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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Saara Sillanpää, Lenka Kramna, Sami Oikarinen, Markku Sipilä, Markus Rautiainen, Janne Aittoniemi, Jussi Laranne, Heikki Hyöty, Ondrej Cinek
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	16S profiling acute otitis media bacteriome profiling mass sequencing next-generation sequencing Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9237ed312b284cc7901c3b10d5292563
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:9237ed312b284cc7901c3b10d5292563
record_format	dspace
spelling	oai:doaj.org-article:9237ed312b284cc7901c3b10d52925632021-11-15T15:21:45ZNext-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 Media10.1128/mSphere.00006-172379-5042https://doaj.org/article/9237ed312b284cc7901c3b10d52925632017-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00006-17https://doaj.org/toc/2379-5042ABSTRACT 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 pneumoniae was detected in 28 (31%) samples, Haemophilus influenzae in 24 (27%), Moraxella catarrhalis in 18 (20%), Staphylococcus spp. in 21 (23%), Turicella otitidis in 5 (5.6%), Alloiococcus otitidis in 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling. S. pneumoniae was the dominant pathogen in 14 (16%) samples, H. influenzae in 15 (17%), M. catarrhalis in 5 (5.6%), T. otitidis in 2, and Staphylococcus auricularis in 2. Weaker signals of Prevotella melaninogenica, Veillonella dispar, and Veillonella montpellierensis were 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. IMPORTANCE Although S. pneumoniae, H. influenzae, and M. catarrhalis have 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.Saara SillanpääLenka KramnaSami OikarinenMarkku SipiläMarkus RautiainenJanne AittoniemiJussi LaranneHeikki HyötyOndrej CinekAmerican Society for Microbiologyarticle16S profilingacute otitis mediabacteriome profilingmass sequencingnext-generation sequencingMicrobiologyQR1-502ENmSphere, Vol 2, Iss 2 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	16S profiling acute otitis media bacteriome profiling mass sequencing next-generation sequencing Microbiology QR1-502
spellingShingle	16S profiling acute otitis media bacteriome profiling mass sequencing next-generation sequencing Microbiology QR1-502 Saara Sillanpää Lenka Kramna Sami Oikarinen Markku Sipilä Markus Rautiainen Janne Aittoniemi Jussi Laranne Heikki Hyöty Ondrej Cinek 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
description	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 pneumoniae was detected in 28 (31%) samples, Haemophilus influenzae in 24 (27%), Moraxella catarrhalis in 18 (20%), Staphylococcus spp. in 21 (23%), Turicella otitidis in 5 (5.6%), Alloiococcus otitidis in 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling. S. pneumoniae was the dominant pathogen in 14 (16%) samples, H. influenzae in 15 (17%), M. catarrhalis in 5 (5.6%), T. otitidis in 2, and Staphylococcus auricularis in 2. Weaker signals of Prevotella melaninogenica, Veillonella dispar, and Veillonella montpellierensis were 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. IMPORTANCE Although S. pneumoniae, H. influenzae, and M. catarrhalis have 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.
format	article
author	Saara Sillanpää Lenka Kramna Sami Oikarinen Markku Sipilä Markus Rautiainen Janne Aittoniemi Jussi Laranne Heikki Hyöty Ondrej Cinek
author_facet	Saara Sillanpää Lenka Kramna Sami Oikarinen Markku Sipilä Markus Rautiainen Janne Aittoniemi Jussi Laranne Heikki Hyöty Ondrej Cinek
author_sort	Saara Sillanpää
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
title_short	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
title_full	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
title_fullStr	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
title_full_unstemmed	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
title_sort	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
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/9237ed312b284cc7901c3b10d5292563
work_keys_str_mv	AT saarasillanpaa nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT lenkakramna nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT samioikarinen nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT markkusipila nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT markusrautiainen nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT janneaittoniemi nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT jussilaranne nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT heikkihyoty nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia AT ondrejcinek nextgenerationsequencingcombinedwithspecificpcrassaystodeterminethebacterial16srrnageneprofilesofmiddleearfluidcollectedfromchildrenwithacuteotitismedia
_version_	1718428144464035840

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

Ejemplares similares