Investigating Microbiome -Targeted Treatments for Chronic Rhinosinusitis: A novel approach to combat dysbiosis in the nasal microbiome
Date
2021
Authors
Menberu, Martha Alemayehu
Editors
Advisors
Vreugde, Sarah
Wormald, Peter-John
Wormald, Peter-John
Journal Title
Journal ISSN
Volume Title
Type:
Thesis
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Statement of Responsibility
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Abstract
Chronic rhinosinusitis (CRS) is a detrimental inflammatory upper airway disorder with
different underlying pathophysiology affecting the mucosa of the nasal cavity and paranasal
sinus. Approximately 12.5% of the western populations is affected by this disease with a large
socioeconomic impact and compromised quality of life. Microbial dysbiosis that is
characterized by alterations in the structure and function of the sino-nasal microbiota
composition, with the predominant reduction of Corynebacteria is recently linked with the
pathogenesis of CRS. As a result, the sino-nasal microbiota has emerged as an attractive
therapeutic target. While knowledge of the nasal microbiome is expanding rapidly, the exact
microbial dynamics at species or strain level, their interactions and potential therapeutic
strategies remain in their infancy. Therefore, understanding the role of both commensal and
pathobiont microbes that exist in the sino-nasal tract, with their dynamic interactions between
microbes and their hosts, as well as the investigation of innovative therapeutics targeting the
microbiota is an active area of current research worldwide. In this thesis, the development of
two novel microbiome-targeted treatment strategies were explored including, the fatty acid
compounds as prebiotics and commensal Corynebacteria as probiotics to restore the normal
microbiota in CRS patients. The first part of this thesis reviews the existing literature relating
to CRS, the complex role of sino-nasal microbiota both in health and CRS, dysbiosis of the
sino-nasal microbiota, and especially focuses on the beneficial function of commensal
microbes and several therapeutic strategies for nasal homeostasis. In the second part of the
thesis we investigate the prebiotic effect of fatty acid (FA) excipients, Tween 80 and its free
fatty acid moiety, Oleic acid on the growth of various nasal commensals including, C. accolens,
C. propinquum, C. pseudodiphtheriticum and S. epidermidis as well as common nasal
pathogens, S. aureus and P. aeruginosa in planktonic and biofilm forms in an in vitro study.
As a result, Tween 80 and Oleic acid demonstrated a significant growth promotion effect on commensal Corynebacteria, mainly C. accolens and reciprocally an antibacterial and
antibiofilm effects against pathogens including, the clinical isolates and reference strains of S.
aureus at FDA-approved concentration of 0.5% or below. Moreover, an increased growth of
C. accolens biofilms induced by Tween 80 or Oleic acid showed a significant growth change
on S. aureus and P. aeruginosa biofilms in an in vitro mixed commensal-pathogen biofilm
model. The findings in this study support the therapeutic potential of FA compounds as
prebiotics for the management of dysbiosis-associated CRS.
The third part of the thesis focuses on evaluating the antimicrobial potential of commensal C.
accolens isolates, which are predominant members of the nasal microbiome against S. aureus
and methicillin-resistant S. aureus (MRSA) isolates from CRS patients. A total of 10 C.
accolens strains were identified based on microbiological, biochemical and molecular tests
among 20 healthy control subjects and used as potential starting strains for exploring the
antimicrobial potential toward S. aureus pathogens. All C. accolens isolates and their secreted
proteins exhibited anti-staphylococcal activity in a dose-dependent manner as determined
through deferred growth inhibition and micro dilution assays. C. accolens strains, in particular
C779, C781 and C787 was found to be the best strains with strong antibacterial and antibiofilm
effects. Subsequently, we were able to show that the effect is due to production of protein-like
substance by C. accolens, which was directly involved in the reduction of planktonic growth,
biofilm biomass and metabolic activity of S. aureus isolates. This finding has led to the
exploration of antibacterial protein products from C. accolens to realize the development of
novel probiotic therapies to promote sinus health. The fourth part of this thesis briefly describes
a detailed proteomic analysis of commonly expressed proteins across 6 C. accolens strains to
identify and characterize antibacterial and other proteins functionally associated with various
probiotic properties. As a result, Acetyltransferase, GNAT family protein was found to be the
strongest positively correlated abundant protein detected in C. accolens associated with strong antibacterial effect. Besides, commonly expressed C. accolens proteins with recognized
antimicrobial activity, including the glycosyl hydrolase family 25 and N-acetylmuramoyl-Lalanine
amidase as well as many other proteins involved in the survival and adhesion probiotic
properties were identified with various abundance level across strains. Thus, all these results
hold significant promise to develop more targeted therapy for maintaining nasal homeostasis.
For the final part of this thesis, we conducted a combination of in vitro and in vivo studies,
aiming to evaluate the probiotic properties of C. accolens nasal isolates. Healthy nasal C.
accolens strains, C779, C781 and C787 was selected for evaluating potential probiotic features
as they demonstrated paramount effect in terms of antimicrobial property. In our in vitro
experiments, these strains displayed a good adhesion ability to human nasal epithelial cells
(HNECs), able to outcompete S. aureus for HNEC adhesion, and dampen S. aureus-dependent
immune activation with no cytotoxic property. Furthermore, Whole genome sequence analysis
confirmed them as non-virulent with no detectable antibiotic resistant gene associated with a
health risk. In a well-designed in vivo experiment in C. elegans, the strains were found to be
safe and able to protect C. elegans from S. aureus induced toxicity, giving us valuable insights
to launch probiotic C. accolens strains and develop novel probiotic therapy in the near future
for the management of CRS linked with microbial imbalance. Ultimately, these studies open
new paths towards the development of clinically recognized microbiome-targeted treatments,
both probiotics and prebiotics for manipulating a stable microbiome ecosystem in CRS. Further
work involving a randomised controlled trial is necessary to evaluate changes in nasal
microbiota composition and in health outcomes before all treatments can be translated into
clinical practice.
School/Discipline
School of Medicine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2022
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