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.
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.
Gene Expression and Evolution in Escherichia Coli Biofilm
(North Dakota State University, 2014) Samanta, Priyankar
Biofilms can be defined as a complex aggregation of bacterial communities that involves many gene regulatory mechanisms, as well as evolutionary processes to increase biodiversity. Specific Aim 1 used a gene regulation approach to identity novel targets for the development of biofilm prevention and treatment techniques. The goal was to determine genes that get expressed early in biofilm development (prevention targets) and genes that get expressed late and in the outer layer of the biofilm (treatment targets). Biofilm formation is regulated by numerous regulators, including the two-component osmoregulator system EnvZ/OmpR, the colanic acid activator rcsCDB and the global regulator FlhD/FlhC. In this study, we determined the temporal and spatial expression of flhD, ompR and rcsB in E. coli k-12 AJW678 biofilm, as well as the gene expression of flhD in isogenic ompR and rcsB mutants. Results indicated that flhD was expressed early, and in the outer layer of the mature biofilm. We concluded that FlhD/FlhC would be the first target for novel prevention and treatment technique. One mechanism to increase biodiversity in biofilm is the insertion of transposon elements, which was investigated as Specific Aim 2. Insertion of IS elements into the flhD promoter resulted in increased motility in numerous E. coli K-12 strains has been shown in previous study. In this study, we recovered isolates from biofilm, where IS1 had inserted in the flhD promoter further downstream than in previously described strains. These isolates showed reduced motility. We also wanted to determine the effect of an IS element insertion on regulation of flhD expression by OmpR and RcsB in biofilm. Temporal and spatial gene expression of three different GFP-tagged flhD promoters was measured. The results indicated that IS5 insertion in the flhD promoter at the published hotspot did not have any effect on regulation of flhD expression by OmpR and RcsB in biofilm.
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.
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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