Culture-independent microbiota analysis is widely used in research and being increasingly used in translational studies. However, methods for standardisation and application of these analyses in clinical trials are limited. Here we report the microbiota analysis that accompanied two phase 2b clinical trials of the novel, non-antibiotic therapy OligoG CF-5/20 for cystic fibrosis (CF) lung infection. Standardised protocols (DNA extraction, PCR, qPCR and 16S rRNA gene sequencing analysis) were developed for application to the Pseudomonas aeruginosa (NCT02157922) and Burkholderia cepacia complex (NCT02453789) clinical trials involving 45 and 13 adult trial participants, respectively. Microbiota analysis identified that paired sputum samples from an individual participant, taken within 2 h of each other, had reproducible bacterial diversity profiles. Although culture microbiology had identified patients as either colonised by P. aeruginosa or B. cepacia complex species at recruitment, microbiota analysis revealed patient lung infection communities were not always dominated by these key CF pathogens. Microbiota profiles were patient-specific and remained stable over the course of both clinical trials (6 sampling points over the course of 140 days). Within the Burkholderia trial, participants were infected with B. cenocepacia (n = 4), B. multivorans (n = 6), or an undetermined species (n = 3). Colonisation with either B. cenocepacia or B. multivorans influenced the overall bacterial community structure in sputum. Overall, we have shown that sputum microbiota in adults with CF is stable over a 2 h time-frame, suggesting collection of a single sample on a collection day is sufficient to capture the microbiota diversity. Despite the uniform pathogen culture-positivity status at recruitment, trial participants were highly heterogeneous in their lung microbiota. Understanding the microbiota profiles of individuals with CF ahead of future clinical trials would be beneficial in the context of patient stratification and trial design.

独立于培养的微生物群分析广泛用于研究，并越来越多地用于转化研究。然而，这些分析在临床试验中的标准化和应用方法是有限的。在这里，我们报告了伴随针对囊性纤维化 (CF) 肺部感染的新型非抗生素疗法 OligoG CF-5/20 的两项 2b 期临床试验的微生物群分析。为铜绿假单胞菌(NCT02157922) 和洋葱伯克霍尔德菌的应用开发了标准化方案（DNA 提取、PCR、qPCR 和 16S rRNA 基因测序分析）complex (NCT02453789) 临床试验分别涉及 45 和 13 名成人试验参与者。微生物群分析发现，来自单个参与者的配对痰样本，在 2 小时内采集，具有可重复的细菌多样性特征。尽管培养微生物学已确定患者在招募时被铜绿假单胞菌或洋葱双歧杆菌复杂物种定植，但微生物群分析显示患者肺部感染群落并不总是由这些关键的 CF 病原体主导。微生物群特征是患者特异性的，并且在两个临床试验过程中保持稳定（140 天过程中的 6 个采样点）。在伯克霍尔德菌试验中，参与者感染了B. cenocepacia( n  = 4)、B. multivoran s ( n  = 6) 或未确定的物种 ( n  = 3)。B. cenocepacia或B. multivorans 的定植影响了痰中的整体细菌群落结构。总体而言，我们已经表明，CF 成人的痰菌群在 2 小时的时间范围内是稳定的，这表明在收集日收集单个样本足以捕捉微生物群的多样性。尽管招募时病原体培养阳性状态一致，但试验参与者的肺微生物群具有高度异质性。在未来的临床试验之前了解 CF 患者的微生物群特征将有助于患者分层和试验设计。