Erisa Budo | ALES Graduate Seminar

Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Erisa Budo. This seminar is open to the general public to attend.

MSc with Drs. Burim Ametaj and Jeroen de Buck.

Zoom link: https://ualberta-ca.zoom.us/j/93166193007?pwd%3Di5mtbGuStELPKX7c5ERfajeb4b3xdt.1

Meeting ID: 931 6619 3007
Passcode: 394848

Thesis Topic: Evaluation of Indolicidin as an Alternative Therapeutic Agent in Murine Models of Mastitis induced by Staphylococcus aureus and Escherichia coli

Abstract: 

Problem: Mastitis, an inflammation of the mammary gland, is a significant health issue in dairy animals caused predominantly by bacterial pathogens such as Staphylococcus aureus and Escherichia coli. The rising incidence of antibiotic-resistant strains has created an urgent need for alternative therapeutic strategies to manage this condition effectively.

Hypothesis and Objective: This study hypothesized that Indolicidin, a cationic antimicrobial peptide, could be an effective therapeutic agent against mastitis by reducing bacterial load. The primary objective was to evaluate the antimicrobial efficacy of Indolicidin in murine mastitis models induced by Staphylococcus aureus and Escherichia coli through bacterial load assessments and observations of cytokine expression.

Experimental Design: Lactating female C57BL/6 mice, 10–12 days postpartum, were used to establish mastitis models through intramammary inoculation with S. aureus or E. coli. Mice were treated with either Rifaximin or Indolicidin at concentrations of 62.5 µg/mL and 125 µg/mL. Bacterial preservation methods were optimized using glycerol solutions to ensure consistent inoculations with low bacterial counts, and the minimum inhibitory concentrations (MICs) of Rifaximin for both pathogens were determined. Antimicrobial efficacy was evaluated by measuring bacterial loads in mammary tissues. Cytokine expressions were examined to provide insights into the inflammatory responses associated with mastitis and the effects of the treatments. Additionally, correlations between mammary gland weight, body weight percentage, and bacterial load were analyzed.

Results: The study found that a 10% glycerol solution effectively preserved bacterial viability for both pathogens. The MICs of Rifaximin were 31.25 µg/mL for E. coli and 15.625 µg/mL for S. aureus, indicating potential within strain variations. An inoculum of 10² CFU of E. coliinduced localized mastitis without systemic infection. Time-kill assays demonstrated that Indolicidin exhibited potent, dose-dependent bactericidal activity against both bacteria, with complete elimination achieved at higher concentrations within four hours. In vivo, some of the lowest bacterial loads in both infection models were observed in mice treated with Indolicidin at 62.5 µg/mL, but these differences were not statistically significant. Increasing the dose to 125 µg/mL in E. coli-infected mice showed no further noticeable trends in bacterial load reduction. Indolicidin at 62.5 µg/mL demonstrated reductions in pro-inflammatory cytokines such as IL-6 and TNF-α in S. aureus-infected mice based on fold changes; however, these reductions were not statistically significant. Rifaximin showed superior efficacy in reducing bacterial load and cytokine levels, with statistically significant reductions in IL-6 and TNF-α in S. aureus-infected mice. In E. coli-infected mice, Indolicidin exhibited dose-dependent effects: the 62.5 µg/mL dose reduced IL-6 and TNF-α levels based on fold changes, though without statistical significance, while the 125 µg/mL dose significantly increased IL-1β and TNF-α, indicating heightened immune stimulation. Correlation analyses indicated that mammary gland enlargement was primarily associated with inflammatory responses rather than bacterial load.

Conclusion: The findings indicate that Indolicidin exhibits potential antimicrobial activity in murine mastitis models, with a potential role in reducing bacterial loads, though statistical significance was not consistently achieved. Observed changes in cytokine expression suggest that further investigation is needed to clarify its effects on inflammation and its suitability as a therapeutic agent. Rifaximin demonstrated greater efficacy in reducing bacterial loads and cytokine levels in the S. aureusinfection model. Future research should focus on optimizing Indolicidin dosing, elucidating its mechanisms of action, and exploring combination therapies to enhance efficacy. This study contributes to developing alternative strategies for mastitis management, aiming to reduce reliance on traditional antibiotics and mitigate the challenges posed by antimicrobial resistance in dairy production.