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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.

16S rRNA Terminal Restriction Fragment Length Polymorphism for the Characterization of the Nasopharyngeal Microbiota