Pérez-Losada Marcos, Alamri Lamia, Crandall Keith A, Freishtat Robert J
Computational Biology Institute, George Washington University, Ashburn, Virginia, United States of America.
Division of Emergency Medicine, Children's National Medical Center, Washington DC, United States of America.
PLoS One. 2017 Jan 20;12(1):e0170543. doi: 10.1371/journal.pone.0170543. eCollection 2017.
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.
鼻咽部是与哮喘等呼吸道疾病相关病原体的储存库。下一代测序（NGS）已被用于描述健康和患病期间婴儿及成人的鼻咽微生物群特征；然而，对于儿童和青少年微生物群的组成和时间动态（即纵向变化）了解较少。在此，我们使用NGS技术来描述哮喘儿童和青少年（6至18岁）的鼻咽微生物群特征，并确定其随时间的稳定性。
从居住在华盛顿特区地区的40名哮喘儿童和青少年中采集相隔5.5至6.5个月的两次鼻咽冲洗液。使用MiSeq平台从样本中收集16S-V4 rRNA基因区域（约250 bp）的序列数据。原始数据在mothur（SILVA123参考数据库）中进行处理，并估计基于操作分类单元（OTU）的α-和β-多样性指标。使用主坐标分析（PCoA）排序和普罗克汝斯分析评估样本间的相关性。使用线性混合效应模型评估微生物多样性和分类单元平均相对比例的差异。还进行了核心微生物群分析，以确定鼻咽部稳定且一致的微生物。
共生成了2,096,584条干净的16S序列，每个样本平均有167个OTU。莫拉克斯氏菌属*、葡萄球菌属*、颗粒链菌属、棒状杆菌属、普雷沃氏菌属、链球菌属*、嗜血杆菌属*、梭杆菌属*和一个奈瑟菌科属的代表序列占总读数的86%。这九个属此前在婴儿和成人的鼻咽部均有发现，但比例不同。上述五个属（*标注）的OTU在95%水平定义了鼻咽核心微生物群。季节间未观察到α-和β-多样性的显著差异，但嗜血杆菌属、莫拉克斯氏菌属、葡萄球菌属和棒状杆菌属的细菌平均相对比例在夏秋季节和年龄组之间存在显著差异（患者间差异）。此外，在分析的40名患者中的35名患者中，不同时间点的OTU在患者个体内也存在显著差异。未来的横断面研究应注意鼻咽微生物群的时间动态。
