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Microrheology and Spatial Heterogeneity of Staphylococcus aureus Biofilms Modulated by Hydrodynamic Shear and Biofilm-Degrading Enzymes And The Interaction of Cationic Peptide G3 on Bacterial Membranes Investigated using 3-dimensional Single Particle Tracking and Solid-State Nuclear Magnetic Resonance

Hart, Jack

[Thesis]. Manchester, UK: The University of Manchester; 2020.

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Abstract

Antibiotic resistance is fast becoming a global health crisis, with the increase in resistant bacteria outpacing the generation and translation of new antibiotics. Bacteria at interfaces can produce a self-made architecture called a biofilm that acts as a physical barrier, significantly reducing the efficacy of antibiotics. The aim of this part of the project was to assess the material properties of developing biofilms non-invasively. Passive microrheology was used investigate biofilms produced by Staphylococcus aureus under various hydrodynamic shears and when exposed to different anti-biofilm enzymes. Biofilms grown under any shear stress were harder (i.e. had a lower creep compliance) than equivalent biofilms grown in stationary media. Furthermore, statistical analysis of the spatial arrangement of bacteria during biofilm growth revealed clustering as a function of height away from the interface surface. The cationic peptide G3 has been shown to be a potential antimicrobial peptide, however the exact mechanism of action is unknown. Two methods were used to probe the interaction of the peptide with the bacteria membrane. Firstly, a novel 3-dimensional tracking method incorporating photoswitchable fluorophores and an adaptive optics-based super-resolution imaging technique was used to investigate the spatial distribution of G3 following exposure in Staphylococcus aureus and Escherichia coli. No preferential localisation of G3 could be observed (i.e. G3 was homogeneously distributed within the cell) for both bacteria. Diffusion kinetics were approximated from short trajectories using a newly developed neural net software package; however no spatial dependences were observed suggesting a weak binding to the membrane. To further investigate the strength of the interaction between G3 and the cell membrane, solid state nuclear magnetic resonance was employed. Using a model phospholipid system, increased disorder (i.e. spatial fluctuations) was observed in bilayers exposed to G3, suggesting a transient interaction with the membrane. From this and the tracking data, we hypothesise that G3 transiently interacts broadly with the entire membrane to cause accumulative strain disruption. No significant clustering or decreases in order parameters suggesting a strong binding with the membrane were observed, however no positive control was assessed in this study.

Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree type:
Doctor of Philosophy
Degree programme:
PhD EPSRC-MRC Centre for Doctoral Training in Regenerative Medicine Physics
Publication date:
Location:
Manchester, UK
Total pages:
165
Abstract:
Antibiotic resistance is fast becoming a global health crisis, with the increase in resistant bacteria outpacing the generation and translation of new antibiotics. Bacteria at interfaces can produce a self-made architecture called a biofilm that acts as a physical barrier, significantly reducing the efficacy of antibiotics. The aim of this part of the project was to assess the material properties of developing biofilms non-invasively. Passive microrheology was used investigate biofilms produced by Staphylococcus aureus under various hydrodynamic shears and when exposed to different anti-biofilm enzymes. Biofilms grown under any shear stress were harder (i.e. had a lower creep compliance) than equivalent biofilms grown in stationary media. Furthermore, statistical analysis of the spatial arrangement of bacteria during biofilm growth revealed clustering as a function of height away from the interface surface. The cationic peptide G3 has been shown to be a potential antimicrobial peptide, however the exact mechanism of action is unknown. Two methods were used to probe the interaction of the peptide with the bacteria membrane. Firstly, a novel 3-dimensional tracking method incorporating photoswitchable fluorophores and an adaptive optics-based super-resolution imaging technique was used to investigate the spatial distribution of G3 following exposure in Staphylococcus aureus and Escherichia coli. No preferential localisation of G3 could be observed (i.e. G3 was homogeneously distributed within the cell) for both bacteria. Diffusion kinetics were approximated from short trajectories using a newly developed neural net software package; however no spatial dependences were observed suggesting a weak binding to the membrane. To further investigate the strength of the interaction between G3 and the cell membrane, solid state nuclear magnetic resonance was employed. Using a model phospholipid system, increased disorder (i.e. spatial fluctuations) was observed in bilayers exposed to G3, suggesting a transient interaction with the membrane. From this and the tracking data, we hypothesise that G3 transiently interacts broadly with the entire membrane to cause accumulative strain disruption. No significant clustering or decreases in order parameters suggesting a strong binding with the membrane were observed, however no positive control was assessed in this study.
Thesis main supervisor(s):
Thesis co-supervisor(s):
Language:
en

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:324056
Created by:
Hart, Jack
Created:
17th March, 2020, 11:37:20
Last modified by:
Hart, Jack
Last modified:
8th April, 2020, 09:25:23

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