Respiratory Medicine Unit, Experimental Medicine Division, Nuffield Department of Medicine, University of Oxfordgrid.4991.5, John Radcliffe Hospital, Oxford, United Kingdom.
National Institute for Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
Microbiol Spectr. 2022 Apr 27;10(2):e0227921. doi: 10.1128/spectrum.02279-21. Epub 2022 Mar 24.
Previous metagenomic studies in asthma have been limited by inadequate sequencing depth for species-level bacterial identification and by heterogeneity in clinical phenotyping. We hypothesize that chronic bacterial airways infection is a key "treatable trait" whose prevalence, clinical phenotype and reliable biomarkers need definition. In this study, we have applied a method for Oxford Nanopore sequencing for the unbiased metagenomic characterization of severe asthma. We optimized methods to compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total sputum DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from highly phenotyped severe asthma during clinical stability. In participants with severe asthma ( = 23) H. influenzae was commonly cultured ( = 8) and identified as the dominant bacterial species by metagenomic sequencing using an optimized method for Illumina MiSeq and Oxford Nanopore. Alongside superior operational characteristics, Oxford Nanopore achieved near complete genome coverage of H. influenzae and demonstrated a high level of agreement with Illumina MiSeq data. Clinically significant infection was confirmed with validated H. influenzae plasmid-based quantitative PCR assay. H. influenzae positive patients were found to have sputum neutrophilia and lower FeNO. In conclusion, using an optimized method of direct sequencing of induced sputum samples, H. influenzae was identified as a clinically relevant pathogen in severe asthma and was identified reliably using metagenomic sequencing. Application of these protocols in ongoing analysis of large patient cohorts will allow full characterization of this clinical phenotype. The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood. Traditional culture lacks sensitivity and current sequencing techniques are limited by operational problems and limited ability to identify pathogens at species level. We optimized a new sequencing technique-Oxford Nanopore technologies (ONT)-for use on human sputum samples and compared it with existing methods. We found ONT was effective for rapidly analyzing samples and could identify bacteria at the species level. We used this to show Haemophilus influenzae was a dominant bacterium in the airways in people with severe asthma. The presence of Haemophilus was associated with a "neutrophilic" form of asthma - a subgroup for which we currently lack specific treatments. Therefore, this technique could be used to target chronic antibiotic therapy and in research to characterize the full breadth of bacteria in the airways.
先前的哮喘宏基因组研究受到物种水平细菌鉴定的测序深度不足和临床表型异质性的限制。我们假设慢性细菌气道感染是一个关键的“可治疗特征”，需要定义其流行率、临床表型和可靠的生物标志物。在这项研究中，我们应用了一种用于牛津纳米孔测序的方法，对严重哮喘进行无偏倚的宏基因组特征分析。我们优化了方法，比较了 Illumina MiSeq、纳米孔测序和 RT-qPCR 在总痰 DNA 提取物上的性能，以及针对临床稳定期间高度表型严重哮喘的诱导痰样本的培养物/MALDI-TOF 分析。在患有严重哮喘的参与者中（ = 23），经常培养流感嗜血杆菌（ = 8），并通过优化的 Illumina MiSeq 和牛津纳米孔测序方法鉴定为优势细菌物种。除了具有优越的操作特性外，牛津纳米孔技术还实现了流感嗜血杆菌的近乎完整基因组覆盖，并与 Illumina MiSeq 数据高度一致。通过验证的流感嗜血杆菌质粒定量 PCR 检测证实了临床显著感染。发现流感嗜血杆菌阳性患者痰液中性粒细胞增多，FeNO 降低。总之，使用诱导痰样本直接测序的优化方法，流感嗜血杆菌被鉴定为严重哮喘的一种临床相关病原体，并通过宏基因组测序可靠地鉴定。在对大型患者队列的正在进行的分析中应用这些方案将允许对这种临床表型进行全面描述。人类气道在健康时曾经被认为是无菌的。现在宏基因组技术表明细菌可能存在，但它们在哮喘中的作用尚不清楚。传统的培养方法缺乏敏感性，而当前的测序技术受到操作问题和在物种水平上识别病原体能力的限制。我们优化了一种新的测序技术-牛津纳米孔技术（ONT）-用于人类痰样本，并将其与现有方法进行了比较。我们发现 ONT 可有效快速分析样本，并能在物种水平上鉴定细菌。我们利用这一点表明流感嗜血杆菌是严重哮喘患者气道中的优势细菌。流感嗜血杆菌的存在与一种“中性粒细胞性”哮喘形式有关，对于这种哮喘形式，我们目前缺乏特定的治疗方法。因此，该技术可用于靶向慢性抗生素治疗，并用于研究气道中细菌的全部范围。
