Matching Items (12)
Filtering by
- All Subjects: Microbiology
- Creators: Haydel, Shelley
Description
The prevalence of antibiotic resistant bacterial pathogens has increased since the introduction of penicillin in the 1940s. Insufficient development of novel antibacterial agents is leaving us with a failing arsenal of therapies to combat these pathogenic organisms. We have identified a clay mineral mixture (designated CB) that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens, yet the antibacterial mechanism of action remains unknown. Antibacterial susceptibility testing of four different clay samples collected from the same source revealed that these natural clays had markedly different antibacterial activity. X-ray diffraction analyses of these minerals revealed minor mineralogical differences across the samples; however, ICP analyses demonstrated that the concentrations of many elements, Fe, Co, Cu, Ni, and Zn in particular, vary greatly across the four clay mixture leachates. Supplementation of a non-antibacterial leachate containing lower concentrations of Fe, Co, Ni, Cu, and Zn to final ion concentrations and a pH equivalent to that of the antibacterial leachate resulted in antibacterial activity against E. coli and MRSA, confirming the role of these ions in the in vitro antibacterial clay mixture leachates. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to CB-L. Supplementation of CB-L with ROS scavengers eliminated oxidative damage in E. coli, but did not rescue the cells from killing, indicating that in vitro killing is due to direct metal toxicity and not to indirect oxidative damage. Finally, we ion-exchanged non-antibacterial clays with Fe, Co, Cu, and Zn and established antibacterial activity in these samples. Treatment of MRSA skin infections with both natural and ion-exchanged clays significantly decreased the bacterial load after 7 days of treatment. We conclude that 1) in vitro clay-mediated killing is due to toxicity associated directly with released metal ions and not to indirect oxidative damage and 2) that in vivo killing is due to the physical properties of the clays rather than metal ion toxicity.
ContributorsOtto, Caitin Carol (Author) / Haydel, Shelley (Thesis advisor) / Stout, Valerie (Committee member) / Roberson, Robby (Committee member) / Sandrin, Todd (Committee member) / Rege, Kaushal (Committee member) / Arizona State University (Publisher)
Created2014
Description
Cystic Fibrosis (CF) is a genetic disorder that disrupts the hydration of mucous of the lungs, which promotes opportunistic bacterial infections that begin in the affected person’s childhood, and persist into adulthood. One of the bacteria that infect the CF lung is Pseudomonas aeruginosa. This gram-negative bacterium is acquired from the environment of the CF lung, changing the expression of phenotypes over the course of the infection. As P. aeruginosa infections become chronic, some phenotype changes are known to be linked with negative patient outcomes. An important exoproduct phenotype is rhamnolipid production, which is a glycolipid that P. aeruginosa produces as a surfactant for surface-mediated travel. Over time, the expression of this phenotype decreases in expression in the CF lung.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.
ContributorsKiermayr, Jonathan Patrick (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Haydel, Shelley (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The prrAB two-component system has been shown to be essential for viability in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To study this system, several prrAB mutants of Mycobacterium smegmatis, a close relative of Mtb, were created for study. These mutants included a deletion mutant complemented with prrA from Mtb controlled by Pmyc1_tetO, a deletion mutant, and a deletion mutant complemented with prrAB from M. smegmatis controlled by the native prrAB promoter sequence (~167 bp upstream sequence of prrAB). In a previous study, the prrAB deletion mutant clumped excessively relative to the wild-type strain when cultured in a nitrogen-limited medium. To address this irregularity, the lipid profiles of these mutants were analyzed through several experimental methods. Untargeted lipidomic profiles were analyzed by Electrospray Ionization Mass Spectrometry (ESI-MS). The ESI-MS data suggested the deletion mutant accumulates triacylglycerol species relative to the wild-type strain. This data was verified by thin-layer chromatography (TLC) and densitometry of the TLC images. The mycolic acid profile of each mutant was also analyzed by TLC but no noteworthy differences were found. High-throughput RNA-Seq analysis revealed several genes involved in lipid biosynthetic pathways upregulated in the prrAB deletion mutant, thus corroborating the ESI-MS and TLC data.
ContributorsOlson, Alexandra Nadine (Author) / Haydel, Shelley (Thesis director) / Bean, Heather (Committee member) / Maarsingh, Jason (Committee member) / School of Social Transformation (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Antibiotic resistant bacteria are a worldwide epidemic threatening human survival. Antimicrobial susceptibility tests (ASTs) are important for confirming susceptibility to empirical antibiotics and detecting resistance in bacterial isolates. Current ASTs are based on bacterial culturing, which take 2-14 days to complete depending on the microbial growth rate. Considering the high mortality and morbidity rates for most acute infections, such long time frames are clinically impractical and pose a huge risk to a patient's life. A faster AST will reduce morbidity and mortality rates, as well as help healthcare providers, administer narrow spectrum antibiotics at the earliest possible treatment stage.
In this dissertation, I developed a nonculture-based AST using an imaging and cell tracking technology. I track individual Escherichia coli O157:H7 (E. coli O157:H7) Uropathogenic Escherichia Coli (UPEC) cells, widely implicated in food-poisoning outbreaks and urinary tract infections respectively. Cells tethered to a surface are tracked on the nanometer scale, and phenotypic motion is correlated with bacterial metabolism. Antibiotic action significantly slows down motion of tethered bacterial cells, which is used to perform antibiotic susceptibility testing. Using this technology, the clinical minimum bactericidal concentration of an antibiotic against UPEC pathogens was calculated within 2 hours directly in urine samples as compared to 3 days using current gold standard tools.
Such technologies can make a tremendous impact to improve the efficacy and efficiency of infectious disease treatment. This has the potential to reduce the antibiotic mis-prescription steeply, which can drastically decrease the annual 2M+ hospitalizations and 23,000+ deaths caused due to antibiotic resistance bacteria along with saving billions of dollars to payers, patients, and hospitals.
In this dissertation, I developed a nonculture-based AST using an imaging and cell tracking technology. I track individual Escherichia coli O157:H7 (E. coli O157:H7) Uropathogenic Escherichia Coli (UPEC) cells, widely implicated in food-poisoning outbreaks and urinary tract infections respectively. Cells tethered to a surface are tracked on the nanometer scale, and phenotypic motion is correlated with bacterial metabolism. Antibiotic action significantly slows down motion of tethered bacterial cells, which is used to perform antibiotic susceptibility testing. Using this technology, the clinical minimum bactericidal concentration of an antibiotic against UPEC pathogens was calculated within 2 hours directly in urine samples as compared to 3 days using current gold standard tools.
Such technologies can make a tremendous impact to improve the efficacy and efficiency of infectious disease treatment. This has the potential to reduce the antibiotic mis-prescription steeply, which can drastically decrease the annual 2M+ hospitalizations and 23,000+ deaths caused due to antibiotic resistance bacteria along with saving billions of dollars to payers, patients, and hospitals.
ContributorsSyal, Karan (Author) / Tao, Nongjian (Thesis advisor) / Haydel, Shelley (Committee member) / Rege, Kaushal (Committee member) / Wang, Shaopeng (Committee member) / Haynes, Karmella (Committee member) / Arizona State University (Publisher)
Created2017
Description
This study examines the effect of the translation of traditional scientific vocabulary into plain English, a process referred to as Anglicization, on student learning in the context of introductory microbiology instruction. Data from Anglicized and Classical-vocabulary lab sections were collected. Data included exam scores as well as pre and post-course surveys on reasoning skills, impressions of biology, science and the course, and microbiology knowledge. Students subjected to Anglicized instruction performed significantly better on exams that assessed their abilities to apply and analyze knowledge from the course, and gained similar amounts of knowledge during the course when compared to peers instructed with standard vocabulary. Their performance in upper-level courses was also better than that of their traditionally educated peers. Hypotheses related to the effect are presented and evaluated; implications for instruction are discussed.
ContributorsRichter, Emily (Author) / Lawson, Anton (Thesis advisor) / Stout, Valerie (Committee member) / Haydel, Shelley (Committee member) / Atkinson, Robert (Committee member) / Arizona State University (Publisher)
Created2011
Description
Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. In fact, there is no currently published assessment of phage abundance on diversity in a key tropical ecosystem such as Amazon peatlands. To better understand phage assemblages in terrestrial ecosystems and how bacteriophages influence organic C cycling to final products like CO2 and CH4, phage communities and phage-like particles were recovered, quantified, and viable phage particles were enriched from pore water from contrasting Amazon peatlands. Here we present the first results on assessing Amazon bacteriophages on native heterotrophic bacteria. Several steps to test for methodological suitability were taken. First, the efficiency of iron flocculation method was determined using fluorescent microscopy counts of phage TLS, a TolC-specific and LPS-specific bacteriophage, and Escherichia coli host pre- and post-extraction method. One-hundred percent efficiency and 0.15% infectivity was evidenced. Infectivity effects were determined by calculating plaque forming units pre and post extraction method. After testing these methods, fieldwork in the Amazon peatlands ensued, where phages were enriched from pore water samples. Phages were extracted and concentrated by in tandem filtering rounds to remove organic matter and bacteria, and then iron flocculation to bind the phages and allow for precipitation onto a filter. Phage concentrates were then used for overall counts, with fluorescent microscopy, as well as phage isolation attempts. Phage isolations were performed by first testing for lysis of host cells in liquid media using OD600 absorbance of cultures with and without phage concentrate as well as attempts with the cross-streaking methods. Forty-five heterotrophic bacterial isolates obtained from the same Amazon peatland were challenged with phage concentrates. Once a putative host was found, steps were taken to further propagate and isolate the phage. Several putative phages were enriched from Amazon peatland pore water and require further characterization. TEM imaging was taken of two phages isolated from two plaques. Genomes of selected phages will be sequenced for identification. These results provide the groundwork for further characterizing the role bacteriophage play in C cycling and greenhouse gas production from Amazon peatland soils.
ContributorsSpring, Jessica Lynette (Author) / Cadillo-Quiroz, Hinsby (Thesis director) / Haydel, Shelley (Committee member) / Misra, Rajeev (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of removing a broad range of antibiotics from the periplasmic space and the outer leaflet of the inner membrane, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, I further characterize the channel protein TolC as a part of the AcrAB-TolC efflux pump complex in Escherichia coli by first determining the specificity of compensatory mutations in TolC against defective AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants, previously generated from defective AcrA or AcrB, against a different mutant AcrB protein; these new mutant combinations were then analyzed by real-time efflux and antibiotic susceptibility assays. A vancomycin susceptible TolC mutant—a phenotype that has been associated with constitutively open TolC channels—was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification by Western blots, and real-time efflux assays to identify residues important for TolC aperture dynamics and protein stability and complex activity. Mutations identified in revertant strains corresponded to residues located in the lower half of the periplasmic domain of TolC; generally, these revertants had poorer efflux than wild-type TolC in the mutant AcrB background, and all revertants had poorer efflux activity than the parental mutant strain.
ContributorsMcFeely, Megan Elizabeth (Author) / Misra, Rajeev (Thesis director) / Haydel, Shelley (Committee member) / Stout, Valerie (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
This project begins with an overview of the female reproductive tract microenvironment. It outlines the microenvironment of the vaginal, cervical, and endometrial epithelium and the interactions with immune cells and hormone cycles. The review also outlines the models currently used to study the female reproductive tract. The second chapter of the thesis is a study of the effects of pathogenic and commensal bacteria P. micra, F. magna, and F. nucleatum on cervical epithelial cells. This study analyzes cytotoxic effects after 24 hour infection of these bacteria. This was assessed through crystal violet staining, conventional pcr of cDNA synthesized from extracted cervical RNA, and LDH analysis. There is also an attempted biofilm assay. It was concluded that bacteria P. micra, F. magna and F. nucleatum have cytotoxic potential. This was not expected as F. magna is largely understood to be a commensal bacteria in the vaginal microbiome.
ContributorsGarza, Camryn Nicole (Author) / Plaisier, Christopher (Thesis director) / Herbst-Kralovetz, Melissa (Committee member) / School of Molecular Sciences (Contributor) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The need for new tuberculocidal drugs is crucial with drug resistance on the rise as the tuberculosis epidemic rages on. One new potential drug target is the PrrAB two component system (TCS) since it does not exist in humans and is essential to viability in Mycobacterium tuberculosis. This project examines Mycobacterium smegmatis, and this nonpathogenic and fast-growing organism possesses two full length PrrAB orthologs, in addition to an orphaned PrrB sensor histidine kinase. While it was determined that PrrAB1 and PrrAB2 are nonessential, the lone PrrB3 is not yet characterized for essentiality. To confirm individual dispensability of PrrAB1 and PrrAB2 and investigate the essentiality of PrrB3 and the full M. smegmatis PrrAB multiplex, we utilized CRISPRi dCas9 to repress the expression (knockdown) of prrAB1 (MSMEG_5662-5663), prrAB2 (MSMEG_0244-0246), and the lone prrB3 (MSMEG_2793) in M. smegmatis independently and simultaneously. Repression of prrAB1 resulted in the greatest growth defect, with a lag of 17 cellular division cycles compared to the control, a strain generated with an empty vector. However, the knockdown of prrAB1 was not lethal to M. smegmatis. The inhibition of all three prrAB orthologs simultaneously, also known as a multiplex knockdown, lagged the control by 13 cellular division cycles. At the 48-hour point, both the single ortholog repression of prrAB1 as well as the whole prrAB system knockdown had a growth defect of 13 replication cycles behind the control. However, the multiplex knockdown stabilized growth at 48 hours, revealing a possible compensatory mechanism in M. smegmatis. Conclusively, we show that the PrrAB TCS is globally inessential for viability in M. smegmatis.
ContributorsHeiligenstein, Piper (Author) / Haydel, Shelley (Thesis director) / Shrivastava, Abhishek (Committee member) / Haller, Yannik (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Mathematical and Natural Sciences (Contributor)
Created2023-12
Description
Ecology has been an actively studied topic recently, along with the rapid development of human microbiota-based technology. Scientists have made remarkable progress using bioinformatics tools to identify species and analyze composition. However, a thorough understanding of interspecies interactions of microbial ecosystems is still lacking, which has been a significant obstacle in the further development of related technologies. In this work, a genetic circuit design principle with synthetic biology approaches is developed to form two-strain microbial consortia with different inter-strain interactions. The microbial systems are well-defined and inducible. Co-culture experiment results show that our microbial consortia behave consistently with previous ecological knowledge and thus serves as excellent model systems to simulate ecosystems with similar interactions. Colony patterns also emerge when co-culturing multiple species on solid media. With the engineered microbial consortia, image-processing based methods were developed to quantify the shape of co-culture colonies and distinguish microbial consortia with different interactions. Factors that affect the population ratios were identified through induction and variations in the inoculation process. Further time-lapse experiments revealed the basic rules of colony growth, composition variation, patterning, and how spatial factors impact the co-culture colony.
ContributorsChen, Xingwen (Author) / Wang, Xiao (Thesis advisor) / Kuang, Yang (Committee member) / Tian, Xiaojun (Committee member) / Brafman, David (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2022