Intracellular staphylococcus aureus in chronic rhinosinusitis.

Abstract

Chronic Rhinosinusitis (CRS) is a heterogeneous disease characterised by recurrent and persistent episodes of nasal obstruction, discharge and facial pain or pressure. Patients suffering from CRS experience considerable morbidity and have impaired qualities of life. The gold standard treatment of cases that fail medical therapy is endoscopic sinus surgery (ESS). Despite the proven efficacy of ESS, the modern sinus surgeon will see a subset of patients who persistently fail any attempt to improve their disease profile. Recent research into CRS had identified bacterial biofilms, in particular those mediated by Staphylococcus aureus to hold a potential role in the aetiopathogenesis of this disease. Patients with biofilms suffer from more severe preoperative symptoms and have worse postoperative outcomes. As a consequence, numerous anti-biofilm therapies have been developed including biofilm dispersal agents and biocidal agents. Despite showing early promise in vitro, the use of these therapeutic agents in vivo has not translated to a conclusive clinical benefit. Recent studies have identified that S. aureus can invade non-professional phagocytic cell types such as epithelium with the ability to survive and replicate intracellularly. This led to the hypothesis that by exploiting the intracellular environment, bacteria may evade host immunity, topical antimicrobial therapy and establish a niche for survival with potential reservoirs for chronic or relapsing Staphylococcal infections. Therefore, this PhD thesis set out to investigate whether intracellular S. aureus plays a disease modifying role in CRS. Chapter 1 critically reviews the context of the work included in this thesis pertaining to CRS, S. aureus, biofilms and intracellular infections. Chapter 2 validates a novel imaging technique using confocal scanning laser microscopy (CSLM) coupled with dual staining of fluorescence in situ hybridisation (FISH) probes and nucleic acid counterstains (propidium iodide, [PI]), to identify the presence of intracellular S. aureus in whole mucosal specimens, with a direct comparison to previously reported techniques of immunohistochemistry (IHC). The study reported the benefits and drawbacks of each technique, and identified specific roles for their use when examining tissue specimens. The major advantage of CSLM-FISH/PI was that simultaneous biofilm analysis was possible in the same piece of tissue. Chapter 3 investigated the unexpected phenomenon of false-positive antibody binding in S. aureus infected tissue specimens when performing IHC in paraffin embedded tissue sections. This was hypothesised to be caused by protein A expression in the bacterial cell wall that continued to bind IgG-class antibodies with high affinity. A methodology was developed and validated to overcome this issue, with significant implications when performing future IHC experiments. Chapter 4 utilised the previously reported CSLM-FISH/PI protocols for intracellular S. aureus detection in a cohort of CRS and control patients. For the first time the association between biofilms and intracellular infection was reported, suggesting that the biofilm may offer a conditioned environment to allow invasion of S. aureus to deeper tissue layers. Chapter 5 followed a wider cohort of patients in their postoperative course in order to ascertain whether a relationship between intracellular infection and disease recalcitrance could be identified. The results found that intracellular S. aureus infection at the time of surgery was significantly associated with failure of medical and surgical therapy in the postoperative patients. This reinforced the theory that the intracellular location provides bacteria with a protective niche where they can avoid host elimination and topical antimicrobial therapy. Chapter 6 investigated whether the concept of bacterial phenotype switching following intracellular infection in airway epithelial cells occurs as a mechanism of allowing these organisms to decrease their virulence and evade innate immunity. It was found that S. aureus reduces production of its superantigenic enterotoxins as a consequence of internalisation; however, this reduction in virulence was reversible after lysing the host cells and a single sub-culture step. Additionally, for the first time we demonstrated that intramucosal organisms harvested from sinonasal biopsies demonstrate altered phenotypic growth patterns and lack of coagulase activity consistent with small colony variants (SCV). This represented another potential explanation for why bacteria are so capable of internalising and persisting in epithelial tissues. The findings of this thesis have provided novel insights alluding to a role of intracellular S. aureus in CRS. The versatility of S. aureus in altering its phenotypic characteristics to take advantage of the local environment makes it troublesome to fully eradicate and significant associations can be made between intracellular infection and recalcitrant disease. Future research should be directed towards identifying novel treatment strategies that can effectively target intracellular organisms.