Characterization of the nasopharyngeal microbiota in health and during rhinovirus challenge

Abstract

Background: The bacterial communities of the nasopharynx play an important role in upper respiratory tract infections (URTIs). Our study represents the first survey of the nasopharynx during a known, controlled viral challenge. We aimed to gain a better understanding of the composition and dynamics of the nasopharyngeal microbiome during viral infection.

Methods: Rhinovirus illnesses were induced by self-inoculation using the finger to nose or eye natural transmission route in ten otherwise healthy young adults. Nasal lavage fluid samples (NLF) samples were collected at specific time points before, during, and following experimental rhinovirus inoculation. Bacterial DNA from each sample (N = 97 from 10 subjects) was subjected to 16S rRNA sequencing by amplifying the V1-V2 hypervariable region followed by sequencing using the 454-FLX platform.

Results: This survey of the nasopharyngeal microbiota revealed a highly complex microbial ecosystem. Taxonomic composition varied widely between subjects and between time points of the same subject. We also observed significantly higher diversity in not infected individuals compared to infected individuals. Two genera - Neisseria and Propionibacterium - differed significantly between infected and not infected individuals. Certain phyla, including Firmicutes, Actinobacteria, and Proteobacteria, were detected in all samples.

Conclusions: Our results reveal the complex and diverse nature of the nasopharyngeal microbiota in both healthy and viral-challenged adults. Although some phyla were common to all samples, differences in levels of diversity and selected phyla were detected between infected and uninfected participants. Deeper, species-level metagenomic sequencing in a larger sample is warranted.

Keywords: Microbiota; longitudinal; nasopharynx; rhinovirus illness; sequencing.

Figure 1

Study timeline including when information or samples were collected from study participants. *Day on which nasal lavage fluid (NLF) samples were obtained prior to rhinovirus inoculation.

Figure 2

Box and whisker plot of read counts at each time point during the study after quality control filtering.

Figure 3

Box and whisker plot of the relative abundance of dominant phyla in infected and not infected individuals by time period. Sample counts for Infected samples: (before (n = 19), during (n = 34), and after (n = 14)). Sample counts for Non-Infected samples: (before (n = 8), during (n = 15), and after (n = 6)).

Figure 4

Rarefaction plots displaying the relationship between the sample size and the number of observed species.

Figure 5

Nasopharyngeal microbiota is similar to neighboring body parts, but distinct from gut and oral cavity. Principal coordinates analysis (PCoA) was performed using the unweighted UniFrac distance matrix using nasal lavage fluid (NLF) samples from pre-inoculation time points. Each sample is represented by a point with NLF (n = 27) in green, gut (n = 45) in dark blue, oral cavity (n = 46) in yellow, external auditory canal (n = 44) in red, nostril (n = 46) in purple, hair (n = 14) in orange, and adenoids (n = 69) in light blue.

Figure 6

Unifrac distance comparisons. (A) Unweighted Unifrac distances between samples from different individuals (inter-individual) and the distances between samples from the same individual (intra-individual). (B) Inter-individual unweighted Unifrac distances compared between infected and not infected subjects.P-values displayed are the results of Mann-Whitney Wilcoxon tests.

Figure 7

Stacked taxonomic bar chart (A) with box and whisker plot (B) - Shannon diversity index (Not infected (n = 29) versus Infected (n = 67). Operational taxonomic units (OTUs) were categorized as “Unclassified”, if RDP classifier could not classify them to at least the family level. Genera that could be classified to the family level, but not the genus level were categorized by the family name, followed by “_Unclassified” to indicate that the genus is not known. All genera comprising less than 1% of the total abundance (whether classified or not) were combined into the “Other <1%” category.