Variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis

Abstract

The nasopharynx is the ecological niche for many commensal bacteria and for potential respiratory or invasive pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Disturbance of a balanced nasopharyngeal (NP) microbiome might be involved in the onset of symptomatic infections with these pathogens, which occurs primarily in fall and winter. It is unknown whether seasonal infection patterns are associated with concomitant changes in NP microbiota. As young children are generally prone to respiratory and invasive infections, we characterized the NP microbiota of 96 healthy children by barcoded pyrosequencing of the V5-V6 hypervariable region of the 16S-rRNA gene, and compared microbiota composition between children sampled in winter/fall with children sampled in spring. The approximately 1,000,000 sequences generated represented 13 taxonomic phyla and approximately 250 species-level phyla types (OTUs). The 5 most predominant phyla were Proteobacteria (64%), Firmicutes (21%), Bacteroidetes (11%), Actinobacteria (3%) and Fusobacteria (1,4%) with Moraxella, Haemophilus, Streptococcus, Flavobacteria, Dolosigranulum, Corynebacterium and Neisseria as predominant genera. The inter-individual variability was that high that on OTU level a core microbiome could not be defined. Microbiota profiles varied strongly with season, with in fall/winter a predominance of Proteobacteria (relative abundance (% of all sequences): 75% versus 51% in spring) and Fusobacteria (absolute abundance (% of children): 14% versus 2% in spring), and in spring a predominance of Bacteroidetes (relative abundance: 19% versus 3% in fall/winter, absolute abundance: 91% versus 54% in fall/winter), and Firmicutes. The latter increase is mainly due to (Brevi)bacillus and Lactobacillus species (absolute abundance: 96% versus 10% in fall/winter) which are like Bacteroidetes species generally related to healthy ecosystems. The observed seasonal effects could not be attributed to recent antibiotics or viral co-infection.The NP microbiota of young children is highly diverse and appears different between seasons. These differences seem independent of antibiotic use or viral co-infection.

Figure 1. Relative abundance of all bacterial phyla found in the NP microbiota of 96 infants 18 months of age.

A cut-off of 0.1% is used for visual differentiation between predominant and less dominant phyla.

Figure 2. Principal component analysis of the individual NP communities.

We observed three individual clusters/axes surrounding a centre of profiles, with in green individual profiles depicting predominantly (>50% of sequences) Moraxella OTU 1, in blue microbiota profiles depicting predominantly H. influenzae OTU 2, and in red microbiota profiles depicting predominantly Streptococcus OTU 3. Mixed phyla profiles (no single OTU is representing >50% of sequences) cluster in the centre of this PCA plot.

Figure 3. Seasonal differences between microbiota profiles of 50 children sampled in fall-winter and 46 children in spring.

The samples are marked by the week number they were obtained (week 48 until week 23). In figure 3a the phyla showing significant association with season of sampling by SAM analysis are depicted. In figure 3b the OTUs showing significant association with season are depicted. The samples are marked by season (blue; fall, green; winter, red; spring), antibiotic use (<1 month: green), presence of viruses (positive: green), presence of multiple viruses (green: 1 virus, red: ≥ 2 viruses), Presence of Human rhinovirus, Adenovirus, Bocavirus, and Para-influenzavirus I-IV (positive: green). Groups of OTUs belonging to specific phyla are depicted with separate colours; Yellow: Firmicutes, Orange: Proteobacteria, Green: Bacteroidetes, Blue: Actinobacteria, Pink: Cyanobacteria.