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- Creators: Barrett, The Honors College
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Description
Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that is the leading cause of chronic infection in the lungs of adults with cystic fibrosis (CF). During chronic lung infections, P. aeruginosa populations adapt genetically to the CF lung, selecting several important mutations required for long-term persistence. These genetic adaptations lead to phenotypic changes that are associated with the transition from early-stage to late-stage chronic CF infection.
The goal of this project was to develop tools for gene transfer between P. aeruginosa clinical isolates. These tools will allow shuffling of early/late stage of infection genes to restore wild-type phenotypes in late chronic infection isolates and create single-phenotype mutants in the early infection strains. This will allow isolation and investigation of single phenotypes in the clinical isolates to identify metabolic biomarkers specifically for detecting the target phenotypes.
The gene transfer mechanisms of transformation by electroporation, transformation by heat shock, and conjugation were tested using the plasmid pMQ30 with a construct to create an in-frame deletion of the rhlR gene (rhlR) via allelic exchange. The disruption of the P. aeruginosa wild-type rhlR gene leads to rhamnolipids-deficient mutant strains; therefore, rhamnolipids production was assessed to validate successful in-frame deletion of the rhlR gene in the P. aeruginosa clinical isolates and laboratory strains. Based on the efficiencies determined from the gene transfer mechanisms tested, the conjugation mechanism was determined to be the most efficient method for gene transfer in P. aeruginosa laboratory strains, and was used to investigate gene transfer in the P. aeruginosa clinical isolates.
The goal of this project was to develop tools for gene transfer between P. aeruginosa clinical isolates. These tools will allow shuffling of early/late stage of infection genes to restore wild-type phenotypes in late chronic infection isolates and create single-phenotype mutants in the early infection strains. This will allow isolation and investigation of single phenotypes in the clinical isolates to identify metabolic biomarkers specifically for detecting the target phenotypes.
The gene transfer mechanisms of transformation by electroporation, transformation by heat shock, and conjugation were tested using the plasmid pMQ30 with a construct to create an in-frame deletion of the rhlR gene (rhlR) via allelic exchange. The disruption of the P. aeruginosa wild-type rhlR gene leads to rhamnolipids-deficient mutant strains; therefore, rhamnolipids production was assessed to validate successful in-frame deletion of the rhlR gene in the P. aeruginosa clinical isolates and laboratory strains. Based on the efficiencies determined from the gene transfer mechanisms tested, the conjugation mechanism was determined to be the most efficient method for gene transfer in P. aeruginosa laboratory strains, and was used to investigate gene transfer in the P. aeruginosa clinical isolates.
ContributorsBhebhe, Charity Ntando (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Jenkins, Carrie (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Abstract:
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
ContributorsElwell, Zachary Andrew (Author) / Blattman, Joseph (Thesis director) / Bean, Heather (Committee member) / Lal, Devyani (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Pseudomonas aeruginosa and Staphylococcus aureus are two key opportunistic pathogens that cause chronic lung infections in cystic fibrosis (CF) patients. Polymicrobial infections with P. aeruginosa and S. aureus are associated with worsened clinical outcomes in CF patients, and unknown still are the mechanisms that cause an increase in patient morbidity and mortality. Studying the interactions between P. aeruginosa and S. aureus is difficult because when co-cultured in vitro, P. aeruginosa drastically outcompetes and eradicates S. aureus cultures. This study explores methods for growing planktonic co-cultures of P. aeruginosa and S. aureus to stationary phase in equal proportions, and this will allow for the examination of changes in quorum-regulated phenotypes.
We grew liquid co-cultures of P. aeruginosa and S. aureus in LB Lennox media and examined their absolute and relative cell densities by plating the co-cultures on selective media. We evaluated the influence of oxygen concentration and co-inoculation vs. staggered inoculation on the ability to achieve a co-cultures with two P. aeruginosa (PA) and two S. aureus (SA) strains. The method that consistently produced PA:SA ratios in the range of 1:1 to 1:100 was to allow a SA mono-culture to reach stationary phase, and then re-suspend the SA cells in fresh media before inoculating with PA. With this method, it is possible to grow both PA and SA to stationary phase, a necessity for studying how PA and SA alter phenotypes in the presence of one another.
P. aeruginosa was found to produce less pyocyanin in the presence of S. aureus, but reduction in pyocyanin expression was depended on the strain of S. aureus. Elastase production differed between the two P. aeruginosa strains as well as between the two S. aureus strains, one increasing and one decreasing in expression. This data indicates that the responses of P. aeruginosa to S. aureus differ depending on both the P. aeruginosa and S. aureus strain present.
We grew liquid co-cultures of P. aeruginosa and S. aureus in LB Lennox media and examined their absolute and relative cell densities by plating the co-cultures on selective media. We evaluated the influence of oxygen concentration and co-inoculation vs. staggered inoculation on the ability to achieve a co-cultures with two P. aeruginosa (PA) and two S. aureus (SA) strains. The method that consistently produced PA:SA ratios in the range of 1:1 to 1:100 was to allow a SA mono-culture to reach stationary phase, and then re-suspend the SA cells in fresh media before inoculating with PA. With this method, it is possible to grow both PA and SA to stationary phase, a necessity for studying how PA and SA alter phenotypes in the presence of one another.
P. aeruginosa was found to produce less pyocyanin in the presence of S. aureus, but reduction in pyocyanin expression was depended on the strain of S. aureus. Elastase production differed between the two P. aeruginosa strains as well as between the two S. aureus strains, one increasing and one decreasing in expression. This data indicates that the responses of P. aeruginosa to S. aureus differ depending on both the P. aeruginosa and S. aureus strain present.
ContributorsWest, Sarah Beth (Author) / Bean, Heather B. (Thesis director) / Misra, Rajeev (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Pyocyanin is a pigment produced by Pseudomonas aeruginosa that acts as a virulence factor in helping this pathogen to establish chronic infection in the lungs of persons with cystic fibrosis (CF). Then, as lung infections become chronic, P. aeruginosa tends to down-regulate pyocyanin production. The effects of environmental conditions, particularly temperature change, on pyocyanin production in P. aeruginosa has not been widely studied in the past. The goals of this project were twofold: First, we aim to identify how environmental conditions potentially present in the CF lungs affect pyocyanin pigment production in P. aeruginosa. Second, through the examination of effects of environmental changes, we aim to identify methods to modulate phenotypes of P. aeruginosa in order to identify putative biomarkers through metabolic analysis. This paper also identifies a newly derived pyocyanin culturing and extraction procedure that yields increased sensitivity for pyocyanin detection.
Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.
Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.
ContributorsWitzel, Lea (Co-author) / Bean, Heather D. (Co-author, Thesis director) / Misra, Rajeev (Committee member) / Haydel, Shelley (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Mycobacterium abscessus (Mabs) is a multidrug-resistant nontuberculous mycobacterium capable of causing persistent pulmonary infection. It most prominently threatens those with cystic fibrosis (CF), a progressive and genetic disorder characterized by an immunocompromised respiratory tract. Current treatments fail to eradicate Mabs, meaning novel alternatives are greatly needed. Antimicrobial peptides (AMPs) are short sequences of amino acids that display broad-spectrum antimicrobial activity and play an important role in innate immunity. To maximize their therapeutic potential, key AMP features can be rationally combined through an iterative engineering process to create synthetic, designed AMPs (dAMPs). In this investigation, two dAMPs, RP554 and RP557, reduced Mabs ATCC 19977 viability by 99.99% and were subjected to further testing. In antimicrobial susceptibility testing with Mabs ATCC 19977, RP554 and RP557 demonstrated bactericidal activity at concentrations 16-32 μM. Complete killing of Mabs ATCC 19977 by RP554 and RP557 occurred rapidly in <24 h. RP554 and RP557 also inhibited 20 Mabs clinical isolates obtained from CF patients. Furthermore, RP554 and RP557 retained anti-Mabs activity after pre-exposure to human serum, indicating potential stability in blood. Conversely, the tested dAMPs did not kill Mabs during in vitro experiments in an artificial sputum medium. Novel antimicrobials, such as the RP554 and RP557 dAMPs, offer therapeutic potential for otherwise resistant bacterial pathogens, including Mabs, that afflict both CF and non-CF patients.
ContributorsBrandt, McKenzie (Author) / Haydel, Shelley (Thesis director) / Bean, Heather (Committee member) / Dermody, Roslyn (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05