Bacterial biofilm matrices were demonstrated through staining with lectins.

<p>Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) (green) and propidium iodide (red) to stain for DNA. Co-localisation of the DNA and lectins are observed (yellow) suggesting that these are part of a biofilm structure. Bacterial biofilm can be observed (arrow) with bacteria being surrounding by a matrix that binds the lectin. Scale 10 µm.</p

Summary of demographic data and the pathogens identified in MEE from 24 children undergoing VTI for rAOM. Pathogens were identified using standard culture and FISH, and viability was determined using BacLight staining.

<p>Biofilm and intracellular bacterial presence were also assessed.</p><p>Abbreviations: EUB – eubacterial probe, Spn – <i>S. pneumoniae</i> (organism and probe), Hi –<i>H. influenzae</i> (organism and probe), Mcat – <i>M. catarrhalis</i> (organism and probe), PA – <i>P. aeruginosa</i> (organism and probe), Sau – <i>S. aureus</i> (organism and probe), AO – <i>A. otitidis</i> (organism). na – Not available, Abx – taking antibiotics currently, U – Unknown, NP – nasopharyngeal culture.</p

Dornase alfa (Pulmozyme®) treatment of MEE results in disintegration of the NET structures.

<p>DNA staining of MEEs from children with rAOM treated with Dornase alfa or left untreated. In the top panel, a control MEE untreated with Dornase alfa where extensive DNA stranding, epithelial cells and bacteria are evident. When treated with Dornase alfa even at concentrations as low as 5ug/ml complete fragmentation of the strands in the MEE are seen and nothing remains on the slide at 1 mg/ml.</p

Most DNA present in the MEE was negative for actin, indicating it was actively produced and not released due to necrosis.

<p>MEE were stained with phalloidin to detect actin presence. MEEs were treated with the nucleic acid stain SYTO9 (green) and TRITC labelled Phalloidin (red). A) No red fluorescence was observed in most samples indicating actin was not present. This compared to B) where some actin is present (red). Even when the samples were positive for actin, this was not extensive, suggesting a combination of active DNA release and the presence of necrotic neutrophils is likely.</p

Multi-species bacterial biofilms containing known otopathogens, were demonstrated in the MEE of children with rAOM.

<p>Representative merged maximum intensity projection confocal image from FISH and Hoechst stained MEE (Child 3). MEE was culture positive for <i>S. aureus</i>. FISH: <i>S. pneumoniae</i> (AF488 green); universal EUB338 probe (AF546 red) <i>H. influenzae</i> (AF633 grey) Hoechst 33342 staining of host cell nuclei (blue). Chains of <i>S. pneumoniae</i> can be observed in this sample as well as those which are intracellular. Other unidentified bacteria (red) can be seen in chains and microcolony structures throughout this MEE.</p

Most DNA present in the MEE was neutrophil-derived.

<p>MEE stained with neutrophil elastase AlexaFluor647 (Red) and propidium iodide (Yellow) and. A) Co-localisation of DNA and neutrophil elastase is present throughout the sample indicating the DNA that is present is neutrophil derived. Scale 50 µm. B) Co-localisation of DNA and neutrophil elastase is present throughout the sample as well as being bacterially associated throughout the sample (arrows). Scale 10 µm.</p

Genome-Wide Association Study to Identify the Genetic Determinants of Otitis Media Susceptibility in Childhood

<div><h3>Background</h3><p>Otitis media (OM) is a common childhood disease characterised by middle ear inflammation and effusion. Susceptibility to recurrent acute OM (rAOM; ≥3 episodes of AOM in 6 months) and chronic OM with effusion (COME; MEE ≥3 months) is 40–70% heritable. Few underlying genes have been identified to date, and no genome-wide association study (GWAS) of OM has been reported.</p> <h3>Methods and Findings</h3><p>Data for 2,524,817 single nucleotide polymorphisms (SNPs; 535,544 quality-controlled SNPs genotyped by Illumina 660W-Quad; 1,989,273 by imputation) were analysed for association with OM in 416 cases and 1,075 controls from the Western Australian Pregnancy Cohort (Raine) Study. Logistic regression analyses under an additive model undertaken in GenABEL/ProbABEL adjusting for population substructure using principal components identified SNPs at <em>CAPN14</em> (rs6755194: OR = 1.90; 95%CI 1.47–2.45; P_adj-PCA = 8.3×10⁻⁷) on chromosome 2p23.1 as the top hit, with independent effects (rs1862981: OR = 1.60; 95%CI 1.29–1.99; P_adj-PCA = 2.2×10⁻⁵) observed at the adjacent <em>GALNT14</em> gene. In a gene-based analysis in VEGAS, <em>BPIFA3</em> (P_Gene = 2×10⁻⁵) and <em>BPIFA1</em> (P_Gene = 1.07×10⁻⁴) in the <em>BPIFA</em> gene cluster on chromosome 20q11.21 were the top hits. In all, 32 genomic regions show evidence of association (P_adj-PCA<10⁻⁵) in this GWAS, with pathway analysis showing a connection between top candidates and the TGFβ pathway. However, top and tag-SNP analysis for seven selected candidate genes in this pathway did not replicate in 645 families (793 affected individuals) from the Western Australian Family Study of Otitis Media (WAFSOM). Lack of replication may be explained by sample size, difference in OM disease severity between primary and replication cohorts or due to type I error in the primary GWAS.</p> <h3>Conclusions</h3><p>This first discovery GWAS for an OM phenotype has identified <em>CAPN14</em> and <em>GALNT14</em> on chromosome 2p23.1 and the <em>BPIFA</em> gene cluster on chromosome 20q11.21 as novel candidate genes which warrant further analysis in cohorts matched more precisely for clinical phenotypes.</p> </div

Top 15 genes from VEGAS gene-based analysis based on GenABEL P-values from the Raine Study discovery GWAS after adjustment for PCs.

a<p>- rs17396317 lies within the pseudogene <i>BPIFA4</i>, 15.258 kb upstream of <i>BPIFA3</i>.</p><p>Ordered by chromosome and physical location.</p

Regional LocusZoom plots of association across the region of chromosome 20 containing the <i>BPIFA</i> genes.

<p>(A) LocusZoom plots of association in the imputed dataset adjusted for PCs across the <i>BPIFA4P-BPIFA3-BPIFA1</i> gene region in the full discovery set of 1491 Raine Study participants. (B) The same region after adjusting for the top SNP rs17396317. The top associated SNP (rs17396317) is represented by purple circle in (A), the colour of all other SNPs is representative of the pairwise r² value with the relevant top SNP using patterns of LD from the Raine Study cohort. Recombination rates are shown by the solid blue line. Physical positions and gene designations are based on NCBI Build 36 of the human genome. Gene designations for NCBI Build 37 are indicated in the panel between (A) and (B).</p

Top 25 genotyped polymorphisms from the discovery GWAS carried out in the Raine Study ordered by chromosome and physical location with analysis adjusted for the first two PCs using GenABEL.

<p>Where MAF  =  minor allele frequency, OR  =  odds ratio, 95% CI = 95% confidence intervals. <b>^a -</b> Gene/Region is based on annotation from Ensembl build 54; intergenic SNPs fall >20 kb from an annotated gene. <b>^b -</b> Odds ratios are shown for the risk allele; <b>^c</b> - rs17396317 lies within the pseudogene <i>BPIFA4</i>, 15.258 kb upstream of <i>BPIFA3</i>.</p