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

Brugger, Silvio D.; Frei, Laurence; Frey, Pascal M; Aebi, Suzanne; Mühlemann, Kathrin; Hilty, Markus (2012). 16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota. PLoS ONE, 7(12), e52241. Lawrence, Kans.: Public Library of Science 10.1371/journal.pone.0052241

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of General Internal Medicine (DAIM) > Clinic of General Internal Medicine > Centre of Competence for General Internal Medicine
04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Infectiology

UniBE Contributor:

Brugger, Silvio and Hilty, Markus

ISSN:

1932-6203

Publisher:

Public Library of Science

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:39

Last Modified:

05 Dec 2014 10:14

Publisher DOI:

10.1371/journal.pone.0052241

PubMed ID:

23284951

Web of Science ID:

000312794500123

BORIS DOI:

10.7892/boris.16030

URI:

https://boris.unibe.ch/id/eprint/16030 (FactScience: 223580)

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