BackgroundThe 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.ResultsA 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0083-8) contains supplementary material, which is available to authorized users.

This study provides novel evidence of a link between S. aureus biofilms and skewing of the T-cell response toward the T-helper(2) pathway that is independent of superantigen activities. Further research is required to confirm the cause-effect relationship of this association.

MGO is only partially responsible for the antibiofilm activity of manuka honey. Infusion of MGO-negative honey with MGO, however, achieves similar cidality to the equivalent MGO-rich manuka honey.

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.

In the sheep model of endoscopic ICA injury, the muscle patch and U-Clip anastomotic device significantly improved survival, reduced blood loss, and achieved primary hemostasis while maintaining vascular patency.

Despite awareness of the epidemiological burden of otitis media and its risk factors in Indigenous children, studies undertaken since 1985 demonstrate that otitis media remains a significant public health concern in this population.

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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.