The bacterial microbiome in chronic rhinosinusitis: Richness, diversity, postoperative changes, and patient outcomes.

BACKGROUND: The bacterial microbiome in chronic rhinosinusitis (CRS) remains poorly understood. Microorganisms are believed to be important contributors to the inflammatory response seen in these patients. OBJECTIVE: To examine the bacterial CRS microbiome by using a pyrosequencing technique and determine the diversity, richness, prevalence, and abundance of bacterial species in these patients. Furthermore, the postoperative changes that occur in the microbiome and correlations with patient outcomes are assessed. METHODS: Swabs were collected from 23 patients with CRS and 11 controls during surgery. Further postoperative swabs were collected in the CRS group. Bacterial DNA was extracted from the swabs and then sequenced by using 16S ribosomal DNA bacterial tag-encoded FLX amplicon pyrosequencing. RESULTS: A total of 456 unique bacterial species were detected. No difference was seen for richness or diversity between the study groups (p > 0.05). Diversity declined after surgery in the CRS group (p = 0.01). Propionibacterium acnes and Staphylococcus epidermidis were the most prevalent species. Several significant differences were determined for prevalence and mean relative abundance (MRA) between the study groups. In particular, Acinetobacter johnsonii was more prevalent and had a higher MRA in the controls. Furthermore, the MRA of this species increased after surgery and was associated with improved quality of life. CONCLUSION: This study characterized the sinonasal microbiome in a group of controls and patients with CRS. Important differences in diversity, prevalence, abundance, and temporal changes were described. Of great interest is the potential association between A. johnsonii and health. These findings provide new insights into the interplay between the microbiome and health in the paranasal sinuses.

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.

The fungal microbiome in chronic rhinosinusitis: richness, diversity, postoperative changes and patient outcomes.

BACKGROUND: Our understanding of fungi in chronic rhinosinusitis (CRS) has been limited by previously employed detection techniques. This study examines the fungal component of the microbiome in CRS patients and controls using a highly sensitive culture-independent molecular technique. The aims of this study include the characterization of fungal richness, prevalence, abundance, temporal changes, and their relationship with patient outcomes. METHODS: Swabs were collected from the sinuses of 23 CRS patients and 11 controls. Collection occurred intraoperatively, and at 6 and 12 weeks postoperatively. DNA was extracted from the swabs and fungal outcomes were determined through 18S ribosomal DNA (rDNA) fungal tag-encoded FLX amplicon pyrosequencing. RESULTS: Fungi were ubiquitous to all patients. A total of 207 fungal genera were detected, with a mean sample richness of 8.18 and 12.14 in the control and CRS groups, respectively. Malassezia was detected in all patients at surgery and was also the most abundant. Postoperatively, fungal richness decreased (p < 0.05) and was associated with declines in the prevalence of Fusarium and Neocosmospora (p < 0.05). Neocosmospora was also less abundant postoperatively (p < 0.05). No correlations were found with quality of life. CONCLUSION: This is the first study to use a highly sensitive pyrosequencing technique to reveal the true diversity of fungi in the sinuses of CRS patients and postoperative changes in richness. The presence of Malassezia, a genus not previously described in the sinuses, is of great interest, and its potential as a disease modifier should see further investigation given its association with atopic disease.

Safety and efficacy of topical bacteriophage and ethylenediaminetetraacetic acid treatment of Staphylococcus aureus infection in a sheep model of sinusitis.

BACKGROUND: Treatment of sinonasal bacterial biofilms continues to be a challenge in modern rhinology. This study's objective was to assess the safety and efficacy of topically applied Cocktail of S. aureus specific phage (CTSA) alone and in combination with ethylenediaminetetraacetic acid (EDTA) for treatment of Staphylococcus aureus biofilms in vivo. METHODS: Using a sheep model of sinusitis, frontal sinuses (n = 6 per treatment) were flushed once daily with a CTSA (2 × 10(6) plaque forming units [PFU]/mL), with or without EDTA (0.075 mg/mL), and compared to a control flush containing saline and heat-inactivated CTSA. Safety was assessed using histology and scanning electron microscopy (SEM) after treatment for 3 days. Efficacy was assessed by quantifying the generation of S. aureus biofilms in the frontal sinuses after 5 days of treatment. Biofilm mass was compared between treatment groups and controls using LIVE/DEAD BacLight staining and confocal scanning laser microscopy to visualize the tissue sections. COMSTAT2 software was used to compute the biofilm mass present on tissue sections. RESULTS: Tissue morphology was conserved, with no significant signs of inflammation, when comparing control and test treatments. Furthermore, SEM analysis indicated test treatments were not toxic or damaging to mucosal cilia. COMSTAT2 quantification of biofilm showed a significant reduction in biofilm levels when comparing the control with CTSA (p = 0.0043), EDTA (p = 0.0095), and CTSA-EDTA (p = 0.0022) treatments. CONCLUSION: Results indicate that CTSA and EDTA are safe and efficacious for short-term topical application against S. aureus infection in a sheep sinusitis model, and have the potential to be translated to a clinical setting.

Probiotic manipulation of the chronic rhinosinusitis microbiome.

BACKGROUND: Staphylococcus aureus (SA) is a key pathogenic component of the chronic rhinosinusitis (CRS) microbiome and is associated with increased disease severity and poor postoperative outcomes. Probiotic treatments potentially offer a novel approach to the management of pathogenic bacteria in these recalcitrant patients through supporting a healthy community of commensal species. This study aims to investigate the probiotic properties of Staphylococcus epidermidis (SE) against SA in a mouse model of sinusitis. METHODS: Twenty C57/BL6 mice received intranasal inoculations of phosphate buffered saline (PBS), SE, SA, or a combination of SE and SA (SE+SA) for 3 days. Following euthanasia, the mouse snouts were harvested and prepared for histological analysis. Counts of periodic acid-Schiff (PAS)-positive goblet cells were the primary outcome measure. RESULTS: Goblet cell counts were significantly higher in both the SA and SE+SA groups compared to those receiving PBS or SE alone (p < 0.05). However, the SE+SA group demonstrated significantly lower goblet cell counts compared to the SA group (p < 0.05). Mice receiving SE alone did not show a significant difference to those receiving PBS (p > 0.05). The presence of SA postinoculation was confirmed by culture in both the SA and SE+SA groups. CONCLUSION: This study confirms the probiotic potential of SE against SA in a mouse model of sinusitis. Although the interactions that occur between many probiotic species and pathogens are yet to be fully understood, studies such as this support further exploration of ecologically-based treatment paradigms for the management of CRS.

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

BACKGROUND: Bacteria 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. METHODS: Sinonasal 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. RESULTS: Microbes 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. CONCLUSIONS: This 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.

The bacteriology of chronic rhinosinusitis and the pre-eminence of Staphylococcus aureus in revision patients.

BACKGROUND: The role of bacteria in the etiopathogenesis of chronic rhinosinusitis (CRS) remains an area of interest. The impact of surgery and factors such as the presence of polyps, asthma, and aspirin sensitivity on the bacterial state are poorly understood. To determine the effect of these factors, this study examines the culture results from a large cohort of CRS patients. METHODS: This retrospective study used the culture results from 513 CRS patients, which were analyzed for species growth and compared to factors such as previous surgery, presence of polyps, aspirin sensitivity, and asthma. Univariate and multivariate logistic regression models were used for statistical analysis. RESULTS: Eighty-three percent (83%) of patients had a positive culture result. The average number of isolates detected per patient was 0.95. S. aureus was the most frequently cultured organism (35%), followed by P. aeruginosa (9%), Haemophilus spp. (7%), and S. pneumonia (5%). Revision patients were more likely to grow S. aureus (p = 0.001), P. aeruginosa (p = 0.044) and have a positive culture (p = 0.001). Asthma was correlated with a positive culture (p = 0.039). No difference was determined between polyp and nonpolyp patients for any of the bacterial outcomes. CONCLUSION: This study highlights important factors in the bacteriology of CRS patients. S. aureus was the most prevalent species identified in our cohort, followed by P. aeruginosa. S. aureus rates of isolation were also significantly higher in patients undergoing revision surgery. No association was found between the presence of nasal polyposis and culture rates.

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

BACKGROUND: 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. METHODS: Forty-eight frontal sinuses were inoculated with Aspergillus fumigatus alone, with 1 of 4 bacteria, or a cilia toxin. Bacterial and fungal biofilm was determined using confocal scanning laser microscopy. Inflammation and cilia integrity were assessed using light microscopy and transmission electron microscopy, respectively. RESULTS: No fungal biofilm formed when inoculated alone. Florid fungal biofilm developed in more than 75% of sinuses associated with bacterial biofilm of all species, except Haemophilus influenzae, which failed to establish bacterial biofilm. Fungal biofilm also established in association with cilia toxin. Significant cilial damage was incited by all bacterial biofilms and cilia toxin, and was associated with fungal proliferation. Fungal biofilm formation did not significantly increase mucosal inflammation or epithelial damage over that caused by the bacteria or cilia toxin alone. CONCLUSION: Bacterial biofilms cause sinonasal mucosal inflammation and epithelial injury, which provides conditions appropriate for fungal biofilm proliferation. The role of cilia in sinonasal mucosal defense against fungal organisms has been demonstrated. Without such an insult, fungal biofilms fail to proliferate in occluded sinuses. Improving cilial recovery postoperatively and treating bacterial biofilms may be key factors in reducing recalcitrance in allergic fungal rhinosinusitis patients.