Theses and Dissertations at Montana State University (MSU)
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Item NMR hydrophilic metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium antimicrobials in Escherichia coli and Bacillus cereus exposed to DABCO and mannose functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2021) Aries, Michelle Lynne; Chairperson, Graduate Committee: Mary J. Cloninger; This is a manuscript style paper that includes co-authored chapters.Novel antibiotics developed using a new scaffold are needed to combat the rising tide of antibiotic resistant bacteria. Multivalent antibiotics are a relatively new approach that have the potential to greatly increase the efficacy of antibiotics while making it difficult for bacteria to develop resistance. Dendrimers are an attractive framework for the multivalent presentation of antibacterial moieties. Quaternary ammonium compounds (QAC) are a positively charged class of membrane disruptors that are attracted to the large negative charge on phospholipid membranes. Nuclear magnetic resonance (NMR) metabolomics is a quantitative method used for comparison of metabolic profiles of wild type and mutated bacterial samples, enabling the study of bacterial response to antimicrobials. Proton (1 H) NMR hydrophilic metabolomics was used to study gram-negative and gram-positive bacteria upon exposure to 1,4-diazabicyclo-2,2,2-octane (DABCO) with a 16-carbon chain tethered onto a mannose functionalized poly(amidoamine) (PAMAM) dendrimer (denoted as DABCOMD), a membrane disrupting multivalent QAC. Stock Escherichia coli (E. coli) (denoted as wild type) and DABCOMD mutated E. coli (denoted as mutants) were collected in the mid log and stationary phases. The same procedures were used for Bacillus cereus (B. cereus) as for E. coli samples (denoted as unchallenged), except that a DABCOMD challenged sample set was added (denoted as challenged). The challenged sample set procedures were identical to the unchallenged, except DABCOMD was included at 33 % of the MIC value in the growth media for growth curve acquisition and sample collection. The greatest differences observed between the metabolic profiles of the wild type and mutated E. coli samples and between the challenged and unchallenged B. cereus samples were in energy-associated metabolites and membrane-related pathways. The mutants in all sample types were associated with higher levels of spent energy molecules (including AMP and NAD+) and peptidoglycan related compounds (including N-acetylglucosamine). Overall, more changes were observed for B. cereus (gram-positive), especially in challenged mutant B. cereus samples, than for E. coli (gram-negative) samples. Since DABCOMD is a positively charged multivalent membrane disruptor, both B. cereus and E. coli mutated to garner protection by altering their peptidoglycan layer composition, which is energetically costly.Item Experimental infection of specific pathogen-free domestic lambs with Mycoplasma ovipneumoniae(Montana State University - Bozeman, College of Agriculture, 2021) Johnson, Thea Haviland; Chairperson, Graduate Committee: Diane Bimczok; Diane Bimczok, Kerri Jones, Cassie Mosdal, Steven Jones, SK, CB, AS, and B. Tegner Jacobson were co-authors of the article, 'Immunoglobulin transfer, survival, and growth in motherless lambs fed a bovine serum-based colostrum replacer' which is contained within this thesis.; Kerri Jones, B. Tegner Jacobson, Julia Schearer, Noah Adams, Isaak Thornton, Cassie Mosdal, Steven Jones, Mark Jutila, Agnieszka Rynda-Apple, Thomas Besser and Diane Bimczok were co-authors of the article, 'Experimental infection of specific-pathogen-free domestic lambs with Mycoplasma ovipneumoniae causes asymptomatic colonization of the upper airways that is resistant to antibiotic treatment' in the journal 'Veterinary microbiology' which is contained within this thesis.Mycoplasma ovipneumoniae (M. ovi) is a respiratory pathogen commonly found in sheep and goats. It is associated with mild to moderate respiratory disease in domestic lambs, but severe pneumonia outbreaks in wild ruminants, specifically bighorn sheep. The goal of our study was to better understand the role of M. ovi as a respiratory pathogen in domestic sheep and to explore potential antibiotic treatment approaches. We first established a flock of specific pathogen-free (SPF) lambs through supervised lambing and motherless rearing in a Large Animal BSL-2 facility. Lambs were fed a colostrum replacer that yielded low mortality, steady weight gain and serum IgG and protein concentrations comparable to those of lambs raised on ewe colostrum. We inoculated the SPF lambs with field isolates of M. ovi and monitored the lambs for eight weeks for colonization with the bacteria, M. ovi-specific antibodies, clinical symptoms, and cellular and molecular correlates of lung inflammation. After eight weeks, lambs were treated with the macrolide antibiotic gamithromycin and observed for an additional four weeks. Stable colonization of the upper respiratory tract with M. ovi was established in all four M. ovi-inoculated, but in none of the four mock-infected lambs. All M. ovi-infected lambs developed a robust antibody response to M. ovi within 2 weeks. However, we did not observe significant clinical symptoms, evidence of lung damage or inflammation in any of the infected lambs. Interestingly, treatment with gamithromycin failed to reduce M. ovi colonization. These observations indicate that, in the absence of co-factors, M. ovi causes asymptomatic colonization of the upper respiratory tract of that is resistant to clearance by the host immune response as well as by gamithromycin treatment in domestic lambs.Item Outcomes of a quality improvement project: integrating sepsis bundles in the rural emergency department(Montana State University - Bozeman, College of Nursing, 2019) Popp, Kierston Christian; Chairperson, Graduate Committee: Casey ColeBACKGROUND: Rural hospitals have a poor adherence to the Surviving Sepsis Campaign guidelines, which includes door-to-antibiotic administration times under 60 minutes leading to a higher risk of mortality (Mohr et al., 2018). The aim of this project was to improve door-to-antibiotic times through the implementation of a sepsis bundle, which would place all necessary orders together. The project was set in a rural emergency department in southwestern Montana. Participants included provider staff at the facility including family nurse practitioners, physician assistants, and medical doctors. METHODS: The FADE (focus, analyze, develop, execute, and evaluate) method of quality improvement was used for this project. Baseline assessment included a review of patient medical records who met sepsis criteria from January-June 2017. Antibiotic administration times were reviewed using data collection from the patient charts. A literature review was conducted to identify appropriate sepsis bundle implementation interventions. INTERVENTIONS: Sepsis bundles were introduced to the provider staff through education and meetings to aid in identifying the need for sepsis bundles in the emergency department. Baseline times were also presented to the staff to provide evidence that the current practices were not meeting goals. A sepsis bundle was chosen by the medical director and the Doctor of Nursing Practice (DNP) student that fit best with the resources available in the emergency department. RESULTS: Three months after the implementation of sepsis bundles, a chart review was performed on all patients that met sepsis criteria. Again, door-to-antibiotic administration times was reviewed. Door-to-antibiotic administration times improved by 40.5 minutes, which is a 22 percent improvement. CONCLUSION: The use of sepsis bundles in the care of the septic patient improved door-to-antibiotic administration times. Although improvement in the quality improvement measures was noted, additional work is needed to achieve Surviving Sepsis Campaign's goal of door-to-antibiotic times of under 60 minutes.Item Characterization of the stability of Pseudomonas aeruginosa ribosomal proteins under stress conditions(Montana State University - Bozeman, College of Letters & Science, 2017) Yanardag, Sila; Chairperson, Graduate Committee: Michael FranklinIn this study, I aimed to standardize western blot methods for probing large and small ribosomal subunits of Pseudomonas aeruginsa grown under different environmetal conditions, and to characterize the stability of ribosomal proteins to bring light to the heterogeneous composition of the population, which is hypothesized as one mechanism for antibiotic tolerance. Long-term studies done with P.aeruginosa PAO1 showed that mRNA transcripts of two proteins, RMF and HPF, are highly abundant at the biofilm-nutrient interface of the thick P.aeruginosa biofilms. Also, it was previously shown by Perez et al. and Williamson et al. (Pérez-Osorio et al., 2010; Williamson et al., 2012) that the cells located at the oxygen limited interphase of the biofilm were metabolically inactive or slow-growing. Akiyama et al. (Akiyama et al., 2017) and Williamson et al. (Williamson et al., 2012) found that HPF is a critical protein for the maintenance of 23S rRNA and overall ribosomal RNA stability after prolonged stress exposure (Akiyama et al., 2017; Williamson et al., 2012). In light of this information, Akiyama et al. (Akiyama et al., 2017) showed that in the absence of the HPF protein, P.aeruginosa cannot protect its ribosome integrity and cannot resuscitate from dormancy after the environmental stressors are gone. Perez et al. (Pérez-Osorio et al., 2010) showed that P.aeruginosa biofilms are heterogeneous in physiology, and it is posited that persister cells of the biofilm are located at the bottom of the biofilm, unaffected by the antibiotic exposure and therefore can repopulate the biofilm (Williamson et al., 2012). Localization of ribosomal subunits and determination of the abundance of ribosomes within the heterogeneous biofilms will provide valuable insights on the mechanisms of persister cell formation, dormancy, and resusication from dormancy. In order to do so, I have isolated two ribosomal proteins, L5 and S13, and HPF. In this study, I generated polyclonal antibodies against those three proteins. I used the antibodies to determine the abundance of these proteins during the normal course of growth of the wild type and Deltahpf mutant strains. Growth analysis in nutrient rich media gave us an understanding of the stability of 70S ribosomes when the bacterium was growing without any stress. Later, the wild type and Deltahpf strain were grown in a carbon and nitrogen-limited environment for seven days to examine the response of the cells to the starvation stress regarding ribosomal stability. Finally, I tested the hypothesis that cells located at the bottom of the biofilm are abundant in the HPF protein, and therefore contain more inactive ribosomes compared to the cells located at the top of biofilm.Item Antibiotic use and acquired bacterial resistance(Montana State University - Bozeman, College of Nursing, 1996) Hollis, Brett Roy; Chairperson, Graduate Committee: Mary K. Dempsey-NoreikaItem The effect of Aureomycin in pelleted or whole grain creep rations fed to suckling lambs : the effect of Thyroprotein, Thiouracil, and Stilbestrol on gains of fattening lambs(Montana State University - Bozeman, College of Agriculture, 1956) Daley, Charles A.Item The effects of ruminally undegradable protein, propionic acid and monensin on puberty and reproductive efficiency in beef heifers(Montana State University - Bozeman, College of Agriculture, 1991) Lalman, David LeonItem Antibiotic penetration through Pseudomonas aeruginosa colony biofilms(Montana State University - Bozeman, College of Engineering, 2001) Walters, Marshall Charles, IIIItem In vitro and in vivo studies on transferable drug resistance in the Enterobacteriaceae(Montana State University - Bozeman, College of Agriculture, 1970) Aden, David PaulItem Monensin effects on digestion of corn or barley high concentrate diets(Montana State University - Bozeman, College of Agriculture, 1995) Surber, Lisa Marie McKinley