Date Published: January 20, 2017
Publisher: Public Library of Science
Author(s): Marcos Pérez-Losada, Lamia Alamri, Keith A. Crandall, Robert J. Freishtat, Paul J Planet.
The nasopharynx is a reservoir for pathogens associated with respiratory illnesses such as asthma. Next-generation sequencing (NGS) has been used to characterize the nasopharyngeal microbiome of infants and adults during health and disease; less is known, however, about the composition and temporal dynamics (i.e., longitudinal variation) of microbiotas from children and adolescents. Here we use NGS technology to characterize the nasopharyngeal microbiomes of asthmatic children and adolescents (6 to 18 years) and determine their stability over time.
Two nasopharyngeal washes collected 5.5 to 6.5 months apart were taken from 40 children and adolescents with asthma living in the Washington D.C. area. Sequence data from the 16S-V4 rRNA gene region (~250 bp) were collected from the samples using the MiSeq platform. Raw data were processed in mothur (SILVA123 reference database) and Operational Taxonomic Units (OTU)-based alpha- and beta-diversity metrics were estimated. Relatedness among samples was assessed using PCoA ordination and Procrustes analyses. Differences in microbial diversity and taxon mean relative proportions were assessed using linear mixed effects models. Core microbiome analyses were also performed to identify stable and consistent microbes of the nasopharynx.
A total of 2,096,584 clean 16S sequences corresponding to an average of 167 OTUs per sample were generated. Representatives of Moraxella*, Staphylococcus*, Dolosigranulum, Corynebacterium, Prevotella, Streptococcus*, Haemophilus*, Fusobacterium* and a Neisseriaceae genus accounted for 86% of the total reads. These nine genera have been previously found in the nasopharynxes of both infants and adults, but in different proportions. OTUs from the five genera highlighted (*) above defined the nasopharyngeal core microbiome at the 95% level. No significant differences in alpha- and beta-diversity were observed between seasons, but bacterial mean relative proportions of Haemophilus, Moraxella, Staphylococcus and Corynebacterium varied significantly between summer-fall and age groups (inter-patient variation). Additionally, OTUs varied significantly within patients between time points in 35 of the 40 patients analyzed. Future cross-sectional studies should be mindful of the temporal dynamics of the nasopharyngeal microbiota.
The application of next-generation sequencing (NGS) technology to microbial communities residing in the respiratory airways (i.e., airway microbiome) has shown that bacteria may play a significant role in the onset, development, and severity of respiratory diseases such as asthma [1–4]. Both metataxonomics and metagenomics (see  for distinction) have demonstrated that airway microbiome composition and structure vary between healthy and diseased individuals and revealed significant associations between representatives of several opportunistic pathogens (Moraxella, Streptococcus, Haemophilus, Neisseria and Staphylococcus), clinical conditions, and stages of asthma [3, 6–12]. Moreover, microbiome research has shown that all of these pathogens are normal and transient residents of the nasopharynx . As such, they are mainly asymptomatic, but through the nasopharynx they can directly spread to other sections of the respiratory tract and potentially cause asthma, otitis media or pneumonia; or invade the bloodstream to cause sepsis and meningitis [8, 13–15].