Matching Items (16)

131378-Thumbnail Image.png

Investigating the Role of the Las and Rhl Quorum Sensing Systems in the Pathogenesis of Pseudomonas aeruginosa

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

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.

Contributors

Agent

Created

Date Created
2020-05

133345-Thumbnail Image.png

The Effectiveness of Inhibition and Biofilm Disruption on Antibiotic Resistant E. coli

Description

The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to

The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase the dosage of currently prescribed antibiotics. This study attempted to combat two forms of antibiotic resistance. The first is the AcrAB efflux pump which is able to pump antibiotics out of the cell. The second is the biofilms that E. coli can form. By using an inhibitor, the pump should be unable to rid itself of an antibiotic. On the other hand, using Tween allows for biofilm formation to either be disrupted or for the biofilm to be dissolved. By combining these two chemicals with an antibiotic that the efflux pump is known to expel, low concentrations of each chemical should result in an equivalent or greater effect on bacteria compared to any one chemical in higher concentrations. To test this hypothesis a 96 well plate BEC screen test was performed. A range of antibiotics were used at various concentrations and with varying concentrations of both Tween and the inhibitor to find a starting point. Following this, Erythromycin and Ciprofloxacin were picked as the best candidates and the optimum range of the antibiotic, Tween, and inhibitor were established. Finally, all three chemicals were combined to observe the effects they had together as opposed to individually or paired together. From the results of this experiment several conclusions were made. First, the inhibitor did in fact increase the effectiveness of the antibiotic as less antibiotic was needed if the inhibitor was present. Second, Tween showed an ability to prevent recovery in the MBEC reading, showing that it has the ability to disrupt or dissolve biofilms. However, Tween also showed a noticeable decrease in effectiveness in the overall treatment. This negative interaction was unable to be compensated for when using the inhibitor and so the hypothesis was proven false as combining the three chemicals led to a less effective treatment method.

Contributors

Agent

Created

Date Created
2018-05

132599-Thumbnail Image.png

Characterization of Lipid Transport Mutants that Overcome the Iron-Transport Defect in Escherichia coli

Description

When limited for iron, Escherichia coli secretes a siderophore, enterobactin, to solubilize and intake extracellular Fe3+ by a TonB-dependent high-affinity pathway. Consequently, E. coli tonB mutants grow poorly on a medium limited for iron. Upon longer incubation, however, faster growing

When limited for iron, Escherichia coli secretes a siderophore, enterobactin, to solubilize and intake extracellular Fe3+ by a TonB-dependent high-affinity pathway. Consequently, E. coli tonB mutants grow poorly on a medium limited for iron. Upon longer incubation, however, faster growing colonies emerge and overcome this growth defect. The work presented in this paper reports and characterizes these faster growing colonies (revertants) in an attempt to dissect the mechanism by which they overcome the TonB deficiency. Genomic analysis revealed mutations in yejM, a putative inner-to-outer membrane cardiolipin transporter, which are responsible for the faster growth phenotype in a tonB mutant background. Further characterization of the revertants revealed that they display hypersensitivity to vancomycin, a large antibiotic that is normally precluded from entering E. coli cells, and leaked periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild type copy of yejM on a plasmid, suggesting that yejM mutations are solely responsible for the observed phenotypes. In the absence of wild type tonB, however, the deletion of all known of cardiolipin synthase genes (clsABC) did not produce the phenotype similar to mutations in the yejM gene, suggesting the absence of cardiolipin from the outer membrane per se is not responsible for the increased outer membrane permeability. These data show that a defect in lipid biogenesis and transport can compromise outer membrane permeability barrier to allow siderophore intake and that YejM may have additional roles other than transporting cardiolipin.

Contributors

Agent

Created

Date Created
2019-05

133289-Thumbnail Image.png

The Inactivation of Pathogens in Contaminated Medications Via Selection Photonic Disinfection

Description

Each year the hospitals in the United States dispose of viable medications worth millions of dollars. These facilities are currently forced to do so not because the medications have expired, or are no longer effective, but rather because to re-use

Each year the hospitals in the United States dispose of viable medications worth millions of dollars. These facilities are currently forced to do so not because the medications have expired, or are no longer effective, but rather because to re-use any leftover medications would allow for the possibility of spreading disease. Once a medications sterile seal has been broken, any remaining contents of its container are considered potential pathogenic biohazards, and must be disposed of. The main objective of this thesis was to explore a potential alternative to simply discarding these lifesaving and often expensive leftover medications. The ultimate goal of this work is to establish a process by which excess drugs could be safely and effectively purified for re-use, subsequently cutting costs, and enhancing medication availability. Pseudomonas aeruginosa (P.a.) and Staphylococcus aureus (S.a) were cultured for their commonality in healthcare-associated infections (HAI's), and allowed to contaminate medication-like compounds. These bacterially inoculated solutions were meant to mimic the contaminated medications mentioned above and were then treated with a novel, physical means of pathogen inactivation named SElective PHOtonic DISinfection (SEPHODIS). Pathogen load reduction was determined through plate count assays both before and after exposure to the SEPHODIS system. structural preservation of medication was established through the use of infrared spectroscopy. The results of these experiments furthered the confidence of SEPHODIS as an efficient means of pathogen inactivation, while promoting promise of a real-world application in the form of medication purification.

Contributors

Agent

Created

Date Created
2018-05

133571-Thumbnail Image.png

Genetic Manipulation of Pseudomonas aeruginosa Clinical Isolates

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

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.

Contributors

Agent

Created

Date Created
2018-05

134374-Thumbnail Image.png

Analysis of the prrAB two-component system regulatory effects on the lipid profile of Mycobacterium smegmatis

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.

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.

Contributors

Agent

Created

Date Created
2017-05

134307-Thumbnail Image.png

The Effects of Environmental Changes on the Rhamnolipid Production in Pseduomonas aeruginosa

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

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 30C versus 37C, but not at 40C. 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.

Contributors

Agent

Created

Date Created
2017-05

134479-Thumbnail Image.png

Comparison of Inflammatory Changes in Ethmoid Mucosa and Nasal Turbinate Tissue: A Histopathological Study

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

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.

Contributors

Agent

Created

Date Created
2017-05

131628-Thumbnail Image.png

The impact of Staphylococcus aureus volatiles on Pseudomonas aeruginosa phenotypes

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

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.

Contributors

Agent

Created

Date Created
2020-05

156388-Thumbnail Image.png

A study of protein-protein interactions in Salmonella typhimurium

Description

The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function

The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.

Contributors

Agent

Created

Date Created
2018