title: Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.
abstract: BACKGROUND
  Chronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection.
  
  
  OBJECTIVE
  To test the hypothesis that chronic OM in humans is biofilm-related.
  
  
  METHODS
  Middle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice.
  
  
  METHODS
  Confocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively.
  
  
  RESULTS
  Of the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation.
  
  
  CONCLUSIONS
  Direct detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pa 15212, USA.
authors.forename: Luanne
authors.initials: L
authors.lastname: Hall-Stoodley
authors.forename: Fen Ze
authors.initials: FZ
authors.lastname: Hu
authors.forename: Armin
authors.initials: A
authors.lastname: Gieseke
authors.forename: Laura
authors.initials: L
authors.lastname: Nistico
authors.forename: Duc
authors.initials: D
authors.lastname: Nguyen
authors.forename: Jay
authors.initials: J
authors.lastname: Hayes
authors.forename: Michael
authors.initials: M
authors.lastname: Forbes
authors.forename: David P
authors.initials: DP
authors.lastname: Greenberg
authors.forename: Bethany
authors.initials: B
authors.lastname: Dice
authors.forename: Amy
authors.initials: A
authors.lastname: Burrows
authors.forename: P Ashley
authors.initials: PA
authors.lastname: Wackym
authors.forename: Paul
authors.initials: P
authors.lastname: Stoodley
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Post
authors.forename: Garth D
authors.initials: GD
authors.lastname: Ehrlich
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Kerschner
mesh_terms: D000293:Adolescent; D018441:Biofilms / Q000254:growth & development; D002648:Child; D002675:Child, Preschool; D002908:Chronic Disease; D004432:Ear, Middle / Q000382:microbiology* / Q000473:pathology; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007223:Infant; D008297:Male; D018613:Microscopy, Confocal; D009092:Mucous Membrane / Q000382:microbiology / Q000473:pathology; D010033:Otitis Media / Q000382:microbiology* / Q000473:pathology; D010034:Otitis Media with Effusion / Q000382:microbiology / Q000473:pathology; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S