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European Archives of Oto-Rhino-Laryngology

, Volume 273, Issue 11, pp 3553–3560 | Cite as

Haemophilus influenzae biofilm formation in chronic otitis media with effusion

  • Helen Van Hoecke
  • Ann-Sophie De Paepe
  • Edward Lambert
  • Jonas D Van Belleghem
  • Piet Cools
  • Leen Van Simaey
  • Pieter Deschaght
  • Mario Vaneechoutte
  • Ingeborg Dhooge
Otology

Abstract

Otitis media with effusion (OME) is a highly prevalent disease in children, but the exact pathogenesis and role of bacteria are still not well understood. This study aimed to investigate the presence of otopathogenic bacteria in the middle ear effusion (MEE) and adenoid of children with chronic OME (COME), and to investigate in vivo whether these bacteria, especially Haemophilus influenzae, are organized as a biofilm in the middle ear fluid. MEE and adenoid samples were collected from 21 patients with COME. Extensive bacterial culturing and genotyping was performed on all middle ear and adenoid samples. Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) was used to visualize possible biofilm structures for a selection of middle ear effusion samples. 34 MEE samples were collected from 21 patients of which 64.7 % were culture positive for bacteria and 47.0 % were culture positive for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and/or Streptococcus pneumoniae. All 21 adenoid samples were culture positive for one or more of these four otopathogens. H. influenzae (35.3 %) and S. pneumoniae (76.2 %) were the most frequently cultured bacteria in the MEE and adenoid samples, respectively. The same bacterial species was found in MEE and adenoid for 84.6 % of the patients and in 81.2 % of the cases where the same species was found in more than one site it involved the same bacterial genotype. FISH and CLSM demonstrated the presence of H. influenzae specific biofilm structures in five of the eight culture positive MEEs that were tested, but in none of the two culture negative MEEs. The findings in this study indicate that the adenoid acts as a reservoir for bacteria in MEE and confirms that biofilms, in at least half of the cases consisting of H. influenzae, are indeed present in the MEE of children with COME. Biofilms may thus play a crucial role in the pathogenesis of COME, which is important in the understanding of this disease and the development of potential future treatment options.

Keywords

Chronic otitis media with effusion (COME) Biofilm Fluorescent in situ hybridization (FISH) Confocal scanning laser microscopy (CLSM) Haemophilus influenzae 

Notes

Compliance with ethical standards

The authors have no conflict of interest. No external funding was provided for this study. The study was approved by the Ethical Committee of Ghent University Hospital (number of approval: B670201214394). Written informed consent was provided by the parents or legal guardians of the study participants.

Supplementary material

405_2016_3958_MOESM1_ESM.docx (123 kb)
Supplementary material 1 (DOCX 122 kb)
405_2016_3958_MOESM2_ESM.avi (1.7 mb)
Figure S1. 3D animations of biofilm structures obtained by CLSM of FISH stained samplesAnimations were prepared with ImageJ software [29]. Legend. Two probes were used for the visualisation of biofilms in MEE samples: EUB388-Alexa555 probe is a universal probe which stains all bacteria green. The H. influenzae specific probe stains H. influenzae bacteria red. The combination of the EUB388-Alexa555 probe and the H. influenzae specific probe results in a yellow colour, which specifically indicates the presence of H. influenzae. DAPI (4’,6-diamidino-2-phenylindole) pigment stains eukaryote nucleoli blue. ORL1 MEE from right ear (AVI 1757 kb)
405_2016_3958_MOESM3_ESM.avi (1.9 mb)
Figure S1. 3D animations of biofilm structures obtained by CLSM of FISH stained samplesAnimations were prepared with ImageJ software [29]. Legend. Two probes were used for the visualisation of biofilms in MEE samples: EUB388-Alexa555 probe is a universal probe which stains all bacteria green. The H. influenzae specific probe stains H. influenzae bacteria red. The combination of the EUB388-Alexa555 probe and the H. influenzae specific probe results in a yellow colour, which specifically indicates the presence of H. influenzae. DAPI (4’,6-diamidino-2-phenylindole) pigment stains eukaryote nucleoli blue. ORL2 MEE from right ear (AVI 1913 kb)
405_2016_3958_MOESM4_ESM.avi (1.7 mb)
Figure S1. 3D animations of biofilm structures obtained by CLSM of FISH stained samplesAnimations were prepared with ImageJ software [29]. Legend. Two probes were used for the visualisation of biofilms in MEE samples: EUB388-Alexa555 probe is a universal probe which stains all bacteria green. The H. influenzae specific probe stains H. influenzae bacteria red. The combination of the EUB388-Alexa555 probe and the H. influenzae specific probe results in a yellow colour, which specifically indicates the presence of H. influenzae. DAPI (4’,6-diamidino-2-phenylindole) pigment stains eukaryote nucleoli blue. ORL12 MEE from left ear (first take) (AVI 1728 kb)
405_2016_3958_MOESM5_ESM.avi (2.4 mb)
Figure S1. 3D animations of biofilm structures obtained by CLSM of FISH stained samplesAnimations were prepared with ImageJ software [29]. Legend. Two probes were used for the visualisation of biofilms in MEE samples: EUB388-Alexa555 probe is a universal probe which stains all bacteria green. The H. influenzae specific probe stains H. influenzae bacteria red. The combination of the EUB388-Alexa555 probe and the H. influenzae specific probe results in a yellow colour, which specifically indicates the presence of H. influenzae. DAPI (4’,6-diamidino-2-phenylindole) pigment stains eukaryote nucleoli blue. ORL12 MEE from left ear (second take) (AVI 2488 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Helen Van Hoecke
    • 1
  • Ann-Sophie De Paepe
    • 2
  • Edward Lambert
    • 2
  • Jonas D Van Belleghem
    • 3
  • Piet Cools
    • 3
  • Leen Van Simaey
    • 3
  • Pieter Deschaght
    • 3
  • Mario Vaneechoutte
    • 3
  • Ingeborg Dhooge
    • 1
  1. 1.Department of OtorhinolaryngologyGhent University HospitalGhentBelgium
  2. 2.Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium
  3. 3.Laboratory Bacteriology Research, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium

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