Lorwai Tan

The microbiome of chronic rhinosinusitis: culture, molecular diagnostics and biofilm detection

BackgroundBacteria and fungi are believed to influence mucosal inflammation in chronic rhinosinusitis (CRS). However their presence and relationship to disease is debated. This study used multiple detection methods to compare microbial diversity and microbial abundance in healthy and diseased sinonasal mucosa. The utility of contemporary detection methods is also examined.MethodsSinonasal mucosa was analyzed from 38 CRS and 6 controls. Bacterial and fungal analysis was performed using conventional culture, molecular diagnostics (polymerase chain reaction coupled with electrospray ionization time-of-flight mass spectrometry) and fluorescence in situ hybridization.ResultsMicrobes were detected in all samples, including controls, and were often polymicrobial. 33 different bacterial species were detected in CRS, 5 in control patients, with frequent recovery of anaerobes. Staphylococcus aureus and Propionibacterium acnes were the most common organisms in CRS and controls, respectively. Using a model organism, FISH had a sensitivity of 78%, and a specificity of 93%. Many species were detected in both CRS and controls however, microbial abundance was associated with disease manifestation.ConclusionsThis study highlights some cornerstones of microbial variations in healthy and diseased paranasal sinuses. Whilst the healthy sinus is clearly not sterile, it appears prevalence and abundance of organisms is critical in determining disease. Evidence from high-sensitivity techniques, limits the role of fungi in CRS to a small group of patients. Comparison with molecular analysis suggests that the detection threshold of FISH and culture is related to organism abundance and, furthermore, culture tends to select for rapidly growing organisms.

A sheep model for the study of biofilms in rhinosinusitis.

Background Bacterial biofilms have been shown in chronic diseases such as cystic fibrosis, cholesteatoma, and otitis media with effusion. Recently, their detection on the mucosal tissue of sinusitis patients has implicated them in the pathogenesis of this condition. We present an animal model using sheep experimentally infected with Staphylococcus aureus to study the possible association between biofilm and sinusitis. Methods Twenty-four sheep underwent bilateral endoscopic sinus surgery to identify their frontal ostia. The frontal sinuses were treated in one of the following ways according to preoperative randomization: (1) ostium left patent, (2) ostium left patent and bacteria instilled, (3) ostium occluded, or (4) ostium occluded and bacteria instilled. The frontal mucosa was harvested at day 7 and examined for biofilm presence using confocal scanning laser microscopy (CSLM) as well as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results All three modalities showed different rates of biofilm detection. Three-dimensional structures that could be interpreted as biofilms were documented in 86% (n = 36) of the sinuses analyzed using SEM. These structures were seen in all four study groups. The detection rate using the other two modalities was much lower with CSLM, showing biofilms in 48% (n = 20) and TEM in only 29% (n = 12) of the sinuses analyzed. Unlike SEM, these two modalities only detected bacterial biofilms in sinuses randomized to bacterial instillation. Conclusion The demonstration of bacterial biofilms in this animal model of sinusitis further supports the hypotheses that biofilms may play a role in the pathogenesis of this condition. There is an obvious discrepancy in the sensitivity and specificity of biofilm detection using the three modalities mentioned. CSLM appears to be the most objective technique. The inherent flaws, sampling error, and subjectivity involved in SEM and TEM make these less reliable in documenting biofilm existence.

Methylglyoxal‐infused honey mimics the anti‐Staphylococcus aureus biofilm activity of manuka honey: Potential Implication in Chronic Rhinosinusitis

Low pH, hydrogen peroxide generation, and the hyperosmolarity mechanisms of antimicrobial action are ubiquitous for all honeys. In addition, manuka honey has been shown to contain high concentrations of methylglyoxal (MGO), contributing the relatively superior antimicrobial activity of manuka honey compared to non‐MGO honeys. In high concentrations, manuka honey is effective in killing Staphylococcus aureus biofilms in vitro. Lower concentrations of honey, however, are desirable for clinical use as a topical rinse in chronic rhinosinusitis in order to maximize the tolerability and practicality of the delivery technique. This study, therefore, was designed to evaluate the contribution of MGO to the biofilm‐cidal activity of manuka honey, and furthermore determine whether the antibiofilm activity of low‐dose honey can be augmented by the addition of exogenous MGO.

Th2 immunological inflammation in allergic fungal sinusitis, nonallergic eosinophilic fungal sinusitis, and chronic rhinosinusitis

Background Noninvasive fungal sinusitis is a heterogenous group of conditions including allergic fungal sinusitis (AFS) and nonallergic eosinophilic fungal sinusitis (NEFS). Th2-mediated cascades have been postulated to be the major inflammatory response in patients with AFS although other mechanisms also may be involved. The detailed mucosal Th2 cytological status of NEFS still has not been studied in great depth. Methods Using a meticulous patient selection algorithm over a 2-year period, infundibular mucosal tissue from patients with AFS, NEFS, chronic rhinosinusitis (CRS), and normal controls was studied (n = 59). Immunohistochemistry for mast cells, eosinophils, and immunoglobulin E (IgE) cells was performed and cell counts per unit area were measured. Results Mast cell, eosinophil, and IgE+ cell numbers were significantly raised in patients with AFS, NEFS, and CRS when compared with controls. There was no significant difference between cell numbers in patients with AFS and NEFS. Conclusion Patients with AFS exhibit a classic Th2 inflammatory response in nasal mucosal tissue with NEFS and CRS patients showing evidence of a similar Th2 cascade, including the presence of IgE+ cells.

Response of Peripheral Blood Lymphocytes to Fungal Extracts and Staphylococcal Superantigen B in Chronic Rhinosinusitis

Background: Previous studies have suggested that chronic rhinosinusitis may result from a hypersensitivity response of the nasal mucosa to the presence of fungal antigens or staphylococcal superantigens in the nasal mucus. Both of these groups of antigens are present so frequently in the nasal mucus of patients with chronic rhinosinusitis that their presence together is likely to be a common event.

Aspergillus fumigatus biofilm on primary human sinonasal epithelial culture

Background Bacterial biofilms have been implicated in chronic rhinosinusitis (CRS). However, direct evidence in support of fungal biofilms in sinus disease is lacking in the literature. This study was designed to develop and characterize an in vitro Aspergillus fumigatus biofilm model on primary human sinonasal epithelial cell culture. Methods Sinonasal biopsy specimens harvested during endoscopic sinus surgery of six CRS patients and three pituitary tumor (control) patients were cultured in Dulbeccos modified Eagle media (DMEM; Invitrogen)/Hams F12 airway media to encourage epithelial cell proliferation. Epithelial cells separated by immunomagnetic beads were seeded in tissue culture-treated Y-shaped microslides. At confluence the primary cultures were inoculated with A. fumigatus spores. Fungus was allowed to germinate and form biofilms under two in vitro conditions: (1) static (no flow through of media) and (2) continuous flow coculture (continuous flow movement of media). At regular intervals cocultures were stained with FUN-1, concanavalin A–alexa fluor 488, and examined by confocal scanning laser microscopy. Comstat software was used to assess biomass and thickness. Results A. fumigatus formed three-dimensional biofilm structures with parallel-packed, cross-linked hyphae and channels/passages. Metabolically active hyphae showed orange–red fluorescing intravacuolar structures. Extracellular matrix (ECM) between/around the hyphae fluoresced intense green. A. fumigatus biofilms development occurred in five stages: (1) conidial attachment to epithelial cells, (2) hyphal proliferation, (3) ECM production, (4) hyphal parallel packing and cross-linking, and (5) channel/pores formation. Mature biofilms showed basal conidial, middle hyphal, and superficial ECM layers. Biofilms formed underflow conditions displayed more robust and faster growth kinetics when compared with that under static conditions, with a thick, stocky, wrinkly/undulating hyphal growth and extensive ECM production. The differences in biomass and average thickness of the cocultures under static and flow conditions were statistically significant after similar periods of incubation (p = 0.0002; p < 0.0001, respectively. Conclusion To our knowledge, this is the first article of an in vitro model characterizing A. fumigatus biofilm formation using primary human sinonasal epithelium under different growth conditions.

Human cathelicidin antimicrobial peptide is up-regulated in the eosinophilic mucus subgroup of chronic rhinosinusitis patients

Background Eosinophilic mucus chronic rhinosinusitis (EMCRS) patients are a subgroup of CRS with a poorer prognosis due to frequent recurrences of disease. The cathelicidin antimicrobial peptide (CAMP) is an important innate defense peptide but its role in CRS is not well characterized. The purpose of this study was to investigate CAMP mRNA and protein expression from EMCRS, CRS, and normal control patients. Methods Biopsy specimens of nasal mucosa and nasal polyps were taken from 59 CRS patients and 9 controls. CAMP mRNA and protein levels were analyzed by real-time quantitative reverse-transcriptase polymerase chain reaction, immunoassay, Western blot, and immunohistochemistry. Results The expression of CAMP mRNA was significantly increased in EMCRS patients compared with CRS patients (p = 0.0004). By immunohistochemistry, expression of CAMP was localized to nasal epithelial, submucosal glands, and inflammatory subepithelial cells. Western blotting confirmed the presence of CAMP in EMCRS, CRS, and control patients. However, we did not detect statistically significant differences in the protein levels in tissue homogenates between EMCRS, CRS, and control patients. Conclusion This study shows expression of CAMP in nasal mucosa supporting its role in innate defenses against inhaled pathogens. Although CAMP mRNA was up-regulated in EMCRS patients, there were no statistically significant differences in protein levels in the nasal mucosa of EMCRS compared with CRS patients and controls.

Bacterial-induced epithelial damage promotes fungal biofilm formation in a sheep model of sinusitis.

Fungal biofilms have been discovered in chronic rhinosinusitis (CRS) patients, but factors contributing to their establishment are obscure. A recent animal study showed bacterial co‐inoculation was required. We examine the role of 4 bacterial species and a cilia toxin on fungal biofilm formation in a sheep sinusitis model. The importance of epithelial integrity on fungal biofilm formation is also examined.

Human lysozyme has fungicidal activity against nasal fungi

Background The cationic antimicrobial peptide lysozyme is the most prevalent innate immune protein in nasal secretions but there is a paucity of research regarding its role in paranasal sinus disease. Lysozyme is generally regarded as an antibacterial agent; however, some data suggest activity toward yeast. This study was designed to determine if lysozyme displays fungicidal activity toward fungi commonly identified in patients with chronic rhinosinusitis (CRS) or fungal sinusitis. Methods Using a colony-forming unit assay the fungicidal activity of lysozyme (0, 0.5, 5, and 50 micromolar; 0- to 7-hour treatment) was tested against strains of Aspergillus fumigatus, the yeast Candida albicans, and other fungi commonly identified in mucin of patients with CRS. Fungi cultured directly from the mucin of two CRS patients were also tested to determine if they were resistant to the fungicidal activity of lysozyme. Results The fungicidal effect of lysozyme was both concentration and time dependent. After 7-hour treatment lysozyme (5 micromolar) had >80% fungicidal activity against A. fumigatus, Penicillium sp., Acremonium sp., C. albicans, and Candida parapsilosis. The fungicidal activity of lysozyme toward Alternaria alternata could not be determined. Lysozyme was also fungicidal toward the clinical isolates A. fumigatus and Aspergillus terreus cultured from the mucin of CRS patients. Conclusion Lysozyme displays fungicidal activity toward many fungi commonly identified in patients with CRS, as well as clinical fungi isolates cultured from the mucin of CRS patients. Additional studies are required to determine the regulation of lysozyme in CRS.

Immunomodulatory effect of cytosine-phosphate-guanosine (CpG)-oligonucleotides in nonasthmatic chronic rhinosinusitis: an explant model.

Background The use of cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) or immunostimulatory sequences (ISSs) in the treatment of airway diseases is gaining interest. Binding of the CpG-ODN ligand to Toll-like receptor 9 (TLR9) triggers a shift from a Th2- to a Th1-type response in the target tissue. In this study, we explored the potential use of CpG-ODN to dampen the predominantly Th2-driven chronic inflammatory state in our cohort of patients. Methods An in vitro explant model comprising of sinonasal tissue from patients with asthma (n = 12) and without asthma (n = 11) were stimulated with CpG-ODN or Staphylococcus aureus enterotoxin B (SEB) or CpG-ODN in combination with SEBfor 48 hours. Ten of the 12 asthma patients had nasal polyps. RNA was extracted for multiplex real-time reverse transcription polymerase chain reaction analysis and the 2−delta deltaCT method used to determine interleukin (IL)-5, p35 IL-12, interferon (IFN) gamma, and TLR9 expression levels. Results CpG-ODN significantly reduced IL-5 mRNA expression in patients without asthma (p = 0.0379) but not in the asthma-associated group. SEB alone caused an increase in IL-5 levels that could be dampened when CpG-ODN was added in combination with SEB. Significant differences in mean IL-5 expression levels between the asthmatic and nonasthmatic categories were detected (Welch t-test; **p = 0.0041). Asthmatic and nonasthmatic patients present as two distinct categories as reflected by significant differences in their IL-5 response to CpG-ODN (F = 11.93; ***p = 0.0008), SEB (F = 41.34; *p = 0.0476) and CpG-ODN with SEB (F = 13.2; *p = 0.0114). In contrast, no significant differences were observed in the expression levels of IL-12, IFN-gamma, and TLR9. Conclusion Localized application of CpG-ODN on its own or in combination with SEB may potentially reduce the expression of the proinflammatory cytokine IL-5 in nonasthmatic patients and may be further developed as an immunotherapeutic agent.