Microbiological Sciences

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Research from the Department of Microbiological Sciences. The department website may be found at https://www.ndsu.edu/micro/

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16S Ribosomal RNA and Phylograms: Characterizing Student Reasoning to Learning Outcomes from the American Society for Microbiology Curriculum
(North Dakota State University, 2017) Grassie, Chelsey Lee
The American Society for Microbiology (ASM) has established a suggested curriculum for introductory microbiology courses that includes a focus on evolution. However, no data is published to describe how proficiently students address the learning outcomes, in part because validated assessments do not exist. Thus, the goal of this project was to develop assessment prompts that capture student understanding about fundamental statement five under the core concept of evolution. In total, 167 written responses were collected from upper-division microbiology courses, with pre-pharmacy and microbiology majors comprising the majority of students (74.6%). Two coders coded all written responses, and five student interviews were conducted. Results indicate that students have not retained instruction on 16S rRNA, or have not been exposed to it in their classes. Additionally, most students have not been exposed to phylograms, and are unfamiliar with genetic distance being represented on a phylogenetic tree. Emergent reasoning techniques are described.
Assessment of Vacuum Steam Pasteurization to Improve Safety and Quality of Low Moisture Foods
(North Dakota State University, 2016) Shah, Manoj Kumar
Low moisture foods such as grains, seeds, spices and flour are part of our daily diet. While they are rich in bioactive compounds, they can be minimally processed or often consumed raw. Several outbreaks due to Salmonella and E. coli O157:H7 have been attributed to low moisture foods. The efficacy of vacuum steam pasteurization in inactivation of Salmonella PT 30, E. coli O157:H7 and E. faecium on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns was determined. The impact of pasteurization on microbial shelf life of whole and milled flaxseed was also monitored along with their water activity. Vacuum steam pasteurization was effective at inactivation of each bacteria, providing >5.0 log CFU/g reduction at temperatures as low as 75 and 85C. Also, there was no negative impact on microbial shelf life or water activity on pasteurized flaxseed as compared to unpasteurized products.
Attachment and Biofilm Formation of Foodborne Pathogens
(North Dakota State University, 2017) Smith, Sara Jean
Outbreaks of Listeria monocytogenes, Salmonella, and Escherichia coli are increasingly attributed to fresh produce. Current control measures have been assessed for decades, with no alternatives adopted. Sources were identified, reducing flhD transcription and biofilm amounts nearly 2-fold. β-phenylethylamine (PEA), reduced growth and biofilm 96% and 70%, respectively. Curli production was assessed and found to be microorganism-, strain-, and/or serotype-dependent. Reporter fusions were constructed, evaluating expression of Listeria cellulose protein (Lcp). Plcp was not impacted by conditions used. Conditions were then used in attachment of L. monocytogenes to stainless steel. Attachment was significantly reduced by 5 ppm chlorine and 2% lysate. Small molecules could be alternatives to current control measures. More research is needed on what induces curli production. Controls confirm that reporter fusions are an effective way to discover signals impacting gene expression. Attachment/expression assays, indicate that something other than Lcp are responsible for changes in attachment to stainless steel.
The B-Cell Role in Autoimmunity
(North Dakota State University, 2023) Mindt, Marissa Marie
This review is focused on B-cell’s role in autoimmunity and was conducted by reading current papers across a variety of journals and compiling the information learned in this process. Specifically, this review paper encompasses an analysis of what autoimmune diseases are, as well as some of the characteristics of autoimmune diseases the introduction section of this paper serves as a crash course on B-cell. The disease write-up is where autoimmune diseases that are affected and mediated by B-cells, such as systemic lupus erythematosus, multiple sclerosis, and Chron’s disease, are explored. The next section will compare the mechanisms behind each of the diseases to explain how B-cells can cause a large variety of symptoms as well as highlight what symptoms overlap and why. Treatment options and current research are covered throughout this paper to display an understanding of how these diseases are combated.
Biofilm Formation of Escherichia coli from Surface Soils is Influenced by Variation in Cell Envelope, Iron Metabolism, and Attachment Factor Genes
(North Dakota State University, 2018) Petersen, Morgan L.
Biofilm formation may increase survival and persistence of Escherichia coli in the highly variable conditions of soil environments, though it remains unknown the extent variation in biofilm formation affects survival. We asked what genetic traits influence biofilm formation in phylogroup D E. coli isolates from surface soils, and are they associated with the soil environment? Biofilm density was analyzed and compared with soil environment characteristics. Isolates produced more biofilm per unit growth at 15°C than 37°C. Biofilm formation was greater in soil isolates than fecal isolates and in soils with moisture and higher calcium and pH levels. A GWAS analysis found variants involved in cell envelope formation and structure were associated with biofilm formed at 37°C, and stress response and iron acquisition variants were associated with biofilm formed at 15°C. Motility variants were associated with a negative effect on biofilm formed and adhesion variants associated with a positive effect.
A Brief Overview of Lung Disease Caused by Environmental Microorganisms, Aspergillus Fumigatus and Saccharopolyspora Rectivirgula
(North Dakota State University, 2020) Berry, Missy Ann
People often take breathing and respiratory health for granted, but our lungs are constantly inundated with damaging agents, making them highly vulnerable to injury and infection. Globally, respiratory diseases are a leading cause of morbidity, mortality, and disability. Many respiratory diseases are preventable; however, avoidance of ubiquitous microorganisms is not realistic. The aim of this paper is to discuss how two environmental microorganisms, Aspergillus fumigatus and Saccharopolyspora rectivirgula, affect cellular activity and cause lung disease. Prevention, control, and cure of respiratory disease is the ultimate goal and understanding the mechanisms of disease progression is the first step.
Brucellosis Epidemiology, Virulence Factors, Control and Molecular Targets to Prevent Bacterial Infectious Diseases
(North Dakota State University, 2012) Mugabi, Robert
Brucellosis is a bacterial zoonosis that infects both professional phagocytic and nonphagocytic cells in the hosts. Brucella intracellular survival is important for its virulence. In a study to establish the seroprevalence and risk factors of brucellosis in livestock in Kazo and Buremba sub-counties of Kiruhura district, Uganda, fifty goat and 112 bovine serum samples were tested for Brucella antibodies. The prevalence of Brucella antibodies in goats and cattle was 26.0% and 38.4% respectively, while individual seroprevalence rates by livestock breeds were 10.7% (cross-breed goats), 45.5% (local goat breeds), 49.1% (cross-breed cattle), 31.0% (local cattle breeds), and 17.4% (exotic cattle breeds) (p = 0.001). Sharing of watering points, using surface water for livestock, presence of wildlife on pasture, lack of vaccination was significantly correlated with Brucella seropositivity in livestock. The molecular study on biofilm in Escherichia coli included in this paper revealed that pflA knock out mutations had a significant effect on biofilm amounts when biofilms formed on D-serine and acetate. The ldhA formed generally high bacterial biofilm amounts on all carbon sources as compared to the wild type.
Characterization of a Novel Cryptosporidium Genotype in Red-Winged Black Birds
(North Dakota State University, 2014) Jesudoss Chelladurai, Jeba Rose Jennifer
Cryptosporidium species cause cryptosporidiosis, characterized by acute gastroenteritis in humans and animals worldwide. Knowledge of the diversity of Cryptosporidium among mammals and birds is incomplete, especially in North American passerines. In this first molecular study of Cryptosporidium in a North American passerine, C. parvum and a novel genotype, called the red-winged black bird genotype were isolated. Genetic characteristics and phylogenetic analyses of the red-winged black bird genotype were described at the 18S rRNA, actin and HSP70 loci, and it was distinct from previously described species and genotypes. The novelty of this genotype was also supported by propagation studies in chickens, zebra finches and cockatiels that failed to produce patent infections. The study adds to our understanding of the co-evolution of the parasite with its hosts and potential sources of C. parvum transmission to susceptible human and animal hosts.
Characterizing Humidity, Sex, and B-Cell Gene Regulation in Fungal Allergic Asthma
(North Dakota State University, 2020) Kusick, Emma Claire
Asthma is a debilitating lung disease that affects nearly 300 million people worldwide. Environments with high humidity and subsequent mold exposure often trigger allergic asthma. Sex differences have been reported in the incidence, prevalence, and severity of asthma. B-lymphocytes are recruited in high numbers to the allergic lung in response to the inhalation of Aspergillus fumigatus mold spores (conidia). In this work, we used a mouse model of allergic fungal asthma to assess environmental humidity, sex, and B-lymphocytes in an inhalational model of allergic fungal asthma. Our results showed that animals sensitized in low humidity conditions had no airway hyperresponsiveness (AHR), inflammation, but an increase in IgG3 antibody production. Males weighed more than females, female mice had more fibrosis and produced more IgG3 Ab, but sex showed no impact on low humidity. C19+ B-lymphocytes differentially downregulated multiple genes related to allergic asthma returning the body to homeostasis.
Characterizing the Impact of Stress Exposure on Survival of Foodborne Pathogens
(North Dakota State University, 2019) Shah, Manoj Kumar
Bacterial pathogens transmitted by the fecal-oral route endure several stresses during survival/growth in host and non-host environments. For foodborne pathogens, understanding the range of phenotypic responses to stressors and the environmental factors that impact survival can provide insights for the development of control measures. For example, the gastrointestinal system presents acidic, osmotic, and cell-envelope stresses and low oxygen levels, but Listeria monocytogenes can withstand these stresses, causing illnesses in humans. Survival/growth characteristics may differ among L. monocytogenes strains under these stressors due to their genetic diversities. Our knowledge of such phenotypic characteristics under bile and salt stresses are inadequate. In this dissertation, variation in growth characteristics was observed among L. monocytogenes strains under bile and osmotic stresses with no evidence of cross-protection, but rather an antagonistic effect was observed with the formation of filaments when pre-exposed to 1% bile and treated with 6% NaCl. This shows that variation in stress adaptability exists among L. monocytogenes strains with the ability to form filaments under these conditions. Similarly, Salmonella survival in soil is dependent on several factors, such as soil, amendment types, moisture, irrigation, and desiccation stress. In this study, the use of HTPP (heat-treated poultry pellets) was investigated as a soil amendment in the survival/growth of Salmonella in soil extracts mimicking runoff events, and in soil cultivated with spinach plants to assess its safety for use for an organic fertilizer. The presence of HTPP in soil increased S. Newport survival with a greater likelihood of its transfer to and survival on spinach plants. Increased microbial loads and rpoS mutant showed decreased growth/survival in soil extracts, however, rpoS was not important for survival in soil under the tested conditions showing possible lack of desiccation stress. These results show that HTPP provided nutrients to the Salmonella for increased growth and survival in soil extracts and soil, respectively, which show that the use of treated BSAAO to soils may still require appropriate mitigation to minimize Salmonella Newport contamination of leafy greens in the pre-harvest environment. Overall, the results in this study increased our understanding of L. monocytogenes and Salmonella phenotypic adaptation to stressful environments.
Cronobacter Sakazakii Characterization and Analysis of Cytotoxicity in Microvascular Endothelial Cells
(North Dakota State University, 2014) Hafner, Hilary Jayne
Contamination of powdered infant formulas by the bacteria Cronobacter sakazakii can pose serious risk to infants and neonates who consume the formula and subsequently develop C. sakazakii related illnesses such as sepsis and meningitis (1). The Gibbs’ lab assesses C. sakazakii isolates’ ability to cross the blood brain barrier and cause meningitis. This thesis research investigated C. sakazakkii cytotoxicity towards microvascular endothelial cells which comprise the first cell line encountered in the barrier. Understanding the mechanisms used to affect these cells will contribute to our understanding of early stages of invasion. Cytotoxicity assays performed for this research found that the cell line used could not sustain confluency when co-cultured with C. sakazakii isolates over periods beyond 24 hours of incubation. In addition, cell-free cytotoxicity assays found that live cells are not necessary to cause damage suggesting a toxin mediated effect.
Current Status of the Biology, Pathogenesis, and Impacts of Ebola Virus
(North Dakota State University, 2021) Alamri, Dalal
Ebola viruses (EV) are single-stranded negative-sense RNA viruses belonging to the Filoviridae family. There are 6 species of Ebola, and four of them can cause Ebola virus disease (EVD) in humans. Ebola viral hemorrhagic fever is one of the deadliest diseases known to infect humans and non-human primates. The primary mode of transmission of Ebola has been identified as direct contact with infected animals, humans and body fluids. The early diagnosis of EVD is difficult because of similarities of the initial disease presentation to influenza-like symptoms such as high fever, myalgia, fatigue, headache, and chills. The most common symptoms that have been reported from previous outbreaks were fever, sore throat, abdominal pain, vomiting, bleeding, diarrhea, and chest pain. Several methods have been used to detect Ebola such as ELISA, conventional RT-PCR, and real-time RT-PCR. Scientists have been working on several therapeutics and vaccines to prevent and treat Ebola.
The Decomposition Ecology and Microbial Forensics of the Postmortem Microbiome
(North Dakota State University, 2021) Ewald, Adam Patrick
Every living organism dies and is decomposed into nutrients and by-products. This is lead initially by the normal flora of the newly deceased host. The postmortem microbiome, so-called “necrobiome”, undergoes temporal changes affected by the environment in the carcass. The increased data on the necrobiome is fueled by recent advances in genome sequencing analysis techniques. Metagenome sequencing analyzes temporal changes in a population. Genotypic information elucidates identity, structural, and functional traits across a biome. Initially, the necrobiome is composed of taxa common to the living tissue. As decomposition progresses, new and unique taxa emerge. Those suited for growth in the specific environment become dominant. Alpha diversity, beta diversity, and community traits are used to analyze the necrobiome. The necrobiome has potential for forensic evidence predictions. This review covers the succession of the necrobiome specific to body location, their effect on the decomposing carcass, and potential forensics uses of the necrobiome.
Drivers of Infectious Disease Outbreaks: How Climate, Environment and Disease Control Programs Influence Occurence of Infectious Disease Outbreaks
(North Dakota State University, 2012) Muleme, Michael
This research study described the factors driving infectious disease outbreaks using Foot-and-mouth disease (FMD) in Uganda and Lyme disease in North Dakota (ND), Minnesota (MN) and Wisconsin (WI) as case studies. Retrospective data on FMD vaccines and outbreaks in Uganda (2001 – 2010) and Lyme disease in ND, MN and WI (1990 – 2011) was used. The time (7.5 weeks) taken to respond to FMD outbreaks, limited serotyping/subtyping (9/121) of outbreaks and the low percentage of cattle vaccinated (2.1 – 21.2%) portray ineffective control programs. Similarly, increase in fall temperature (P = 0.0189) and annual precipitation (P = 0.0250) were associated with increased human Lyme cases. Shrub land coverage and human population also increased in WI, MN and ND while forest coverage increased in ND. These favor tick and deer proliferation leading to increased human exposure to Lyme Borreliosis. Therefore ineffective disease control programs, climate and environment factors influence infectious disease occurrence.
The Effects of Acetoacetic Acis on Bacterial Growth and Biofilm Formation of Cronobacter Sakazakii, Serratia Marcescens, and Yersinia Enterocolitica
(North Dakota State University, 2016) Murphy, Jennifer Marie
Prevention of bacterial biofilms is an area of research currently being investigated by many research teams. The ability of a chemical to be incorporated into a material that could be used in a medical or food production setting could be of a major value. In this study, we explored the ability of acetoacetic acid (AAA) to reduce biofilm amounts and bacterial growth. We also looked at the transcription of early and late expressed virulence genes in the presence of AAA. We conclude that AAA is a plausible candidate for preventing biofilm formation as we saw a reduction in of biofilm amounts and growth in C. sakazakii, S. marcescens and Y. enterocolitca. We also concluded that AAA was effecting the transcription of virulence genes.
The Effects of Agricultural Factors on Foodborne Bacterial Pathogen Isolation, Attachment, and Survival
(North Dakota State University, 2020) Kraft, Autumn Lea
The pathogens enterohemorrhagic Escherichia coli (EHEC), Salmonella enterica, and Listeria monocytogenes are prominent causes of foodborne illness. If they reach the pre- or post-harvest environment of produce, they can survive for extended periods of time and transfer to post-harvest processing to reach consumers. There are a multitude of factors that influence this survival, bacterial attachment, and isolation, which have been identified for pre- and post-harvest conditions. However, how these pathogens respond to the changes in these systems requires more work to inform effective control methods. To compare the efficacy of an isolation method in different types of matrices, we used immunomagnetic separation (IMS) to selectively isolate strains of EHEC representing non-O157 serogroups from feces, ground beef, and lettuce. The differing matrices and the presence of native microbes seemed to interrupt isolation, with O111 and O145 beads performing the poorest. To understand how EHEC attachment is influenced by food processing-relevant conditions, we investigated the effects of PBS, 4.5% NaCl, and lettuce exudates on attachment to stainless steel (SS). These simulated limited nutrients, osmotic pressure, and alternative carbon sources that EHEC can encounter during food processing. Initial association to SS was reduced under the 4.5% NaCl for all EHEC strains, but this did not translate to reduced attachment. Variation in attachment was observed in lettuce exudates only, and this variation was driven by strains. Finally, we evaluated pathogen survival in the pre-harvest environment. We simulated an aqueous environment that EHEC, Salmonella, and L. monocytogenes might encounter during pre-harvest. Soils were collected from distinctly different environments and extracts were created by leeching water-soluble components from soil. Extracts were inoculated with pathogens and they were monitored to see if the varying chemistries or microbiomes influenced survival. Initially, survival was reduced in the low-nutrient extract, particularly when native microbes were present. Overall, work done here identified factors like matrix or specific environmental conditions and their effects on the isolation, attachment, and survival. These data provide the basis for further work to improve accuracy of IMS-based detection in complex matrices, determine strain-specific mechanisms for EHEC attachment, and assess associations between soil microbiome composition and pathogen survival.
Effects of Intensive Agricultural Management Practices on Soil Microbial Assembly and Recruitment
(North Dakota State University, 2022) Sciences, Microbiological; Nath, Debdutta
Soil microbial communities play an important role in ecosystems in various ways to promote healthy and fertile soil. However, intensive agricultural practices with excessive tillage and fertilizer applications can affect the abundance and community structure of microbial communities in soil as well as their assembly and recruitment by plant roots. Using amplicon sequencing and microscopy, we have examined bacterial and fungal communities under different tillage and fertilizer treatments in a 34-year-old field-trial at the Carrington Research Extension Center of NDSU. We observed that fertilizer application has a significantly stronger effect than tillage on soil properties, as well as the overall soil microbial abundance and community structure. Significantly higher mycorrhizal colonization was found under organic manure application. Overall, the results of this study can improve our understanding of the effects of fertilizer application on soil microbial communities and how management practices can be optimized to reduce the imprints of intensive agriculture.
Efficacy of Chlorine Dioxide Fumigation on the Spores of Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees
(North Dakota State University, 2019) Mahdi, Osama Salih
Honeybees (Apis mellifera) play a critical role in agricultural pollination. However, their health and numbers are in decline. A major cause of this decline is bacterial diseases, of which American foulbrood disease (AFB) is particularly important and troubling. Since the causative agent, Paenibacillus larvae, is spore forming, it can resist antibiotics, many disinfectants, and environmental stresses. We provide protocols and methods for the growth, maintenance, sporulation, and germination of P. larvae. Also, this study investigates the sporicidal activity of ClO2 on P. larvae spores. The gas efficacy depends on treatment time and gas level. The effective level was 645-811 ng/ml ClO2 for 30 min, 191-198 ng/ml for 1 hour, 21-18 ng/ml for 2 h and 7-16 ng/ml ClO2 for 4 h. For decontamination of contaminated surfaces, 214- 245 ng/ml ClO2 for 1 h and 191- 200 ng/ml ClO2 for 2 h completely inactivate the spores.
Emerging Infectious Diseases with Limited Treatment Options: The Case of Ebola Hemorrhagic Fever in Uganda and Shiga Toxin Producing Escheria Coli in the United States
(North Dakota State University, 2012) Gemmeda, Rahel Dubiwak
Emerging infectious diseases are diseases that newly emerge in a population or change the frequency or spatial distribution of their occurrence. Ebola Hemorrhagic Fever (EHF) and Shiga Toxin-producing Escherichia coli (STEC) infections are among diseases that emerged in the 1970s. The two diseases have limited treatment options with no vaccines. This paper is based on two case studies. The first case study utilized data from the 2007/2008 EHF outbreak in Uganda and investigated the epidemiological and clinical aspects of the outbreak. The second case study was based on a study done on STEC isolates collected from beef cattle at the North Dakota State University Research Extension Center in Dickinson. The study investigated the prevalence of the common pathogenic STEC serotypes. The driving factors for the emergence of EHF and STEC, their prevention and control strategies and their challenges were discussed based on the case studies.
Emerging Infectious Diseases with Vaccination as a Major Control Approach: The Case of the African 2 (Af2) Clonal Complex of Mycobacterium Bovis in Uganda and Pneumococcal Vaccine for Streptococcus Pneumoniae in the United States
(North Dakota State University, 2016) Miller, Timothy Robert
Respiratory infectious diseases are among the leading cause of morbidity and mortality in the world. This paper presents two respiratory diseases, Mycobacterium bovis and Streptococcus pneumoniae, which cause significant global health issues despite the advancement of vaccines. In Uganda, M. bovis isolates (n=27) were PCR-tested for a specific chromosomal deletion (RDAf2) and spoligotyped for specific characterization of the Af2 clonal complex. Seventeen (63%) isolates contained the Af2 clonal complex. The high prevalence indicates Af2 clonal complex is of epidemiological interest and a likely cause of bovine tuberculosis in Kampala, Uganda. Ten vervet serum (varying age groups) levels were measured (Luminex) for the immunoglobulin G (IgG) antibody response against the 23-valent, Pneumovax 23® (PN23), pneumococcal capsular polysaccharide serotypes when simultaneously administered with F1/V plague vaccine and Influenza vaccine. Subjects 9-11 years and 25-26 years have shown a slightly better immune response (11%) to PN23 serotypes compared to 19-23 year subjects.

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