Table_2_A Novel Description of the Human Sinus Archaeome During Health and Chronic Rhinosinusitis.docx

Human microbiome studies remain focused on bacteria, as they comprise the dominant component of the microbiota. Recent advances in sequencing technology and optimization of amplicon sequencing protocols have allowed the description of other members of the microbiome, including eukaryotes (fungi) and, most recently, archaea. There are no known human-associated archaeal pathogens. Their diversity and contribution to health and chronic respiratory diseases, such as chronic rhinosinusitis (CRS), are unknown. Patients with CRS suffer from long-term sinus infections, and while the microbiota is hypothesized to play a role in its pathogenesis, the exact mechanism is poorly understood. In this cross-sectional study, we applied a recently optimized protocol to describe the prevalence, diversity and abundance of archaea in swab samples from the middle meatus of 60 individuals with and without CRS. A nested PCR approach was used to amplify the archaeal 16S rRNA gene for sequencing, and bacterial and archaeal load (also based on 16S rRNA genes) were estimated using Droplet Digital™ PCR (ddPCR). A total of 16 archaeal amplicon sequence variants (ASVs) from the phyla Euryarchaeota and Thaumarchaeota were identified. Archaeal ASVs were detected in 7/60 individuals, independent of disease state, whereas bacterial ASVs were detected in 60/60. Bacteria were also significantly more abundant than archaea. The ddPCR method was more sensitive than amplicon sequencing at detecting archaeal DNA in samples. Phylogenetic trees were constructed to visualize the evolutionary relationships between archaeal ASVs, isolates and clones. ASVs were placed into phylogenetic clades containing an apparent paucity of human-associated reference sequences, revealing how little studied the human archaeome is. This is the largest study to date to examine the human respiratory-associated archaeome, and provides the first insights into the prevalence, diversity and abundance of archaea in the human sinuses.

人类微生物组研究始终聚焦于细菌，因其构成了微生物群的主体成分。近年来，测序技术的进步和扩增子测序方案的优化使得对微生物群其他成员的描述成为可能，包括真核生物（真菌）以及最近发现的古菌。目前尚未发现与人类相关的古菌病原体。古菌的多样性和其对健康以及慢性呼吸道疾病，如慢性鼻炎鼻窦炎（CRS）的贡献尚不明确。患有CRS的患者遭受长期的鼻窦感染，尽管假设微生物群在疾病发病机制中发挥作用，但其确切机制仍不甚明了。在本项横断面研究中，我们应用了一种最近优化的方案，以描述60名患有和未患有CRS的个体中鼻中隔拭子样本中古菌的流行率、多样性和丰度。采用套式PCR方法扩增古菌的16S rRNA基因进行测序，并利用Droplet Digital™ PCR（ddPCR）估计细菌和古菌负荷（基于16S rRNA基因）。共鉴定出来自Euryarchaeota和Thaumarchaeota门类的16个古菌扩增子序列变体（ASVs）。古菌ASVs在7/60的个体中检测到，与疾病状态无关，而细菌ASVs在60/60的个体中均被检测到。细菌的数量也显著多于古菌。ddPCR方法在检测样本中古菌DNA方面比扩增子测序更为敏感。构建了系统发育树，以可视化古菌ASVs、分离株和克隆之间的进化关系。ASVs被放置在包含明显缺乏人类相关参考序列的系谱群中，揭示了人类古菌组研究之匮乏。这是迄今为止最大规模的研究，旨在考察人类呼吸相关古菌组，并首次提供了关于人类鼻窦中古菌的流行率、多样性和丰度的见解。