Date of Award

Spring 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Claudia Muratori

Committee Member

P. Thomas Vernier

Committee Member

Olga Pakhomova

Committee Member

Piotr Kraj

Abstract

This research explores the impact of nanosecond pulsed electric fields (nsPEF) on two fronts: their immune stimulatory effects and their potential as a novel strategy to enhance the sensitivity of Methicillin-resistant Staphylococcus aureus (MRSA) to clinically relevant antibiotics. While pulsed electric fields have been reported to have an immune stimulatory effect, the mechanisms responsible for these effects have yet to be determined.

Our investigation addresses the rising concern of MRSA derived skin and soft tissue infections (SSTIs). Consistent with other publications, we found that nsPEF alone cause modest inactivation of planktonic MRSA. We then investigated the effects of nsPEF in combination with commonly used antibiotics for the treatment of SSTI: vancomycin, doxycycline and daptomycin. Notably, the combination of nsPEF with daptomycin demonstrates a significant increase in bacterial inactivation compared to each monotherapy, irrespective of the treatment order. Conversely, when combining nsPEF with doxycycline or vancomycin, the treatment order emerges as crucial factor influencing the level of inactivation. Cells treated with nsPEF prior to antibiotic exposure show an increase in MRSA sensitivity to these drugs, while the opposite order does not improve the efficacy of the combined treatment. Furthermore, co-treatment of nsPEF and vancomycin effectively treats MRSA growing in biofilms, structures known for their increased resistance to antimicrobials

In parallel we investigated whether cellular perturbation by nsPEF triggered the NLRP3 inflammasome. Inflammasomes are intracellular innate immune platforms activated by damageand pathogen- associated stress. Their activation is responsible for the processing and release of proinflammatory cytokines of the IL-1 family, constituting one of the first line of defense against pathogens including S. aureus. We present evidence that nsPEF trigger the formation of the NLRP3 inflammasome, through visualization of the inflammasome-adaptor protein (ASC), the activation caspase-1 and the release of IL-1β in primary and immortal macrophages. Most interestingly, our study suggests that nsPEF can trigger the activation of multiple inflammasomes in response to the stimuli generated during and after pulse treatment.

In summary, these findings support the central idea guiding our current research: that nsPEF have a dual effect. Specifically, they enhance the susceptibility of bacteria to antibiotics while concurrently boosting the host immune responses against MRSA.

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DOI

10.25777/fd0q-4985

ISBN

9798382772745

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