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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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.
Genetic Disruption of VIP Signaling Alters Intestinal Microbial Structure and Immunity
(North Dakota State University, 2018) Bains, Manpreet
Vasoactive intestinal peptide (VIP) regulates clock gene expression in the brain that synchronizes diurnal feeding behaviors in mammals. In the gastrointestinal (GI) tissues, VIP influences host nutrient absorption from ingested food, and regulates host metabolic functions. VIP signaling ensures efficient nutrient absorption by influencing ghrelin and leptin expression to balance caloric intake. Importantly, obese humans have elevated plasma VIP levels, supporting its association with fat mass accumulation. In contrast, VIP deficiency leads to weight loss and reduced adiposity, while disrupting epithelial cell nutrient absorption, tight junctions and mucus secretion. Moreover, VIP regulates host glucose metabolism as VIP knockout mice are pre-diabetic with elevated blood glucose and insulin levels. In addition to metabolism, VIP is anti-inflammatory and when knocked out results in exacerbated inflammatory bowel disease (IBD) pathology. The GI track is also home to ≈40 trillion bacteria, called the gut microbiota, which unlock additional calories from fiber for the host. Microbiota dysbiosis is caused by dysfunction in biological systems downstream from VIP signaling, including dysregulated expression of host clock genes, metabolic hormones, immune-relevant mediators and metabolic and inflammatory disease states, like obesity and IBD. It is not known, however, whether the VIP signaling axis contributes to the maintenance of the gut microbiota structure and diversity. We hypothesized that VIP deficiency will cause gut dysbiosis, lower bacterial diversity and reduce its energy extraction potential. To this end, we isolated fecal samples from VIP knockout mice (VIP-/-) and employed 16S rRNA sequencing. VIP deficiency (VIP-/- and VIP-/+) resulted in marked gut microbial compositional changes and reduced bacterial diversity compared to male and female VIP+/+ littermates (n=48). Increased abundance of Bacteroides, Parabacteroides and Helicobacter genera (gram-negative, GN), with reductions of Lachnospiraceae NK4A136, Oscillibacter and Ruminiclostridium genera (gram-positive, GP), were the driving force for the observed increase in the GN/GP ratio. A predicted algorithm program, called PICRUSt, showed changes in microbial metabolism consistent with elevated lipopolysaccharide metabolism and reduced intake of fiber in VIP-/- mice. These data support that VIP regulates intestinal homeostasis by maintaining microbiota balance, diversity and energy harvesting potential, while upholding an anti-inflammatory tone by limiting lipopolysaccharide biosynthesis.
Glycan Triggers of Life Cycle Development in the Apicomplexan Parasite Cryptosporidium
(North Dakota State University, 2017) Edwinson, Adam
Cryptosporidium is an apicomplexan parasite that causes the diarrheal disease cryptosporidiosis, an infection that can become chronic and life threating in immunocompromised and malnourished individuals. Development of novel therapeutic interventions is critical as current treatments are entirely ineffective in treating cryptosporidiosis in populations at the greatest risk for disease. Repeated cycling of host cell invasion and replication by sporozoites results in the rapid amplification of parasite numbers and the pathology associated with the disease. Little is known regarding the factors that promote the switch from invasion to replication of Cryptosporidium, or the mechanisms underlying this change, but identification of replication triggers could provide potential targets for drugs designed to prevent cryptosporidiosis. The focus of this dissertation was to identify potential triggers and the mechanisms underlying the transition from invasive sporozoite to replicative trophozoites in Cryptosporidium. We demonstrate glycoproteins secreted by host cells promote the transition from invasion to replication in Cryptosporidium, and free Gal-GalNAc triggers nuclear division in linear and rounded sporozoites. The proportion of rounded and multinucleated cells in response to the host secretions differed between C. parvum and C. hominis, the two species of greatest concern to human health, with C. parvum sporozoites progressing towards rounding and replication faster. We demonstrated the use of glycomimetic polymers for studying Cryptosporidium biology. Slides imprinted with increasing densities of Gal and GalNAc had the greatest proportion of trophozoite development. Gal and GalNAc glycomimetic polymers were able to cause parasite replication; however the effect was less than what was observed in the presence of secreted host glycoproteins and free Gal-GalNAc. We also characterized a rhomboid protease (ROM) in Cryptosporidium which we name CpROM2, providing information regarding expression and localization of the protease during C. parvum development. Using inhibitors specific for ROM activity we show ROMs play a critical role in excystation, but are also required for the transition from invasion to replication in C. parvum. These findings have strengthened our understanding of how Cryptosporidium transitions from invasion to replication by identifying host glycoproteins and Gal-GalNAc as triggers for trophozoite development via a ROM driven mechanism.
Heterogeneity in Phenotypic Response of Foodborne Pathogens to Control Measures
(North Dakota State University, 2019) Malekmohammadi, Sahar
Foodborne pathogens are estimated to cause 48 million foodborne illnesses through consumption of contaminated food annually. Designing efficient control measures is vital to reducing foodborne illnesses. The modern trend toward preserving foods is using combinations of stresses (hurdle model). However, bacterial adaptation to one stress has the potential to increase resistance to subsequent stress, which is known as cross-protection. Due to recent outbreaks, contamination of ready-to-eat (RTE) foods by L. monocytogenes is a major food safety concern. L. monocytogenes nisin resistance increases when first exposed to NaCl and other stresses, such as low pH. In addition to environmental stressors, specific genomic elements can confer nisin resistance, such as the stress survival islet (SSI-1). We wanted to determine if SSI-1 was associated with salt-induced nisin resistance. Examining 48 L. monocytogenes strains when exposed to nisin and salt revealed that nisin resistance of L. monocytogenes strains increased when first exposed to NaCl. Deletion of SSI-1 demonstrated the role of SSI-1 in saltinduced nisin resistance. These data suggest that inducible nisin resistance in L. monocytogenes can be influenced by environmental conditions and the genetic composition of the strain, which should be considered when selecting control measures for RTE foods. Contamination of low moisture foods (LMFs) (aw <0.85) by Salmonella is a major concern, as Salmonella can survive for a long time on LMFs. A common method to control Salmonella on LMFs is thermal treatment. LMFs can be stored for long periods of time before thermal treatment. There is a possibility of cross-protection when Salmonella is exposed to low aw conditions followed by thermal treatment. 32 Salmonella strains were exposed to flaxseed for 24 weeks. Serovar Agona had a significantly lower death rate compared to Enteritidis and Montevideo (adj. p<0.05). At 24 weeks post inoculation, Agona had significantly higher thermal resistance than Enteritidis (adj. p<0.05). Specific genomic elements can confer osmotic resistance, such as proU and mgtC. ΔmgtC had a higher death rate than wild type. However, deleting proU did not change survival rate. This study broadens our knowledge about heterogeneity of bacterial responses to stressors, which will help to design efficient control measures.
Interactions of Aspergillus fumigatus and Pseudomonas aeruginosa Contribute to Respiratory Disease Severity and Death
(North Dakota State University, 2019) Steffan, Breanne
The lung was recently identified to consist of a complex microenvironment made up of microorganisms that interact with one another and the host cells via direct and indirect interactions. As a result, understanding the dynamic of the microbiome in chronic respiratory diseases has become the focus of pulmonary researches. In cystic fibrosis (CF), chronic infections are a comorbidity associated with the genetic disorder. Recently, it was noted that the interactions of the fungus, Aspergillus fumigatus, and the bacterium, Pseudomonas aeruginosa together contribute to more severe disease outcomes in CF patients. In vitro co-cultures show that P. aeruginosa and A. fumigatus can affect one another’s growth and pathogenicity, but very few studies have attempted to model interactions of these microorganisms in vivo. Based on clinical and basic research, we developed a co-exposure model in which we could compare non-allergic and allergic animals co-exposed to Pseudomonas aeruginosa and Aspergillus fumigatus. While both groups had significant neutrophilia and production of acute phase response cytokines and chemokines, the allergic co-exposed group had a greater mortality with 34.8% of the animals expiring by 24h in comparison to 12.5% for the non-allergic co-exposed animals and 100% survival in the controls. A contributing factor to the more severe disease outcomes in the allergic co-exposed group is the increase in eosinophilic inflammation and IL-17A production, which only occurs when both microorganisms are viable. In addition, it was found that viable P. aeruginosa but not A. fumigatus causes interstitial inflammation, significant neutrophilia, and even death during co-exposures. The decline in health of animals co-exposed to the fungus and bacteria could be attributed not only to the host’s inflammatory response, but also to the spatial and temporal co-localization in the lung. To address this, we performed in vitro studies finding an aggregation of the microorganisms that could also be identified in vivo. This current research emphasizes the need for in vivo studies on polymicrobial interactions.
Investigation of Nutrients as Treatments of Bacterial Biofilms
(North Dakota State University, 2019) Schroeder, Meredith Lynn
Using nutrients bacteria utilize to grow as treatments of bacterial biofilms have been investigated. This dissertation examines the problem of the prevention and treatment of biofilms by: (i) testing the effect of the nutrient β-phenylethylamine (PEA) on growth, ATP content of biofilm, and biofilm biomass, (ii) testing the effect of ethyl acetoacetate (EAA) and other small molecules on growth, ATP content of biofilm, and biofilm biomass, and (iii) investigating three applications of PEA and EAA. PEA could be used as both, prevention and treatment for bacterial biofilms. We observed a reduction of growth, ATP content of biofilm, and biofilm biomass of Escherichia coli K-12 AJW678 with increasing concentrations regardless of when PEA was added. PEA and EAA were able to reduce growth, ATP content of biofilm, and biofilm biomass for multiple bacterial strains, but the efficacy of the nutrients were strain dependent. PEA was found to effectively reduce growth, ATP content of biofilm, and biofilm biomass when used in multiple applications. When PEA was physically integrated in polyurethane, we observed at least a 20 % reduction of ATP content of biofilm for all the bacterial strain tested. To mimic a clinically relevant environment, biofilms were formed in silicone tubing and treatment of PEA and EAA were administered similar to antibiotic lock treatments (ALT). The PEA and EAA treatments reduced ATP content and biofilm biomass of multiple pathogens. PEA was also effective on ATP content, biofilm biomass, and cell counts when used in a microfluidic system.
Lung Mucosal Response to Repeated Inhalational Insults with Immunomodulatory Agents in a Murine Model of Fungal Asthma: Airway Epithelium Takes the Center Stage
(North Dakota State University, 2013) Pandey, Sumali
Asthma is a debilitating disease of the lungs affecting 235 million people worldwide. Fungus-associated asthma leads to a particularly severe type of disease, and exposure to environmental fungi and their products is unavoidable due to the ubiquitous nature of fungal species. Besides being allergenic, fungi are opportunistic pathogens, and anti-fungal and/or allergic pathways may be modified through repeated inhalation of immunomodulatory agents, affecting the outcome of fungus-induced asthma. Our aim in this project was to investigate the extent to which repeated inhalation of immunomodulatory agents influence the lung mucosal responses in a naïve murine host or in one that had been sensitized to fungal proteins (allergic). The immunomodulatory substances chosen hold relevance to human inhalational exposure, and included live or irradiation-killed Aspergillus fumigatus (a fungi) spores, deoxyxnivalenol (a mycotoxin), and fluticasone propionate (an inhalationally administered corticosteroid, commonly prescribed for allergic asthma). In a naïve host, inhalation of live A. fumigatus spores showed pathological features of fungal asthma. However, in an allergen-sensitized lung, both dead and live A. fumigatus spores established fungal airway disease, albeit to different extents. Next, we tested the effect of deoxynivalenol in an allergic host and found that its repeated inhalation did not affect pulmonary disease pathology, but did lead to a dose- and time- dependent increase in mucosal and systemic total IgA. Finally, we tested the effect of fluticasone propionate, and found that it did not influence the development of fungal airway disease, but did induce dynamic changes in lung physiology and antibody titers. Besides mimicking human inhalational exposures, inhalation ensures direct interaction of the inhaled substances with airway epithelium, which plays an important role in defense against inhaled substances and in asthma pathophysiology. By analyzing various mechanisms involved in murine lung-mucosal response to the inhaled substances, a critical involvement of airway epithelium as an orchestrator of immune responses is highlighted, and this would inform mechanism-based future studies. In conclusion, this project is likely to aid in establishing evidence based standards for fungus-related exposures and in making informed therapeutic decisions for fungus-associated diseases.
Phenotypic and Genomic Assessment of Listeria monocytogenes Virulence
(North Dakota State University, 2019) Cardenas Alvarez, Maria Ximena
Listeria monocytogenes is the etiological agent of listeriosis in humans and ruminants causing bacteremia, central nervous system (CNS) infections, abortion, and gastroenteritis among other clinical outcomes. Recent studies have integrated whole genome sequence (WGS), epidemiology data, and host susceptibility to provide evidence for variation in virulence among strains, as a small number of hypervirulent clones have been found linked to a high proportion of human and ruminant invasive listeriosis cases, however, still little is known about variation in virulence across different L. monocytogenes subgroups. To assess and compare the genetic diversity of clinical listeriosis isolates from ruminants in the Upper Great Plains states, we used multilocus sequence typing (MLST) and found that the variation in virulence potential varies among clonal complex (CC), which is reflected in the epidemiology of L. monocytogenes. Based on these results, we evaluated the strains’ virulence potential in Galleria mellonella through larvae survival, LD50, and cytotoxicity, and monitored health index scores and bacterial concentrations post-infection as quantifiable indicators of virulence and immunogenicity. Our findings suggest that strains belonging to CC14, as well as isolates from MN infections are hypervirulent in G. mellonella, as they need a lower bacterial concentration to cause disease and produce a low-level infection that could help in evading the host immune response. We also identify genomic elements associated with strains causing three different clinical outcomes: bacteremia, central nervous system infections, and maternal-neonatal infections. By analyzing 232 whole genome sequences from invasive listeriosis cases, we identified orthologous genes of phage phiX174, transfer RNAs and type I restrictionmodification (RM) system genes along with SNPs in loci associated with environmental adaptation such as rpoB and the phosphotransferase system (PTS) associated with one or more clinical outcomes. Novel genetic variants may be associated with a particular virulence phenotype, as it is likely that strains causing the same clinical outcome share unique genetic elements. Variation in virulence among L. monocytogenes subgroups may confer an increased ability to cross host barriers or higher adaptability to food processing environments, thus the investigation of strainspecific genetic features can impact the design of prevention and management plans for listeriosis.
Rhomboid Proteases and Surface Adhesins During Cryptosporidium Development
(North Dakota State University, 2013) Tabe, Ebot Sahidu
Cryptosporidium parvum, a primary cause of cryptosporidiosis in humans and livestock worldwide, has a complex life cycle that includes an environmental oocyst stage, and stages of merogony, gametogony, and sporogony that are completed in a single host. Development within the host takes place in a protected intracellular but extracytoplasmic niche at the apical surface of epithelial cells. The life cycle can be described as having alternating extracellular invasive and intracellular replicative stages. With no effective chemotherapeutics, understanding the mechanism of host cell invasion by this pathogen is paramount. The first aim dissertation was to identify functions of sporozoite surface proteins and rhomboid proteases (CpROMs) during motility and invasion of host cells. We demonstrate that two CpROMs distinctively and collectively cleaved five thrombospondin-family proteins (TSPs) and a mucin-like glycoprotein in a heterologous assay. Further, there was differential co-expression and co-localization of the CpROMs and their substartes during in vitro life cycle development; CpROM4 and CpTSP10 proteins colocalized to the anterior, middle and posterior of sporozoites and in developing intracellular stages while CpROM5 and TRAP-C1 colocalized to intact and non-intact oocyst walls, the anterior of sporozoites, and intracellular stages as early as 2 h post infection. CpTSP7, also localized to the oocyst wall, the anterior and posterior of sporozoites and intracellular stages from 6 h post infection. Similar to CpTSP10, CpTSP9 was not present in the oocyst wall; however, it was expressed in sporozoites and intracellular stages from 6 h post infection. Short synthetic peptides derived from adhesive ectodomains in thrombospondins including a TRAP-C1 apple domain (TAAP), thrombospondin type I domains in CpTSP7 (7TS) and CpTSP9 (9TS), and a kringle domain in CpTSP10 (10K1) as well as their corresponding antibodies demonstrated competitive and neutralization inhibition effect of C. parvum infection of host cells. Polyclonal antibodies against TAAP caused sporozoites to agglutinate in a concentrationdependent manner, suggesting a contribution to reduced infectivity. In conclusion, the specificity and expression profiles of CpROM4 and CpROM5 indicate that they have distinct functions in shedding surface adhesins during excystation, motility, invasion, and intracellular development.
RNA Viral Prophylaxis: Problems and Potential Solutions
(North Dakota State University, 2019) Singh, Gagandeep
Over 80% of the newly emerging infectious diseases are caused by RNA viruses. Major global problems associated with the development of vaccines against the RNA virus are their high genetic and antigenic diversity. Hence, effective control of epidemics with newly emerging RNA viruses require improved vaccines which are either specific to the new strain or broadly effective even when new viral strains emerge. The main focus of this dissertation is to develop epidemic vaccines using these two approaches. Using a newly emerged swine enteric virus called porcine epidemic diarrhea virus (PEDV) as a model, our first goal was to develop a quick and easy method for rapid response vaccines with potential applicability to a range of RNA viruses. We hypothesized that the methods which can disrupt genomic RNA without impacting the structural integrity of the virus would result in attenuated vaccine with minimum replication in the host while inducing immune responses. As hypothesized, developed rapid response PEDV vaccine induced complete protection against the virulent challenge virus, while vaccine viral shedding was not detected in vaccinated pigs. To address the second problem of rapid viral evolution leading to vaccines becoming obsolete, we used swine influenza virus (SIV) as a model to develop and test a universal vaccine composed of peptides encoding conserved antigenic epitopes which are present in most influenza A viruses. Importantly, a novel amphiphilic invertible polymer (AIP) was used to address the well-recognized problem of poor antigenicity of peptides. We hypothesized that peptides encoding conserved epitopes when conjugated with an AIP will induce strong immune responses and protect against challenge virus. While the conserved epitopes were previously tested by others in mice, we were the first to test a combination of these epitopes in pigs. Pigs vaccinated with the peptide polymer vaccine mounted strong antibody responses against the epitopes indicating that the delivery system was effective. However, protection against replication of the challenge virus was delayed. In summary, the methods developed and tested in this body of work significantly contribute to the area of emergency response management in infectious disease outbreaks.
Transmission and Pathogenesis of Swine Torque-Teno Virus 1 (TTSuV1)
(North Dakota State University, 2019) Ssemadaali, Marvin Apollo
Torque-teno viruses (TTVs) are small ubiquitous non-enveloped single-stranded circular DNA viruses. Since their discovery in a post-transfusion hepatitis patient, they have been isolated in several vertebrate hosts with over 90% prevalence, including swine. They have been detected in the environment, water sources, human drugs, vaccine and blood product as contaminants. Intriguingly, the role of TTVs in human disease causation is still not fully understood. Several epidemiological studies have associated TTVs to human diseases, like cancers, hepatitis, and autoimmune diseases, but no clear link between infection and clinical disease has been demonstrated yet. In contrast, experimental studies done in pigs demonstrated that swine TTVs (TTSuVs) could an act as sole pathogens. Other studies also demonstrated that TTSuVs could exacerbate symptoms of other viral pathogens in coinfections. Here, we showed that TTSuV1 could be zoonotic, as we detected TTSuV1 DNA in human serum samples. We also showed that TTSuV1 could replicate in human immune cells, and consequently suppress their ability to respond to immune stimuli. Further in-vivo studies, to elucidate host immune regulation by TTSuVs, showed a delayed antibody response and minimal viremia. Also, we found that viral sensing could be limited to interferon-inducing sensors (DHX36), while upregulation of PD-1 could demonstrate how these viruses may establish chronic infections. In another study, we showed the use of our novel recombinant TTSuV1 culture system to study the synergistic interactions between TTSuV1 and porcine circovirus 1 (PCV1). When both viruses were cultured together in-vitro, their respective viral titers were increased, compared to the single virus infections. We also demonstrated that increased in-vitro replication of TTSuV1 could be relying on expression of PCV1 replicase. In addition, molecular mechanisms were used to explain this synergistic relationship; a strong promoter activity by the putative major promoter of TTSuV1 was shown to be blocked PCV1 and TTSuV1 replicase proteins, but protein-DNA interaction assays need further optimizations to demonstrate physical interaction between these viruses. In conclusion, our result showed new information about TTSuV1 transmission, pathogenesis, host innate immune regulation, and their role in coinfections.

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