IU Indianapolis ScholarWorks :: Browsing by Subject "Pseudomonas aeruginosa"

Browsing by Subject "Pseudomonas aeruginosa"

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Antibiotic Treatment of Pseudomonas aeruginosa Biofilms Stimulates Expression of mgtE, a Virulence Modulator
(2012-08-07) Redelman, Carly Virginia; Anderson, Gregory G.; Blazer-Yost, Bonnie.; Bauer, Margaret.
Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.
Bacterial Pyocyanin Inducible KRT6A Accelerates Closure of Epithelial Defect Under Conditions of Mitochondrial Dysfunction
(Elsevier, 2023) Ghatak, Subhadip; Hemann, Craig; Boslett, James; Singh, Kanhaiya; Sharma, Anu; El Masry, Mohamed S.; Abouhashem, Ahmed Safwat; Ghosh, Nandini; Mathew-Steiner, Shomita S.; Roy, Sashwati; Zweier, Jay L.; Sen, Chandan K.; Surgery, School of Medicine
Repair of epithelial defect is complicated by infection and related metabolites. Pyocyanin is one such metabolite which is secreted during Pseudomonas aeruginosa infection. Keratinocyte migration is required for the closure of skin epithelial defects. The current work sought to understand pyocyanin-keratinocyte interaction and its significance in tissue repair. SILAC proteomics identified mitochondrial dysfunction as the top pathway responsive to pyocyanin exposure in human keratinocytes. Consistently, functional studies demonstrated mitochondrial stress, depletion of reducing equivalents, and ATP. Strikingly, despite all the above, pyocyanin markedly accelerated keratinocyte migration. Investigation of underlying mechanisms revealed a new function of KRT6A in keratinocytes. KRT6A was pyocyanin inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing including compromised expression of apical junction proteins. This work recognizes KRT6A for its role of enhancing keratinocyte migration under conditions of threat posed by pyocyanin. Qualitatively deficient junctional proteins under conditions of defensive acceleration of keratinocyte migration explains why an infected wound close with deficient skin barrier function as previously reported.
Defining and identifying early-onset lung disease in cystic fibrosis with cumulative clinical characteristics
(Wiley, 2022) Huang, Leslie; Lai, HuiChuan J.; Antos, Nicholas; Rock, Michael J.; Asfour, Fadi; Howenstine, Michelle; Gaffin, Jonathan M.; Farrell, Philip M.; Pediatrics, School of Medicine
Background: Because of the heterogeneity in cystic fibrosis (CF) lung disease among young children, a clinical method to identify early-onset lung disease is needed. Objective: To develop a CF early-onset lung disease (CFELD) scoring system by utilizing prospectively collected longitudinal data on manifestations in the first 3 years of life. Design: We studied 145 infants born during 2012-2017, diagnosed through newborn screening by age 3 months, and followed to 36 months of age. Cough severity, pulmonary exacerbations (PEx), respiratory cultures, and hospitalizations were collected at each CF center visit (every 1-2 months in infancy and quarterly thereafter). These data were used to construct the CFELD system and to classify lung disease into five categories: asymptomatic, minimal, mild, moderate, and severe. Results: The most frequent manifestation of CF early lung disease was MD-reported PEx episodes, PEx hospitalizations, and positive Pseudomonas aeruginosa cultures. Parent-reported cough severity was correlated with the number of respiratory hospitalizations (r = 0.48, p < 0.0001). The distribution of CFELD categories was 10% asymptomatic, 17% minimal, 29% mild, 33% moderate, and 12% severe. The moderate and severe categories occurred threefold higher in pancreatic insufficient (PI, 49%) versus sufficient subjects (16%), p < 0.0001. In addition to PI, gastrointestinal and nutrition-related hospitalizations, plasma cytokines interleukin (IL)-6 and IL-10, duration of CFTR modulator therapy, and type of health insurance were significant predictors of CFELD scores. Conclusion: The CFELD scoring system is novel, allows systematic evaluation of lung disease prognosis early, and may aid in therapeutic decision-making particularly in the initiation of CFTR modulator therapy.
Development of a New in vitro System for Cystic Fibrosis Research
(Office of the Vice Chancellor for Research, 2013-04-05) Coffey, Barbara M.; Anderson, Gregory G.
Individuals with cystic fibrosis (CF) have a life expectancy of 40 years and require daily treatments to mitigate the effects of the disease. CF impacts organs throughout the body, especially the lungs, where thick mucus builds up, impairs breathing, and provides an environment for bacterial growth. Chronic lung infection is the leading cause of mortality in CF. The majority of CF lung infections are caused by Pseudomonas aeruginosa, a common bacterium which typically does not cause disease in healthy individuals. In the CF lung, however, P. aeruginosa burrows into the thick mucus layer, evades the immune system, and resists antibiotic therapy by encasing itself in a protective matrix called a biofilm. Laboratory methods for studying biofilm are not true replicas of the CF lung environment, leaving a knowledge gap between how bacteria grow in a test tube (in vitro) and how they grow in the lungs of a person with CF. The focus of this work is to develop an improved laboratory model which combines artificial sputum (as a surrogate for mucus in the CF lung) and cultured CF airway epithelial cells. To assess the potential of this model, we have performed experiments to compare P. aeruginosa in artificial sputum versus standard laboratory media. Results demonstrate that P. aeruginosa in artificial sputum exhibits differences in growth, biofilm formation, toxin production, cytotoxicity, and protein expression, compared to results in standard media. These data suggest that our model system can contribute new information to the understanding of CF airway infection. The aim of future studies is to use this system to identify sputum components and bacterial proteins which have not been recognized previously by standard methods. It is our ultimate goal to contribute knowledge leading to improved longevity and quality of life for people with CF.
DNA alternate polymerase PolB mediates inhibition of type III secretion in Pseudomonas aeruginosa
(Elsevier, 2021) Chakravarty, Shubham; Ramos-Hegazy, Layla; Gasparovic, Abigail; Anderson, Gregory G.; Biology, School of Science
Opportunistic pathogen Pseudomonas aeruginosa uses a variety of virulence factors to cause acute and chronic infections. We previously found that alternate DNA polymerase gene polB inhibits P. aeruginosa pyocyanin production. We investigated whether polB also affects T3SS expression. polB overexpression significantly reduced T3SS transcription and repressed translation of the master T3SS regulator ExsA, while not affecting exsA mRNA transcript abundance. Further, polB does not act through previously described genetic pathways that post-transcriptionally regulate ExsA. Our results show a novel T3SS regulatory component which may lead to development of future drugs to target this mechanism.
Does Magnesium Transport Protein (MgtE) Contribute to the Antibiotic Resistance of Pseudomonas aeruginosa?
(Office of the Vice Chancellor for Research, 2013-04-05) Umwali, Audrey; Anderson, Gregory G.
Pseudomonas aeruginosa is an environmental and opportunistic bacterial pathogen that is resistant to antibiotic treatment when it forms biofilms in the lungs of patients with cystic fibrosis. Biofilms are densely packed communities of bacteria embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS is a polymeric cluster composed of extracellular DNA, proteins and polysaccharides. Based on previous studies, in a low Mg2+ environment, P. aeruginosa wild-type is less or non-resistant to antibiotics and in a high Mg2+ environment, P. aeruginosa is more resistant to antibiotics. The purpose of this project was to find out if the magnesium transport protein (MgtE) is a contributor to the antibiotic resistance of P. aeruginosa .This was accomplished by using two different strains of P.aeruginosa; PA14 wild-type and GGA52 mutant (without the magnesium transporting protein). Four antibiotics were used; gentamicin, tobramycin, ciproflaxin and imipenem. The minimum inhibitory concentration (MIC) of each antibiotic was determined by culturing the bacteria strains on LB agar plates and use Etest strips to observe growth. N-minimal media supplemented with varying magnesium concentration was used to test if Mg2+ increased or reduced the antibiotic resistance at the MIC of P. aeruginosa as well as counting bacterial colonies. The mutant strain (GGA52) is expected to be less resistant than the wild type strain (PA14) because it does not have MgtE. If these predictions are true, then MgtE is an important contributor to the antibiotic resistance of P. aeruginosa. These results can be helpful in understanding the mechanism of antibiotic resistance of P. aeruginosa in patients with cystic fibrosis.
THE EFFECTS OF QUORUM SENSING ANTAGONISTS ON BIOFILM
(Office of the Vice Chancellor for Research, 2012-04-13) Guzman, Juan M.; Anderson, Gregory G.
Cystic fibrosis (CF) is a recessive genetic disorder that causes the for-mation of thick mucus plugs in the lungs of approximately 30,000 people in the United States and 60,000 individuals world-wide. Pseudomonas aeruginosa, an opportunistic bacterial pathogen, is able to colonize the mu-cus plugs and form antibiotic resistant biofilms. These microbial colonies, known as biofilms, cause serious problems for individuals living with CF. P. aeruginosa biofilms are able to cause chronic infections in the lungs of CF patients leading to increased morbidity and mortality. Using a modified bio-film assay, we tested the effects of modified chemical compounds and amino acids on P. aeruginosa biofilm dispersion. A previous study performed on P. aeruginosa, found that treatment of d- and l- amino acids resulted in biofilm dissemination. Through additional experiments, we will identify modified chemical compounds that induce biofilm dispersion. This research will in-crease our knowledge of P. aeruginosa biofilm dispersion, and allow us to explore new forms of treatment and therapy for CF patients with chronic in-fections that could be life threatening.
Electroceutical Treatment of Pseudomonas aeruginosa Biofilms
(Springer Nature, 2019-02-14) Dusane, Devendra H.; Lochab, Varun; Jones, Travis; Peters, Casey W.; Sindeldecker, Devin; Das, Amitava; Roy, Sashwati; Sen, Chandan K.; Subramaniam, Vish V.; Wozniak, Daniel J.; Prakash, Shaurya; Stoodley, Paul; Surgery, School of Medicine
Electroceutical wound dressings, especially those involving current flow with silver based electrodes, show promise for treating biofilm infections. However, their mechanism of action is poorly understood. We have developed an in vitro agar based model using a bioluminescent strain of Pseudomonas aeruginosa to measure loss of activity and killing when direct current was applied. Silver electrodes were overlaid with agar and lawn biofilms grown for 24 h. A 6 V battery with 1 kΩ ballast resistor was used to treat the biofilms for 1 h or 24 h. Loss of bioluminescence and a 4-log reduction in viable cells was achieved over the anode. Scanning electron microscopy showed damaged cells and disrupted biofilm architecture. The antimicrobial activity continued to spread from the anode for at least 2 days, even after turning off the current. Based on possible electrochemical ractions of silver electrodes in chlorine containing medium; pH measurements of the medium post treatment; the time delay between initiation of treatment and observed bactericidal effects; and the presence of chlorotyrosine in the cell lysates, hypochlorous acid is hypothesized to be the chemical agent responsible for the observed (destruction/killing/eradication) of these biofilm forming bacteria. Similar killing was obtained with gels containing only bovine synovial fluid or human serum. These results suggest that our in vitro model could serve as a platform for fundamental studies to explore the effects of electrochemical treatment on biofilms, complementing clinical studies with electroceutical dressings.
Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination
(Wiley Blackwell (Blackwell Publishing), 2013-02) Anderson, Gregory G.; Kenney, Thomas F.; Macleod, David L.; Henig, Noreen R.; O'Toole, George A.; Department of Biology, School of Science
Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4:1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L(-1) , while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L(-1) FTI and 16 mg L(-1) tobramycin. Fosfomycin (1024 mg L(-1)) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L(-1)) and tobramycin (4 mg L(-1)) and high concentrations of fosfomycin (1024 mg L(-1)) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides.
Going After Lipotoxins to Reduce Inflammation in the Airway of Cystic Fibrosis Patients
(Office of the Vice Chancellor for Research, 2013-04-05) Akhand, Saeed S.; Anderson, Gregory G.
People with cystic fibrosis (CF) typically have chronic lung infections, predominantly with Pseudomonas aeruginosa. Lung inflammation, in connection with bacterial colonization, is one of the major factors contributing to the morbidity and mortality of CF patients. Recent studies suggest that a common mutation among CF P. aeruginosa isolates (in the gene mucA) results in high-level expression of lipoproteins which stimulates a pro-inflammatory reaction in cultured CF-derived airways cell (CFBE). Our previous work in this area has revealed that a strain containing a mutation in the putative lipotoxin gene PA4326 is dramatically less toxic to CFBE. We hypothesize that lipotoxins lead to airway structure damage by causing epithelial cell death and tissue destruction, possibly as a downstream effect of immune stimulation. Our results demonstrate that deletion of the PA4326 gene does not affect growth, motility, adhesion, or biofilm development. However, this mutant strain produces 59.1% less pyocyanin compared to the non-mutant strain. Pyocyanin is a bacterial toxin that triggers airway inflammation by stimulating the immune system to produce the signaling molecule IL-8. Thus, our data suggest a possible clue about the decreased toxicity of the PA4325 mutant. The aim of future work is to confirm the role of this lipotoxin gene in the inflammatory process and to elucidate the underlying mechanism of its function. Our long term goal is to characterize other lipotoxins and to develop a novel inhibitor of lspA (a bacterial gene required for lipotoxin production) as an anti-inflammatory strategy to slow down the airway damage and hence improve the longevity and quality of life for people with CF.

Browsing by Subject "Pseudomonas aeruginosa"

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