water spray treatment. In study 2, the left and right side skin (10 X 7 cm) of broiler carcasses were sampled by 10 swabings and 10 stomachings, and finally ground. Sixty five and 83 % of MAB populations on broiler skin could not be recovered after 10 consecutive stomachings and swabbings, respectively, but were recovered after grinding. The first stomaching or swabbing recovered most of the loosely associated bacteria but not tightly associated bacteria, which were recovered by grinding. In general, stomaching was more effective than swabbing for bacteria recovery from broiler skin. In study 3, microbial reductions and structural changes in broiler skin were evaluated after trisodium phosphate dip (TWD/TSP), hot water dip (TWD/HWD), and their combination (TSP/HWD) with (TSP/HWD/B)/without brushing. Microbial analysis, scanning electron microscopy and histological staining showed that TSP/HWD caused greater reduction in MAB, E. coli, total coliforms, and Salmonella with deeper penetration into the stratum compactum layer of the skin dermis than single application of TSP or hot water dip. TSP/HWD/B yielded lowest skin population of MAB whereas penetration into the dermis was similar to TSP/HWD but with sloughing off of part of stratum compactum in some areas. Both TSP/HWD and TSP/HWD/B changed skin color, with an increase in yellowness being the most prominent. In study 4, loose and tight associated GFP tagged Salmonella were quantified and imaged after refrigerated incubation of inoculated broiler skins for up to 48 h. GFP tagged Salmonella were not able to associate tightly with broiler skin during exposure for up to 48 h at 4oC as application of two stomachings (1 min each) removed almost 88% of the Salmonella cells from the inoculated skin. Confocal imaging of broiler skins, either dipped (2 min) or stomached (2 min) in inoculated buffer, revealed that Salmonellae were present on the surface and inside crevices at average depths of 10 and 68 μm, respectively, after dipping, with some cells pushed to average depths of 62 and 132 μm, respectively, after stomaching. Trapped water seemed to hold Salmonellae deep inside the crevices.
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