The role of bacterial biofilms in chronic rhinosinusitis.

This thesis embodies research investigating the role that bacterial biofilms play in the pathogenesis of chronic rhinosinusitis (CRS). It focuses on their detection on the sinus mucosa of CRS patients and the implications of their presence. Finally, it addresses deficiencies in the innate immune system that may predispose to their development in this condition. Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self-produced matrix and irreversibly attach to a surface. Their extreme resistance to both the immune system as well as medical therapies has implicated them as playing a potential role in the pathogenesis of many chronic diseases. Although their role in many diseases is now well established, their objective presence and importance in CRS remains largely unknown. Chapter 1 of this thesis reviews the current literature pertaining to CRS and biofilms and critically evaluates the small body of research relating to this topic. Chapter 2 describes the development of a sheep model to study the role of bacterial biofilms in rhinosinusitis. It compares the use of traditional electron microscopy (EM) and more recent confocal scanning laser microscopy (CSLM) in the detection of biofilms on the surface of sinus mucosa. The results of this study inferred a causal relationship between biofilms and the macroscopic changes that accompany rhinosinusitis. Furthermore it illustrated the superiority that CSLM has over EM in the imaging of biofilms on sinus mucosa Chapter 3 and 4 outline the results of human studies utilizing the more objective CSLM to evaluate the prevalence of bacterial biofilms on the sinus mucosa of CRS patients and their effect on post-operative mucosal healing. The results of these studies demonstrated a biofilm prevalence of approximately 50% in the CRS population studied and suggested, that biofilm presence may predispose to adverse post-operative outcomes following sinus surgery. Chapter 5 and 6 describe experiments examining the level of the innate immune system’s anti-biofilm peptide lactoferrin, in patients with CRS. Lactoferrin was found to be downregulated at both an mRNA and protein level in the majority of CRS patients, with biofilm positive patients demonstrating the most significant reduction. In summary, this thesis provides further evidence that bacterial biofilms play a major role in the pathogenesis and disease persistence in a subset of CRS patients. Deficiencies in components of the innate immune system, such as lactoferrin, may play an important role in the predisposition of certain individuals to the initial development of bacterial biofilms.Thesis (Ph.D.) -- University of Adelaide, School of Medicine 200

Data from: Sinonasal microbiome sampling: a comparison of techniques

Background: The role of the sino-nasal microbiome in CRS remains unclear. We hypothesized that the bacteria within mucosal-associated biofilms may be different from the more superficial-lying, free-floating bacteria in the sinuses and that this may impact on the microbiome results obtained. This study investigates whether there is a significant difference in the microbiota of a sinonasal mucosal tissue sample versus a swab sample. Methods: Cross-sectional study with paired design. Mucosal biopsy and swab samples were obtained intra-operatively from the ethmoid sinuses of 6 patients with CRS. Extracted DNA was sequenced on a Roche-454 sequencer using 16S-rRNA gene targeted primers. Data were analyzed using QIIME 1.8 software package. Results: At a maximum subsampling depth of 1,100 reads, the mean observed species richness was 33.3 species (30.6 for swab, versus 36 for mucosa; p > 0.05). There was no significant difference in phylogenetic and non-phylogenetic alpha diversity metrics (Faith’s PD_Whole_Tree and Shannon’s index) between the two sampling methods (p > 0.05). The type of sample also had no significant effect on phylogenetic and non-phylogenetic beta diversity metrics (Unifrac and Bray-Curtis; p > 0.05). Conclusion: We observed no significant difference between the microbiota of mucosal tissue and swab samples. This suggests that less invasive swab samples are representative of the sinonasal mucosa microbiome and can be used for future sinonasal microbiome studies

Characterization of B-cell subpopulations in patients with chronic rhinosinusitis

Background: Recent research suggest that B and plasma cells may play an important role in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). The purpose of this study was to subcharacterize the B cell response in the sinus mucosa of control and CRS patients. Methods: Representative tissue samples and peripheral blood samples were obtained from controls, CRS without nasal polyps (CRSsNP) and CRSwNP. Using single-cell suspension flow cytometry these samples were analyzed for overall and stage-specific B and plasma cell percentages. Results: Both atopic and nonatopic CRSwNP patients showed an increase in local numbers of naive, active, and memory B cells compared to controls. CRSsNP patients only showed local elevations of naive B cells. Plasma cells were only significantly elevated in the sinus tissue of atopic CRSwNP patients. These local tissue increases did not correlate with increased numbers of circulating B cells. Conclusion: This study provides further evidence of an important role of B cells in CRSwNP patients. The local increase appears to be independent of a systemic response. (C) 2013 ARS-AAOA, LLC

Chronic Rhinosinusitis, S. aureus Biofilm and Secreted Products, Inflammatory Responses, and Disease Severity

Chronic rhinosinusitis (CRS) is a persistent inflammation of the nasal cavity and paranasal sinuses associated with tissue remodelling, dysfunction of the sinuses’ natural defence mechanisms, and induction of different inflammatory clusters. The etiopathogenesis of CRS remains elusive, and both environmental factors, such as bacterial biofilms and the host’s general condition, are thought to play a role. Bacterial biofilms have significant clinical relevance due to their potential to cause resistance to antimicrobial therapy and host defenses. Despite substantial medical advances, some CRS patients suffer from recalcitrant disease that is unresponsive to medical and surgical treatments. Those patients often have nasal polyps with tissue eosinophilia, S. aureus-dominant mucosal biofilm, comorbid asthma, and a severely compromised quality of life. This review aims to summarise the contemporary knowledge of inflammatory cells/pathways in CRS, the role of bacterial biofilm, and their impact on the severity of the disease. Here, an emphasis is placed on S. aureus biofilm and its secreted products. A better understanding of these factors might offer important diagnostic and therapeutic perceptions for recalcitrant disease

<i>Corynebacterium accolens</i> Has Antimicrobial Activity against <i>Staphylococcus aureus</i> and Methicillin-Resistant <i>S. aureus</i> Pathogens Isolated from the Sinonasal Niche of Chronic Rhinosinusitis Patients

Corynebacterium accolens is the predominant species of the healthy human nasal microbiota, and its relative abundance is decreased in the context of chronic rhinosinusitis (CRS). This study aimed to evaluate the antimicrobial potential of C. accolens isolated from a healthy human nasal cavity against planktonic and biofilm growth of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) clinical isolates (CIs) from CRS patients. Nasal swabs from twenty non-CRS control subjects were screened for the presence of C. accolens using microbiological and molecular techniques. C. accolens CIs and their culture supernatants were tested for their antimicrobial activity against eight S. aureus and eight MRSA 4CIs and S. aureus ATCC25923. The anti-biofilm potential of C. accolens cell-free culture supernatants (CFCSs) on S. aureus biofilms was also assessed. Of the 20 nasal swabs, 10 C. accolens CIs were identified and confirmed with rpoB gene sequencing. All isolates showed variable antimicrobial activity against eight out of 8 S. aureus and seven out of eight MRSA CIs. Culture supernatants from all C. accolens CIs exhibited a significant dose-dependent antibacterial activity (p S. aureus and MRSA CIs. This inhibition was abolished after proteinase K treatment. C. accolens supernatants induced a significant reduction in metabolic activity and biofilm biomass of S. aureus and MRSA CIs compared to untreated growth control (p C. accolens exhibited antimicrobial activity against S. aureus and MRSA CIs in both planktonic and biofilm forms and holds promise for the development of innovative probiotic therapies to promote sinus health

APTC-C-SA01: A Novel Bacteriophage Cocktail Targeting Staphylococcus aureus and MRSA Biofilms

The high infection and mortality rate of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the urgent development of new treatment strategies. Bacteriophages (phages) have several advantages compared to antibiotics for the treatment of multi-drug-resistant bacterial infections, and thus provide a promising alternative to antibiotics. Here, S. aureus phages were isolated from patients and environmental sources. Phages were characterized for stability, morphology and genomic sequence and their bactericidal activity against the biofilm form of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA was investigated. Four S. aureus phages were isolated and tested against 51 MSSA and MRSA clinical isolates and reference strains. The phages had a broad host range of 82&ndash;94% individually and of &gt;98% when combined and could significantly reduce the viability of S. aureus biofilms. The phages had a latent period of &le;20 min and burst size of &gt;11 plaque forming units (PFU)/infected cell. Transmission electron microscopy (TEM) identified phages belonging to the family of Myoviridae. Genomic sequencing indicated the lytic nature of all four phages, with no identified resistance or virulence genes. The 4 phages showed a high complementarity with 49/51 strains (96%) sensitive to at least 2/4 phages tested. Furthermore, the frequency of bacteriophage insensitive mutant (BIM) generation was lower when the phages were combined into the phage cocktail APTC-C-SA01 than for bacteria exposed to each of the phages alone. In conclusion, APTC-C-SA01, containing four lytic S. aureus phages has the potential for further development as a treatment against MSSA and MRSA infections

Cytokine-Induced Modulation of SARS-CoV2 Receptor Expression in Primary Human Nasal Epithelial Cells

Background: Viral entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) via the spike protein enables endocytosis into host cells using the ACE2 receptor and TMPRSS2. The frequent upper respiratory tract symptoms of COVID-19 and the localization of the virus to the nasopharynx, the most common site of swabbing, indicate that the sinonasal mucosa may play an important role in SARS-CoV2 infection and viral replication. Methods: This paper investigates the presence of ACE2 receptor and TMPRESS2 expression in the primary human nasal epithelial cells (HNECs) from the following: chronic rhinosinusitis without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP) and control (non-CRS) patients, and maps the expression changes when exposed to Th1, Th2, Th17-associated cytokines. Results: We found that ACE2 and TMPRSS2 expression was higher in control HNECs than CRSwNP HNECs, and that both ACE2 and TMPRSS2 were downregulated further by Th2 cytokines in CRSwNP HNECs. Conclusions: This indicates an immune dysregulated state of CRSwNP mucosa, which normally contributes to a chronic inflammatory state, and might support an altered susceptibility to SARS-CoV2 infection and transmission

APTC-EC-2A: A Lytic Phage Targeting Multidrug Resistant E. coli Planktonic Cells and Biofilms

Escherichia coli (E. coli) are common bacteria that colonize the human and animal gastrointestinal tract, where they help maintain a balanced microbiome. However, some E. coli strains are pathogenic and can cause serious infectious diseases and life-threatening complications. Due to the overuse of antibiotics and limited development of novel antibiotics, the emergence of antibiotic-resistant strains has threatened modern medicine, whereby common infections can become lethal. Phage therapy has once again attracted interest in recent years as an alternative treatment option to antibiotics for severe infections with antibiotic-resistant strains. The aim of this study was to isolate and characterize phage against multi-drug resistant E. coli isolated from clinical samples and hospital wastewater. For phage isolation, wastewater samples were collected from The Queen Elizabeth Hospital (Adelaide, SA, Australia) followed by phage enrichment as required. Microbiological assays, electron microscopy and genomic sequencing were carried out to characterize the phage. From the 10 isolated E. coli phages, E. coli phage APTC-EC-2A was the most promising and could lyse 6/7 E. coli clinical isolates. APTC-EC-2A was stable at a broad pH range (3&ndash;11) and could lyse the host E. coli at temperatures ranging between 30&ndash;50 &deg;C. Furthermore, APTC-EC-2A could kill E. coli in planktonic and biofilm form. Electron microscopy and genomic sequencing indicated the phage to be from the Myoviridae family and of lytic nature. In conclusion, the newly isolated phage APTC-EC-2A has the desired properties that support its potential for development as a therapeutic agent against therapy refractory E. coli infections

Rarefaction plots.

<p>(2A) Rarefaction plots of all 12 samples (one curve per sample) showing curves reaching asymptote at the cut-off of 1100 reads. (2B) Rarefaction curves (number of observed species on Y axis) for mucosal tissue (blue curve) versus swab (red curve). Richness at the 1100 cut-off was 36 for mucosal tissue, versus 30.6 for swab. (p > 0.05). (2C) Rarefaction curves (Faith’s Phylogenetic Diversity “PD_Whole_Tree” on Y axis) for mucosal tissue (blue curve) versus swab (red curve). (p > 0.05). (2D) Rarefaction curves (Shannon’s index on Y axis) for mucosal tissue (blue curve) versus swab (red curve).</p