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

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

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

Sensitivity, specificity and ROC curve areas of PCR assays for study-defined NTHi.

<p>Sensitivity, specificity and ROC curve areas of PCR assays for study-defined NTHi.</p

Genbank reference strains.

<p>Sequence information was obtained from 6 reference strains (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a> – December 2010) to facilitate phylogenetic speciation. na = not applicable.</p

Sequencing phylogeny of study NTHi isolates.

<p>Radiation trees are presented for (A) 598 bases of the 16S rRNA gene, (B) 545 bases of the <i>recA</i> gene, and (C) the concatenation of these sequences (1143 bases). The trees are rooted by <i>H. parainfluenzae</i> T3T1 as indicated by yellow dots. Blue dots represent isolates that cluster with <i>H. influenzae</i> reference strains, orange dots represent closely related phylogenetic variants, red dots represent likely Hh isolates and green dots represent variants related to <i>H. parainfluenzae</i>. Colours were assigned based on the phylogenetic grouping of the concatenated sequences in radial tree C.</p

Study primers and probes.

<p>All product sizes are based on the NTHi Rd KW20 genome.</p>*<p>product size varies considerably across strains.</p>**<p><i>hpd</i> probes were labelled with Hex at 5′-end, SpC3 at 3′-end, and Black Hole Quencher (BHQ) at the internal “T”. F (forward primer); R (reverse primer); P (probe); HRM (high resolution melt).</p