Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target

Ruth B Thornton,Lea-Ann S Kirkham,Selma P Wiertsema,Peter C Richmond,Shyan Vijayasekaran,Paul J Rigby,Harvey L Coates,Jean-Pierre Gorvel

doi:10.1371/journal.pone.0053837

Ruth B Thornton, Lea-Ann S Kirkham + Show 6 more

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https://doi.org/10.1371/journal.pone.0053837

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Abstract

BackgroundBacteria 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.MethodsIn 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.ConclusionsBacterial 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.

Highlights

Recurrent acute otitis media 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), Recurrent acute otitis media (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 [3,4,5,6]
The presence of neutrophil extracellular traps (NETs) and/or bacterial DNA in chronic OM may allow for novel treatments that breakdown DNA such as Dornase alfa used in the treatment of CF

Summary

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 [3,4,5,6]. 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) [8,9,10,11,12]. Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis

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