Date of Award

Spring 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

Program/Concentration

Biomedical Sciences

Committee Director

Erin B. Purcell

Committee Member

Lesley H. Greene

Committee Member

Lisa M. Shollenberger

Committee Member

Piotr J. Kraj

Abstract

Biofilm production plays a crucial role in bacterial antibiotic tolerance and novel approaches are needed to combat it. The stringent response (SR), activated by nucleotide alarmones (pp)pGpp, is crucial for bacterial transcriptional reprogramming. In Clostridioides difficile, the SR is implicated in antibiotic survival, contributing to infection recurrence. Despite its known roles in sporulation and biofilm production in other bacterial species, the broader influence of the SR on C. difficile physiology remains undiscovered. This study deletes the C. difficile SR gene relQ, revealing the pivotal role of RelQ in regulating SR-dependent phenotypes in this organism. The absence of RelQ disrupts growth regulation in unstressed conditions and exhibits varied responses to sublethal antibiotic stresses. In contrast to the wild-type strain, the relQ knockout fails to increase biofilm production under sublethal antibiotic or oxidative stress, indicating the necessity of RelQ in stress-induced biofilm formation. relQ deletion slows planktonic spore accumulation but paradoxically accelerates it in biofilms. The effects of relQ deletion on biofilm and sporulation were apparent even when other clostridial SR genes were not deleted, emphasizing the importance of RelQ in regulating C. difficile stress survival and physiology.

Staphylococcus aureus, typically a skin commensal, becomes an opportunistic pathogen when the protective skin barrier has been compromised. Methicillin-resistant strains pose challenges in managing skin and soft-tissue infections, which are often comprised of biofilms. These infections are treated with antibiotics including vancomycin, but clinical resistance has emerged. This study combines nanosecond pulsed electric fields (nsPEFs) with vancomycin to deactivate methicillin-resistant Staphylococcus aureus (MRSA). When combined with vancomycin, nsPEFs enhance bacterial inactivation and lower inhibitory vancomycin concentrations. Exponentially growing MRSA experiences mild inactivation with nsPEFs alone but shows robust inactivation with combinatorial treatment, especially when nsPEFs are administered before vancomycin. Biofilm-derived cells exhibit greater resistance to nsPEFs than planktonic cells, but augmenting vancomycin treatment with nsPEF shows a promising trend toward greater efficacy. Subjecting intact biofilms to combinatorial nsPEF and vancomycin treatment could be a valuable avenue for future research.

This work explores innovative methods to diminish the protection against extracellular stress conferred by biofilm production in both Clostridioides difficile and methicillin-resistant Staphylococcus aureus.

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In Copyright. URI: http://rightsstatements.org/vocab/InC/1.0/ This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).

DOI

10.25777/0fem-ew87

ISBN

9798382770499

ORCID

0009-0006-9005-2438

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