Nasopharyngeal Microbiome Diversity Changes over Time in Children with Asthma

Background
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.
Methods
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.
Results and Discussion
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.

研究背景
鼻咽部是哮喘等呼吸道疾病相关病原体的储存库。下一代测序（Next-generation sequencing, NGS）已被用于表征健康与疾病状态下婴幼儿及成人的鼻咽部微生物组；然而，目前对于儿童及青少年鼻咽部微生物组的组成与时间动态（即纵向变化）的了解仍较为有限。本研究采用NGS技术，对6至18岁哮喘患儿及青少年的鼻咽部微生物组进行特征分析，并探究其随时间的稳定性。

研究方法
本研究从居住于华盛顿特区地区的40名哮喘患儿及青少年中，采集了两份间隔5.5至6.5个月的鼻咽灌洗液样本。采用MiSeq测序平台对样本的16S-V4核糖体RNA（rRNA）基因区域（约250 bp）进行测序，获取序列数据。原始数据通过mothur软件结合SILVA123参考数据库进行处理，并估算了基于操作分类单元（Operational Taxonomic Units, OTU）的α多样性与β多样性指标。采用主坐标分析（Principal Coordinates Analysis, PCoA）排序与普氏分析（Procrustes analysis）评估样本间的相关性。采用线性混合效应模型评估微生物多样性与类群平均相对丰度的差异。此外还开展了核心微生物组分析，以鉴定鼻咽部稳定且一致存在的微生物类群。

结果与讨论
本研究共获得2,096,584条有效16S序列，每份样本平均对应167个OTU。莫拉克斯氏菌属（Moraxella*）、葡萄球菌属（Staphylococcus*）、多洛西格兰氏菌属（Dolosigranulum）、棒状杆菌属（Corynebacterium）、普雷沃氏菌属（Prevotella）、链球菌属（Streptococcus*）、嗜血杆菌属（Haemophilus*）、梭杆菌属（Fusobacterium*）以及奈瑟菌科（Neisseriaceae）的某一属类群的序列占总测序读段的86%。上述9个菌属此前已在婴幼儿与成人的鼻咽部中被检出，但占比存在差异。上述带*标记的5个菌属对应的OTU，在95%阈值下构成了鼻咽部核心微生物组。不同季节间的α多样性与β多样性均未出现显著差异，但嗜血杆菌属、莫拉克斯氏菌属、葡萄球菌属与棒状杆菌属的细菌平均相对丰度在夏秋季与年龄组间存在显著差异（即患者间变异）。此外，在40名分析对象中，有35名患者的OTU组成在不同时间点间存在显著差异。未来的横断面研究应关注鼻咽部微生物组的时间动态特征。