16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gen...

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Autores principales:	Silvio D Brugger, Laurence Frei, Pascal M Frey, Suzanne Aebi, Kathrin Mühlemann, Markus Hilty
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/59329a28c8584c69b9fca502c7220b6c
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spelling	oai:doaj.org-article:59329a28c8584c69b9fca502c7220b6c2021-11-18T08:04:16Z16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.1932-620310.1371/journal.pone.0052241https://doaj.org/article/59329a28c8584c69b9fca502c7220b6c2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284951/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.Silvio D BruggerLaurence FreiPascal M FreySuzanne AebiKathrin MühlemannMarkus HiltyPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e52241 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Silvio D Brugger Laurence Frei Pascal M Frey Suzanne Aebi Kathrin Mühlemann Markus Hilty 16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
description	A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.
format	article
author	Silvio D Brugger Laurence Frei Pascal M Frey Suzanne Aebi Kathrin Mühlemann Markus Hilty
author_facet	Silvio D Brugger Laurence Frei Pascal M Frey Suzanne Aebi Kathrin Mühlemann Markus Hilty
author_sort	Silvio D Brugger
title	16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
title_short	16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
title_full	16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
title_fullStr	16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
title_full_unstemmed	16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
title_sort	16s rrna terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/59329a28c8584c69b9fca502c7220b6c
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16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

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