Investigating Microbiome -Targeted Treatments for Chronic Rhinosinusitis: A novel approach to combat dysbiosis in the nasal microbiome

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.