title: Next-Generation Sequencing Combined with Specific PCR Assays To Determine the Bacterial 16S rRNA Gene Profiles of Middle Ear Fluid Collected from Children with Acute Otitis Media.
abstract: The aim of the study was to analyze the bacteriome of acute otitis media with a novel modification of next-generation sequencing techniques. Outpatient children with acute otitis media were enrolled in the study, and middle ear fluids were collected during 90 episodes from 79 subjects aged 5 to 42 months (median age, 19 months). The bacteriome profiles of middle ear fluid samples were determined by a nested-PCR amplification of the 16S rRNA gene (V4 region), followed by mass sequencing. The profiling results were compared to the results of specific PCR assays targeting selected prevalent pathogens. Bacteriome profiling using nested amplification of low-volume samples was aided by a bioinformatic subtraction of signal contaminants from the recombinant polymerase, achieving a sensitivity slightly lower than that of specific PCR detection.Streptococcus pneumoniaewas detected in 28 (31%) samples,Haemophilus influenzaein 24 (27%),Moraxella catarrhalisin 18 (20%),Staphylococcusspp. in 21 (23%),Turicella otitidisin 5 (5.6%),Alloiococcus otitidisin 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling.S. pneumoniaewas the dominant pathogen in 14 (16%) samples,H. influenzaein 15 (17%),M. catarrhalisin 5 (5.6%),T. otitidisin 2, andStaphylococcus auricularisin 2. Weaker signals ofPrevotella melaninogenica,Veillonella dispar, andVeillonella montpellierensiswere noted in several samples. Fourteen samples (16%) were not explainable by bacterial pathogens; novel causative agents were not detected. In conclusion, unbiased bacteriome profiling helped in depicting the true mutual quantitative ratios of ear bacteria, but at present, its complicated protocol impedes its routine clinical use.IMPORTANCEAlthoughS. pneumoniae,H. influenzae, andM. catarrhalishave been long established as the most important pathogens in acute otitis media using culture and specific PCR assays, the knowledge of their mutual quantitative relations and possible roles of other bacteria is incomplete. The advent of unbiased bacteriome 16S rRNA gene profiling has allowed the detection of nearly all bacteria present in the sample, and it helps in depicting their mutual quantitative ratios. Due to the difficulties in performing mass sequencing in low-volume samples, only a few bacteriome-profiling studies of otitis media have been published, all limited to cases of chronic otitis media. Here, we present a study on samples obtained from young children with acute otitis media, successfully using a strategy of nested PCR coupled with mass sequencing, and demonstrate that the method can confer quantitative information hardly obtainable by other methods.
authors.affiliation: Department of Otorhinolaryngology and Head and Neck Surgery, Tampere University Hospital and School of Medicine, University of Tampere, Tampere, Finland.
authors.forename: Saara
authors.initials: S
authors.lastname: Sillanpää
authors.affiliation: Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Kramna
authors.affiliation: Department of Virology, School of Medicine, University of Tampere, Tampere, Finland.
authors.forename: Sami
authors.initials: S
authors.lastname: Oikarinen
authors.affiliation: Department of Otorhinolaryngology and Head and Neck Surgery, Tampere University Hospital and School of Medicine, University of Tampere, Tampere, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Sipilä
authors.affiliation: Department of Otorhinolaryngology and Head and Neck Surgery, Tampere University Hospital and School of Medicine, University of Tampere, Tampere, Finland.
authors.forename: Markus
authors.initials: M
authors.lastname: Rautiainen
authors.affiliation: Fimlab Laboratories, Tampere, Finland.
authors.forename: Janne
authors.initials: J
authors.lastname: Aittoniemi
authors.affiliation: Department of Otorhinolaryngology and Head and Neck Surgery, Tampere University Hospital and School of Medicine, University of Tampere, Tampere, Finland.
authors.forename: Jussi
authors.initials: J
authors.lastname: Laranne
authors.affiliation: Department of Virology, School of Medicine, University of Tampere, Tampere, Finland; Fimlab Laboratories, Tampere, Finland.
authors.forename: Heikki
authors.initials: H
authors.lastname: Hyöty
authors.affiliation: Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Cinek
keywords: 16S profiling; acute otitis media; bacteriome profiling; mass sequencing; next-generation sequencing