Matching Items (20)

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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.

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2020-05

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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.

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2017-05

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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.

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2017-05

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Effects of Antibacterial Mineral Leachates on the Cellular Ultrastructure, Morphology, and Membrane Integrity of Escherichia Coli and Methicillin-Resistant Staphylococcus Aureus

Description

Background: We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus

Background: We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates.

Methods: We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Bac light LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity.

Results: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates.

Conclusions: These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.

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Date Created
2010-09-16

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In Vitro Antibacterial Activity and In Vivo Efficacy of Hydrated Clays on Mycobacterium Ulcerans Growth

Description

Background: Buruli ulcer, caused by Mycobacterium ulcerans, is a localized skin lesion that can progress to extensive ulceration and necrosis if left untreated. Unpublished studies of hydrated clays for therapeutic, topical treatment of Buruli ulcer suggest that specific clay mineral products

Background: Buruli ulcer, caused by Mycobacterium ulcerans, is a localized skin lesion that can progress to extensive ulceration and necrosis if left untreated. Unpublished studies of hydrated clays for therapeutic, topical treatment of Buruli ulcer suggest that specific clay mineral products may have beneficial effects on wound healing. In this study, we evaluated the in vitro antibacterial activity of a panel of clay mixtures and their derivative leachates against M. ulcerans and assessed the in vivo efficacy of topically-applied, hydrated clays on Buruli ulcer progression in mice infected with M. ulcerans.

Methods: M. ulcerans 1615 was incubated with 10 % suspensions of CB07, CB08, CB09, CB10, and BY07 clay mixtures, and survival was determined over 28 days. For animal experiments, we examined the effect of topical hydrated clay therapy on Buruli ulcer progression in vivo in mouse tails subcutaneously infected with M. ulcerans 1615.

Results: The CB07, CB08, and CB09 clays exhibited bactericidal activity against M. ulcerans after 7, 14, 21, and 28 days of incubation. In contrast, clay leachates exhibited inhibitory, bacteriostatic effects on M. ulcerans growth in vitro. After establishing an ulcerative M. ulcerans infection for three months, ulcerated regions of the tails were treated once daily (five consecutive days per week) for 22 days with hydrated CB09 clay poultices. Mice in the clay treatment group exhibited healing as assessed by gross morphological changes and a reduction in M. ulcerans present in the wounds.

Conclusions: These data reveal that specific clays exhibit in vitro bactericidal activity against M. ulcerans and that hydrated clay poultices may offer a complementary and integrative strategy for topically treating Buruli ulcer disease.

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Date Created
2016-01-30

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Metal Ions, Not Metal-Catalyzed Oxidative Stress, Cause Clay Leachate Antibacterial Activity

Description

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. 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 the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions.

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Date Created
2014-12-11

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Identification of Mycobacterium smegmatis and antibiotic resistance through the utilization of high titer mycobacteriophage concentrations and MALDI-TOF MS

Description

The diagnosis of bacterial infections based on phage multiplication has the potential for profound clinical implications, particularly for antibiotic-resistant strains and the slow-growing Mycobacterium tuberculosis. The possibility of hastening the diagnosis of antibiotic-resistant mycobacterial infections was accomplished via the study

The diagnosis of bacterial infections based on phage multiplication has the potential for profound clinical implications, particularly for antibiotic-resistant strains and the slow-growing Mycobacterium tuberculosis. The possibility of hastening the diagnosis of antibiotic-resistant mycobacterial infections was accomplished via the study of Mycobacterium smegmatis, a generally non-pathogenic, comparatively fast growing microorganism to M. tuberculosis. These proof-of-concept studies established that after transduction of M. smegmatis cells with bacteriophages, MALDI-TOF MS could be used to detect increased amounts of phage proteins. Recording the growth of M. smegmatis over an 8-hour period, starting with very low OD600 measurements, simulated bacterial loads in clinical settings. For the purposes of MALDI-TOF MS, the procedure for the most effective lethal exposure for M. smegmatis was determined to be a 1-hour incubation in a 95°C water bath. Successful precipitation of the lytic mycobacteriophages D29 and Giles was performed using chloroform and methanol and overlaid with 1-2 μL of α-cyano-4-hydoxycinnaminic acid, which allowed for more distinct and repeatable MALDI-TOF MS spectra. Phage D29 was found to produce an m/z peak at 18.477 kDa, which may have indicated a 2+-charged ion of the 34.8 kDa minor tail protein. The Giles proteins that were identified with MALDI-TOF MS have not been directly compared to protein values reported in the scientific literature. However, the MALDI-TOF MS spectra suggested that distinct peaks existed between M. smegmatis mc2155 and mycobacteriophages, indicating that successful infection with lytic phage and replication thereafter may have occurred. The distinct peaks between M. smegmatis and the phage can be used as indicators of the presence of mycobacteria. At this point, the limits of detection of each phage must be elucidated in order for MALDI-TOF MS spectra to be successfully implemented as a mechanism to rapidly detect antibiotic-resistant mycobacteria.

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2015-05

Characterization of the structure and interactions of the AcrAB-TolC multi-drug efflux pump in Escherichia coli

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 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.

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Date Created
2016-05

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Assessing School of Life Sciences freshmen satisfaction in the Life Sciences Career Paths mentoring program

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Abstract The BIO 189 Life Sciences Career Paths course is a seminar course that is intended to acclimate incoming freshmen into the School of Life Sciences (SOLS). While there are instructors who organize and present in the class, upper division

Abstract The BIO 189 Life Sciences Career Paths course is a seminar course that is intended to acclimate incoming freshmen into the School of Life Sciences (SOLS). While there are instructors who organize and present in the class, upper division undergraduate students are primarily responsible for facilitating lectures and discussions and mentoring the freshmen. Prior research has demonstrated that the mentor-mentee relationship is a very important predictor of success and retention within all university first-year programs. While past studies focused on the student mentor-mentee relationships, there is limited research that measures student satisfaction within freshmen seminar courses, especially in areas of science, technology, engineering, and mathematics (STEM). The purpose of this project is to survey students about their perception of the BIO 189 course. The effort of the project is on pre-health students, as they initiate their undergraduate careers and attempt to achieve acceptance into professional school four years later. Analysis of Likert scale surveys distributed to 561 freshmen revealed that students with an emphasis on "medicine" in their majors preferred a BIO 189 course geared to pre-health interests whereas students seeking an emphasis on research (ecology and cell biology/genetics) sought a BIO 189 course focused on internship and employment opportunities. Assessment of the mentor-mentee relationship revealed that students (n = 561) preferred one-on-one meetings with mentors outside of class (44%) compared to those who preferred interaction in class (30%). A sizable 61.68% of students (n = 548) were most concerned with attaining favorable GPAs, highlighting strong emphasis on academic performance. Overall, 61% of respondents (n = 561) expressed satisfaction with SOLS resources and involvement opportunities, which was hypothesized. These results give substantial insight into the efficacy of a first-year success seminar-mentoring program for college freshmen in STEM.

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Date Created
2016-12

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Early Assessment of Phage Communities in Amazon Peatland Soils

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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

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.

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Date Created
2016-05