Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target Ruth B. Thornton 1,2 *, Selma P. Wiertsema 1,2 , Lea-Ann S. Kirkham 1,2 , Paul J. Rigby 3 , Shyan Vijayasekaran 1,4,5 , Harvey L. Coates 1,4,5 , Peter C. Richmond 1,2 1 School of Paediatrics and Child Health, The University of Western Australia, Perth, Australia, 2 Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Perth, Australia, 3 Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Perth, Australia, 4 Department of Otolaryngology, Head and Neck Surgery, Princess Margaret Hospital for Children, Perth, Australia, 5 Department of Otolaryngology, Head and Neck Surgery, The University of Western Australia, Perth, Australia Abstract Background: Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis. Methods: In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment. Conclusions: Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment. Citation: Thornton RB, Wiertsema SP, Kirkham L-AS, Rigby PJ, Vijayasekaran S, et al. (2013) Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target. PLoS ONE 8(2): e53837. doi:10.1371/journal.pone.0053837 Editor: Jean-Pierre Gorvel, Universite de la Mediterranee, France Received September 14, 2012; Accepted December 3, 2012; Published February 5, 2013 Copyright: ß 2013 Thornton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: GlaxoSmithKline Australia, University of Western Australia Priming Grant, University of Western Australia near miss safety net grant and Telethon Trust Research Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: R.B. Thornton, L.S. Kirkham and S.P. Wiertsema have received travel funding from GlaxoSmithKline. S.P. Wiertsema has received institutional funding for investigations into bacteriology in children with recurrent acute otitis media. H.L. Coates and S. Vijayasekaran consult for GlaxoSmithKline Biologicals. P. Richmond has received Institutional funding from GSK for investigator-led epidemiological studies in otitis media and has received travel support from GSK, Wyeth and other vaccine companies to present scientific data and chair workshops. L.K. is a co-inventor on patents PCT/GB2007/002242, PCT/GB2005/001792 and PCT/GB2005/001774. None of the authors have shares or paid employment with any pharmaceutical company. Other authors have no competing interests to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: ruth.thornton@uwa.edu.au Introduction Recurrent acute otitis media (rAOM) affects between 10–20% of children [1,2] and is a major reason for children requiring surgery, predominantly for ventilation tube insertion (VTI). Biofilm and intracellular infection have been demonstrated on the middle ear mucosa of children with chronic suppurative otitis media (CSOM), rAOM and chronic otitis media with effusion (OME) and are mechanisms of bacterial persistence in the middle ear causing recalcitrance to treatment and disease recurrence [3– 6]. Biofilms on mucosal surfaces are a result of interactions between bacteria and the host and these differ physiologically from biofilms formed on inert surfaces [7]. DNA, both host and bacterially derived, is important in biofilm formation, stabilisation and persistence of nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae in vitro and in the chinchilla model of otitis media (OM) [8–12]. DNA may be derived from active immune mechanisms such as neutrophil extracellular traps (NETs) [13], necrotic neutrophil presence [14], or produced directly by the bacteria [8–11]. NETs consist of a DNA backbone embedded with antimicrobial peptides and enzymes [15] and are important in confining infections and killing bacteria [16]. Some pathogens, including S. pneumoniae and NTHi can resist NET killing [9,11,15– 17] and NTHi has been shown to actively elicit NET formation, using the DNA as a protective niche [18]. High levels of NETs PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e53837