The tyrocidines in the creation of antimicrobial cellulose and sterilizing materials
Date
2020-04
Authors
Journal Title
Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: The rising resistance of pathogenic bacteria is of great concern, especially since resistance has
been reported for five of the six common hospital acquired infections. Furthermore,
continual infections occurring in the food industry can become costly to the companies
and negatively impact consumers. Modified antimicrobial and antifouling materials and
surfaces can be used limit the propagation of microorganisms on various surfaces and
minimise the occurrence of infection and spoilage. These materials can prevent pathogenic cell
adhesion or kill cells with the release of active compounds or kill cells on contact.
Active compounds used to functionalise materials can include silver nanoparticles,
antimicrobially active polymers, antibiotics, enzymes and chemically synthesised peptides. Due
to the increased demand for more environmentally friendly practises, naturally produced
antimicrobial peptides are considered. Antimicrobial peptides have a broad spectrum of activity
and resistance is less likely to develop due to their membranolytic mode of action.
Tyrocidines and analogues (Trcs) are antimicrobial cyclodecapeptides with a
potent broad spectrum of activity against Gram-positive bacteria, filamentous fungi,
human pathogens Candida albicans and malaria parasite Plasmodium falciparum. The
peptides have been shown to adsorb onto various surfaces while maintaining activity, with
a selectivity towards cellulose. The goal for this study was therefore to determine the
application in commercial materials, the robustness of the cellulose-peptide material and
conditions that dictate the cellulose-peptide interaction, as well as the molecular descriptors in
peptide interaction.
In order to facilitate antimicrobial screening of Trcs treated materials, a high throughput solid
surface assay was developed that gave comparable results to that observed with an
industrial standard assay. The assay was developed for detection of antimicrobial
activity within four hours, by utilising a cell viability or metabolic active dye, resazurin. A
key factor that allowed for the fast detection was the use of ten times more cells per cm² as what
is used in other solid surface assays, which allowed for the selection of only the best
performing antimicrobial materials. Optimisation was confirmed with four model organisms
Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa
against materials that contained five different antimicrobial agents (gentamicin,
bacitracin, ampicillin, gramicidin S
and tetracycline).
Screening of association of a Trc mixture containing 11 cyclodecapeptides (Trc mix) to
laboratory and commercial materials showed that the peptides in this mixture maintained activity
and showed full inhibition of L. monocytogenes by peptide treated cellulose-based materials and plastics. Trc mix treated cellulose proved to be robust in terms of cell exposure (killing up to
90% of 10⁷ cells/cm² within 10 minutes of contact time), temperature and solvents. The
only solvents that resulted in decreased activity were 1% m/v SDS and 70% v/v
acetonitrile. Comparison between cellulose treated with tyrocidine and other antimicrobial
peptides, showed the tyrocidine-cellulose outperforming the other peptides in
terms of inhibition of
L. monocytogenes and E. coli.
Study into the aggregation of Trcs using different percentages acetonitrile, as a
denaturant of larger hydrophobic driven oligomers, showed the formation of two distinct
types of oligomer groups: one group stabilised with hydrogen bonding formed at higher
acetonitrile concentrations and one driven by hydrophobic interactions formed in aqueous
solutions. The hydrophobic driven self-assembly structures were temperature stable and
attributed to the temperature and solvent stability of the Trc-treated cellulose. It was
also proposed to be a key factor in the association between Trc and cellulose, apart
from the possible Maillard reaction that drive covalent linkages of some of the
peptides in the initial seeding layer on the cellulose. Trcs showed a concentration
dependent association linked to the formation of an optimal aggregate size that allows for
association. Interaction with various cellulose derivates (glucose, cellobiose, hydropropyl
cellulose) showed conformational changes of the peptide resulting in higher activity against L.
monocytogenes and higher haemolytic activity. Studies into the molecular interaction, using FTIR
and NMR, between cellulose and Trcs with the cellulose derivatives as cellulose models
showed the amino acids exposed to the solvent environment to be most effected by the presence of
the saccharides and therefore involved in the peptide:cellulose interaction.
The hypothesis is that the peptide forms self-assembled structures driven by hydrophobic
interactions in aqueous solutions allowing the formation of hydrogen bonds between the
hydroxyl groups on cellulose and Orn⁹, Asn⁵, and Gln⁶ followed by Trp⁴ and Tyr⁷. Upon
association the peptide oligomers would form a layered sheet like structure on the
cellulose surface with above mentioned amino acids associated to the cellulose and exposed to the
solvent environment. Thereby allowing for further self-assembly and/or antimicrobial
activity as Orn⁹ and Trp⁴ are both key residues in the activity of Trcs. Furthermore, the peptide
can arrange and re-arrange its conformation based on the environment to better suit association.
The creation of functional materials by harnessing Trcs’ natural ability to associate to surfaces
is more environmentally friendly but also creates an antimicrobial material that is robust in terms
of activity and solvent/environment exposure. Therefore, materials functionalised with Trcs hold
great promise in preventing surface colonization by resistant pathogens while still being “green”
in its environmental footprint.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.
Description
Thesis (PhD)--Stellenbosch University, 2020.
Keywords
Tyrocidines, Cellulose, Peptide antibiotics, Antimicrobial peptides, UCTD