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- All Subjects: Microbiology
- Creators: School of Life Sciences
Description
The rise in community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections and the ability of the organism to develop resistance to antibiotics necessitate new treatment methods for MRSA. Geopolymers (GPs) are cheap, porous materials that have demonstrated adsorptive capabilities. In this study, GPs were investigated for their ability to adsorb whole MRSA cells and MRSA secreted proteins [culture filtrate proteins (CFPs)] as a complementary method of controlling MRSA infections. GPs have been synthesized with variable pore sizes (meso/macro scale) and further modified with stearic acid (SA) to increase surface hydrophobicity. Four GPs (SA-macroGP, macroGP, SA-mesoGP, and mesoGP) were incubated with whole cells and with CFPs to quantify GP adsorption capabilities. Following MRSA culture incubation with GPs, unbound MRSA cells were filtered and plated to determine cell counts. Following CFP incubation with GPs, unbound CFPs were separated via SDS-PAGE, stained with SYPRO Ruby, and analyzed using densitometry. Results indicate that macroGP was the most effective at adsorbing whole MRSA cells. Visual banding patterns and densitometry quantitation indicate that SA-mesoGP was the most effective at adsorbing CFP. Ultimately, GP-based products may be further developed as nonselective or selective adsorbents and integrated into fibrous materials for topical applications.
ContributorsGanser, Collin (Co-author, Co-author) / Haydel, Shelley E. (Thesis director) / Seo, Don (Committee member) / Borges, Chad (Committee member) / School of Earth and Space Exploration (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Yellow-bellied marmots (Marmota flavivent) are semi-fossorial ground-dwelling sciurid rodents native to the western United States. They are facultatively social and live in colonies that may contain over 50 individuals. Marmot populations are well studied in terms of their diet, life cycle, distribution, and behavior, however, knowledge about viruses associated with marmots is very limited. In this study we aim to identify DNA viruses by non-invasive sampling of their feces. Viral DNA was extracted from fecal material of 35 individual marmots collected in Colorado and subsequently submitted to rolling circle amplification for circular molecule enrichment. Using a viral metagenomics approach which included high-throughput sequencing and verification of viral genomes using PCR, cloning and sequencing, a diverse group of single-stranded (ss) DNA viruses were identified. Diverse ssDNA viruses were identified that belong to two established families, Genomoviridae (n=7) and Anelloviridae (n=1) and several others that belong to unclassified circular replication associated encoding single-stranded (CRESS) DNA virus groups (n=19). There were also circular DNA molecules extracted (n=4) that appear to encode one viral-like gene and are composed of <1545 nt. The viruses that belonged to the family Genomoviridae clustered with those in the Gemycircularvirus genus. The genomoviruses were extracted from 6 samples. These clustered with gemycircularvirus extracted from arachnids and feces. The anellovirus, extracted from one sample, identified here has a genome sequence that is most similar to those from other rodent species, lagomorphs, and mosquitos. The CRESS viruses identified here were extracted from 9 samples and are novel and cluster with others identified from avian species. This study gives a snapshot of viruses associated with marmots based on fecal sampling.
ContributorsKhalifeh, Anthony (Author) / Varsani, Arvind (Thesis director) / Kraberger, Simona (Committee member) / Dolby, Greer (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Persons with cystic fibrosis (CF) are highly susceptible to lung infections caused by the opportunistic pathogens Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA). By age 20, ~16% of CF patients have co-infections with these two bacteria, and this number grows as the patients age1. PA-SA co-infections are associated with worsened clinical outcomes in CF patients, but the reasons are not well understood. One hypothesis is that SA influences the production of PA virulence factors and other chronic infection phenotypes. Previous work in our lab investigated the effects of SA on PA quorum-regulated phenotypes when they are grown as planktonic co-cultures. We are expanding on this result by testing whether SA can influence PA phenotypes without being in direct contact, and without being able to exchange soluble secreted factors. In this study, we hypothesized that SA produces volatile organic compounds (VOCs) that cause changes in PA phenotypes leading to a down-regulation of motility and protease production, and increased antibiotic resistance. To test this hypothesis, we exposed two laboratory strains of PA to the VOCs produced by pre-grown lawns of two strains of SA, and measured PA motility by conducting swarming, swimming, and twitching assays, measuring protease production, as well as antibiotic sensitivity. After exposing PA to a pre-grown lawn of SA, there was a significant difference in some phenotypes compared to controls. There were significant decreases in swarming motility, twitching motility, and protease production, and an increase in a bright green pigment (possibly siderophores) when PA was exposed to SA. The degree of phenotypic alterations was dependent on both the PA strain and the SA strain being tested. Exposure to SA VOCs also altered PA sensitivity to ciprofloxacin, though one strain caused an increase in susceptibility while the other SA strain caused an increase in resistance. These data demonstrate that SA VOCs can influence PA phenotypes in vitro, which may have relevance for CF patients who are co-infected with these two bacteria.
ContributorsLopez, Brianna Marie (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Spaceflight and spaceflight analogue culture enhance the virulence and pathogenesis-related stress resistance of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). This is an alarming finding as it suggests that astronauts may have an increased risk of infection during spaceflight. This risk is further exacerbated as multiple studies indicate that spaceflight negatively impacts aspects of the immune system. In order to ensure astronaut safety during long term missions, it is important to study the phenotypic effects of the microgravity environment on a range of medically important microbial pathogens that might be encountered by the crew. This ground-based study uses the NASA-engineered Rotating Wall Vessel (RWV) bioreactor as a spaceflight analogue culture system to grow bacteria under low fluid shear forces relative to those encountered in microgravity, and interestingly, in the intestinal tract during infection. The culture environment in the RWV is commonly referred to as low shear modeled microgravity (LSMMG). In this study, we characterized the stationary phase stress response of the enteric pathogen, Salmonella enterica serovar Enteritidis (S. Enteritidis), to LSMMG culture. We showed that LSMMG enhanced the resistance of stationary phase cultures of S. Enteritidis to acid and thermal stressors, which differed from the LSSMG stationary phase response of the closely related pathovar, S. Typhimurium. Interestingly, LSMMG increased the ability of both S. Enteritidis and S. Typhimurium to adhere to, invade into, and survive within an in vitro 3-D intestinal co-culture model containing immune cells. Our results indicate that LSMMG regulates pathogenesis-related characteristics of S. Enteritidis in ways that may present an increased health risk to astronauts during spaceflight missions.
ContributorsKoroli, Sara (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, C. Mark (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Following the journey through the sewerage system, wastewater is subject to a series of purification procedures, prior to water reuse and disposal of the resultant sewage sludge. Biosolids, also known as treated sewage sludge, deemed fit for application on land, is a nutrient-rich, semisolid byproduct of biological wastewater treatment. Technological progression in metagenomics has allowed for large-scale analysis of complex viral communities in a number of samples, including wastewater. Members of the Microviridae family are non-enveloped, ssDNA bacteriophages, and are known to infect enterobacteria. Members of the Genomoviridae family similarly are non-enveloped, ssDNA viruses, but are presumed to infect fungi rather than eubacteria. As these two families of viruses are not relatively documented and their diversity poorly classified, this study aimed to analyze the presence of genomoviruses and the diversity of microviruses in nine samples representative of wastewater in Arizona and other regions of the United States. Using a metagenomic approach, the nucleic acids of genomoviruses and microviruses were isolated, assembled into complete genomes, and characterized through visual analysis: a heat chart showing percent coverage for genomoviruses and a circular phylogenetic tree showing diversity of microviruses. The heat map results for the genomoviruses showed a large presence of 99 novel sequences in all nine wastewater samples. Additionally, the 535 novel microviruses displayed great diversity in the cladogram, both in terms of sub-family and isolation source. Further research should be conducted in order to classify the taxonomy of microviruses and the diversity of genomoviruses. Finally, this study suggests future exploration of the viral host, prior to entering the wastewater system.
ContributorsSchreck, Joshua Reuben (Author) / Varsani, Arvind (Thesis director) / Rolf, Halden (Committee member) / Misra, Rajeev (Committee member) / School of Film, Dance and Theatre (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with increased morbidity and mortality in cystic fibrosis (CF) patients. To adapt to the CF lung environment, P. aeruginosa undergoes multiple genetic changes as it moves from an acute to a chronic infection. The resultant phenotypes have been associated with chronic infection and can provide important information to track the patient’s individualized disease progression. This study examines the link between the accumulation of QS genetic mutations and phenotypic expression in P. aeruginosa laboratory strains and clinical isolates. We utilized several plate-based and colorimetric assays to quantify the production of pyocyanin, rhamnolipids, and protease from paired clinical early- and late-stage chronic infection isolates across 16 patients. Exoproduct production of each isolate was compared to the mean production of pooled isolates to classify high producing (QS-sufficient) and low producing (QS-deficient) isolates. We found that over time P. aeruginosa isolates exhibit a reduction in QS-related phenotypes during chronic infections. Future research of the QS regulatory networks will identify whether reversion of genotype will result in corresponding phenotypic changes in QS-deficient chronic infection isolates.
ContributorsKaranjia, Ava Vispi (Author) / Bean, Heather (Thesis director) / Haydel, Shelley (Committee member) / Davis, Trenton (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and high copy number in the protist genome. Recent studies have revealed the widespread occurrence of intragenomic (intra-individual) polymorphism in many protists, an understudied phenomenon which contradicts the assumed homogeneity of the 18S throughout an individual genome. This thesis quantifies and analyzes the level of intragenomic and intraspecific 18S sequence variability in three Trichonympha species (T. campanula, T. collaris, T. postcylindrica) from Zootermopsis termites. Single-cell DNA extractions, PCR, cloning, and sequencing were performed to obtain 18S rRNA sequence reads, which were then analyzed to determine levels of sequence divergence among individuals and among species. Intragenomic variability was encountered in all three species. However, excluding singleton mutations, sequence divergence was less than 1% in 53 of the 56 compared individuals. T. collaris exhibited the most substantial intragenomic variability, with sequence divergence ranging from 0 to 3.4%. Further studies with more clones per cell are needed to elucidate the true extent of intragenomic variability in Trichonympha.
ContributorsBobbett, Bradley (Author) / Gile, Gillian (Thesis director) / Liebig, Juergen (Committee member) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The changes in marine ecological conditions brought on by warming and stratification of the oceans have radically shifted many marine environments around the globe. This project aimed to better characterize the aggregation behavior of the abundant picocyanobacterium Prochlorococcus marinus, which is hypothesized to dominate over other phytoplankton as the primary autotroph in increasingly warmer and nutrient poor oceans. This aggregation, believed to be mediated through the secretion of sticky Transparent Exopolymeric Substances (TEP), might be key for Prochlorococcus to sink throughout the ocean and serve as a source of carbon to other communities within its environment. Considering the relatively low concentration of TEP secreted by Prochlorococcus when on its own, this study explored the synergistic effect that heterotrophic bacteria and inorganic minerals in the surrounding seawater may have on the aggregation of P. marinus. This was done by inoculating P. marinus and the model heterotroph Marinobacter adhaerens HP15 individually and mixed in cylindrical roller tanks with the addition of ballasting clay minerals into roller tanks to simulate constant sinking for 7 days. The aggregates which formed after rolling were quantified and their sinking velocities and excess densities were measured. Our results indicate that the most numerous and densest aggregates formed when Prochlorococcus was in the presence of both M. adhaerens and kaolinite clay particles. I will discuss how methodology, particularly cell number, may play a role in the enhanced aggregation that I found when Prochlorococcus was cultured together with the Marinobacter.
ContributorsAouad, Samer Ghassan (Author) / Neuer, Susanne (Thesis director) / Cadillo-Quiroz, Hinsby (Committee member) / Cruz, Bianca (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most common form of treatment is chemotherapy followed by radiation, which is insufficient to cure stage four cancers[3]. Salmonella enteric has long been shown to have inherent tumor targeting properties and have been able to penetrate and exist in all aspects of the tumor environment, something that chemotherapy is unable to achieve. This lab has developed a genetically modified Salmonella typhimurium (GMS) which is able to deliver DNA vaccines or synthesized proteins directly to tumor sites. These GMS strains have been used to deliver human TNF-related apoptosis inducing ligand (TRAIL) protein directly to tumor sites, but expression level was limited. It is the hope of the experiment that codon optimization of TRAIL to S. typhimurium preferred codons will lead to increased TRAIL expression in the GMS. For preliminary studies, BALB/c mice were subcutaneously challenged with CT-26 murine colorectal cancer cells and treated with an intra-tumor injection with either PBS, strain GMS + PCMV FasL (P2), or strain GMS + Pmus FasL). APC/CDX2 mutant mice were also induced to develop human colon polyps and treated with either PBS, strain GMS + vector (P1), P2, or P3. The BALB/c mouse showed statistically significant levels of decreased tumor size in groups treated with P2 or P3. The APC/CDX2 mouse study showed statistically significant levels of decreased colon polyp numbers in groups treated with P3, as expected, but was not significantly significant for groups treated with P1 and P2. In addition, TRAIL was codon optimized for robust synthesis in Salmonella. The construct will be characterized and evaluated in vitro and in vivo. Hopefully, the therapeutic effect of codon optimized TRAIL will be maximal while almost completely minimizing any unintended side effects.
ContributorsCrawford, Courtney Rose (Co-author) / Crawford, Courtney (Co-author) / Kong, Wei (Thesis director) / Shi, Yixin (Committee member) / Fu, Lingchen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to be produced. The HIV-1 envelope glycoprotein41 and the Gag structural protein have been identified to be particularly important in HIV-1 transcytosis and cytotoxic lymphocyte response, respectively. Enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of glycoprotein (dgp41) comprising the membrane proximal external region (MPER), transmembrane domain and cytoplasmic tail may present a unique and safe way of presenting these proteins in a state mimicking their natural formation. Another form of presenting the immunogenic glycoprotein41, particularly the MPER component, is by presenting it onto the N-terminal of an IgG molecule, thereby creating an IgG fusion molecule. In our lab, both VLPs and IgG fusion molecules are highly expressed and purified within GnGn Nicotiana benthamiana. The results indicated that these recombinant proteins can be assembled properly within plants and can elicit an immune response in mice. This provides a preliminary step in using such Gag/dpg41 VLPs and RIC as present a safe, effective, and inexpensive HIV vaccine.
ContributorsGarcia, Izamar (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05