Staphylococcus aureus from patients with chronic rhinosinusitis show minimal genetic association between polyp and non-polyp phenotypes

Background: Staphylococcus aureus has a high prevalence in chronic rhinosinusitis (CRS) patients and is suggested to play a more etiopathogenic role in CRS patients with nasal polyps (CRSwNP), a severe form of the CRS spectrum with poorer surgical outcomes. We performed a microbial genome-wide association study (mGWAS) to investigate whether S. aureus isolates from CRS patients have particular genetic markers associated with CRS with nasal polyps (CRSwNP) or CRS without nasal polyps (CRSsNP). Methods: Whole genome sequencing was performed on S. aureus isolates collected from 28 CRSsNP and 30 CRSwNP patients. A mGWAS approach was employed using large-scale comparative genomics to identify genetic variation within our dataset. Results: Considerable genetic variation was observed, with >90,000 single nucleotide polymorphisms (SNPs) sites identified. There was little correlation with CRS subtype based on SNPs and Insertion/Delection (Indels). One indel was found to significantly correlate with CRSwNP and occurred in the promoter region of a bacitracin transport system ATP-binding protein. Additionally, two variants of the highly variable superantigen-like (SSL) proteins were found to significantly correlate with each CRS phenotype. No significant association with other virulence or antibiotic resistance genes were observed, consistent with previous studies. Conclusion: To our knowledge this study is the first to use mGWAS to investigate the contribution of microbial genetic variation to CRS presentations. Utilising the most comprehensive genome-wide analysis methods available, our results suggest that CRS phenotype may be influenced by genetic factors other than specific virulence mechanisms within the S. aureus genome

Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data

Published: 5 May 2015Background: The rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites. Results: A workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types. Conclusions: The application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts.Jake Jervis-Bardy, Lex E X Leong, Shashikanth Marri, Renee J Smith, Jocelyn M Choo, Heidi C Smith-Vaughan, Elizabeth Nosworthy, Peter S Morris, Stephen O'Leary, Geraint B Rogers and Robyn L Mars

Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR

Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

Next generation sequencing and the microbiome of chronic rhinosinusitis: a primer for clinicians and review of current research, its limitations, and future directions

Objective: Microbiomics in chronic diseases, including chronic rhinosinusitis (CRS), have undergone rapid advances in recent times. The introduction of Next Generation Sequencing (NGS) technology has produced significant clinical insights regarding the bacteriology of these conditions. We review studies that have used 16S rRNA sequencing to specifically investigate the microbiota profiles of patients with CRS in a variety of contexts. Methods: Literature review using the CINAHL, MEDLINE, PUBMED, and the Cochrane databases. Papers utilizing 16S-sequencing technology on CRS specimens published between January 1, 1995, and October 31, 2015, were included. Studies limited to only healthy controls were excluded. Results: Consistent with published studies using non-NGS techniques, the main genera commonly identified from the sinuses of CRS patients included Staphylococcus, Propionibacterium, and Corynebacterium. The microbiome of CRS patients had lower bacterial diversity compared to controls in a number of studies. Also consistent with non-NGS-based studies, Staphylococcus was implicated as an important genus, with highly colonized patients having worse surgical outcomes. Conflicting reports of antibiotic effects on the CRS microbiome were observed. Sampling methods were well investigated, many of the studies reviewed failed to include important methodological detail. Conclusion: While 16S sequencing is a novel microbiological laboratory method, current studies have confirmed our existing understanding of bacteriology of CRS without providing significant additional clinical insight. Complementing 16S studies with more complex NGS methods while developing robust clinical studies aimed at shifting the disrupted CRS microbiome will provide researches with the opportunity to derive further clinical insight and develop new therapeutic targets.Jake Jervis Bardy and Alkis J. Psalti

Do biofilms contribute to the initiation and recalcitrance of chronic rhinosinusitis?

Chronic rhinosinusitis is a common disease whose underlying aetiopathogenesis has not been completely understood. Amongst a range of other potential environmental triggers in this disease, a role has recently been proposed for bacterial biofilms. Adopting the biofilm paradigm to explain the initiation and maintenance of this disease may help to clarify previous inconsistencies in this disease that have resulted in the role of bacteria being questioned. Of particular interest is the association of bacterial biofilms with recalcitrant disease states. Over the last five years, research has progressed rapidly since biofilms were first identified on the surface of diseased sinonasal mucosa. Their presence there has now been associated with more severe disease that is often recalcitrant to current management paradigms. Technological advances are allowing accurate characterization of the bacterial and fungal species within these biofilms, which would appear to be an important step in improving our understanding of how these bacterial communities might interact with the host to cause disease. This is an unanswered, yet highly important, question in this field of research that will undoubtedly be an area of investigation in the near future. As the body of evidence suggesting biofilms may be involved in this disease grows, research interest has switched to the development of antibiofilm therapies. Given the unique properties of bacteria existing in this form, biofilm eradication strategies will need to incorporate novel medical therapies into established surgical practices as we attempt to improve the outcomes of our most difficult patients.Andrew Foreman, Joshua Jervis-Bardy, Peter-John Wormal

Impaired mucosal healing and infection associated with Staphylococcus aureus after endoscopic sinus surgery

Background: Differentiating normal nasal discharge and postoperative crust from infection after endoscopic sinus surgery (ESS) can be difficult. We investigated whether bacteria cultured at operation was predictive for postoperative mucosal infection—defined as frank pus, thick mucus, and/or abnormal crusting seen on endoscopic examination associated with positive microbiology. Methods: The records of 48 patients with chronic rhinosinusitis (CRS) with infection at the time of ESS were retrospectively reviewed. Results of intraoperative cultures were compared with those taken at the time of postoperative mucosal infection. Results: Fourteen of 16 patients (87.5%) with intraoperative infection with Staphylococcus aureus progressed to postoperative mucosal infection with S. aureus, whereas patients who cultured “other” bacteria intraoperatively progressed to postoperative mucosal infection in 6/19 cases (31.6%), with S. aureus cultured in only 3/19 cases (15.8%; p = 0.0001). Conclusion: S. aureus infection at ESS predicts for abnormal, S. aureus-associated mucosal healing and infection post-ESS. Although a prospective trial is warranted, these findings suggest a future role for aggressive anti-S. aureus therapy peri- and/or postoperatively in patients who culture positive for this organism to improve postsurgical outcomes.Joshua Jervis-Bardy, Andrew Foreman, John Field, Peter John Wormal