Effects of Propidium Monoazide (PMA) Treatment on Mycobiome and Bacteriome Analysis of Cystic Fibrosis Airways during Exacerbation

Introduction and PurposePropidium monoazide (PMA)-pretreatment has increasingly been applied to remove the bias from dead or damaged cell artefacts, which could impact the microbiota analysis by high-throughput sequencing. Our study aimed to determine whether a PMA-pretreatment coupled with high-throughput sequencing analysis provides a different picture of the airway mycobiome and bacteriome.Results and DiscussionWe compared deep-sequencing data of mycobiota and microbiota of 15 sputum samples from 5 cystic fibrosis (CF) patients with and without prior PMA-treatment of the DNA-extracts. PMA-pretreatment had no significant effect on the entire and abundant bacterial community (genera expressed as operational taxonomic units (OTUs) with a relative abundance greater than or equal to 1%), but caused a significant difference in the intermediate community (less than 1%) when analyzing the alpha biodiversity Simpson index (p = 0.03). Regarding PMA impact on the airway mycobiota evaluated for the first time here; no significant differences in alpha diversity indexes between PMA-treated and untreated samples were observed. Regarding beta diversity analysis, the intermediate communities also differed more dramatically than the total and abundant ones when studying both mycobiome and bacteriome. Our results showed that only the intermediate (or low abundance) population diversity is impacted by PMA-treatment, and therefore that abundant taxa are mostly viable during acute exacerbation in CF. Given such a cumbersome protocol (PMA-pretreatment coupled with high-throughput sequencing), we discuss its potential interest within the follow-up of CF patients. Further studies using PMA-pretreatment are warranted to improve our “omic” knowledge of the CF airways.引言与研究目的 单叠氮碘化丙啶（Propidium monoazide, PMA）预处理现已愈发广泛地应用于消除死细胞或受损细胞所带来的分析偏差——这类偏差会干扰高通量测序对微生物组的分析。本研究旨在探究：将PMA预处理与高通量测序分析相结合，是否会改变气道真菌组（mycobiome）与细菌组（bacteriome）的分析结果。 结果与讨论 我们对5名囊性纤维化（cystic fibrosis, CF）患者的15份痰液样本的真菌群落与细菌群落进行了深度测序分析，并对比了对其DNA提取物分别进行与不进行PMA预处理的测序数据。PMA预处理对整体及高丰度细菌群落（以相对丰度≥1%的操作分类单元（operational taxonomic units, OTUs）所代表的菌属）无显著影响，但在分析α多样性辛普森指数时，会对中等丰度细菌群落（相对丰度<1%）造成显著差异（p=0.03）。本研究首次评估了PMA对气道真菌群落的影响，结果显示经PMA预处理与未预处理的样本之间，α多样性指数并无显著差异。在β多样性分析中，无论是针对真菌组还是细菌组，中等丰度群落的差异均较整体群落与高丰度群落更为显著。本研究结果表明，仅中等（或低）丰度类群的多样性会受PMA预处理影响，由此可知，囊性纤维化患者急性加重期的高丰度分类群大多处于存活状态。鉴于PMA预处理联合高通量测序的实验流程较为繁琐，我们探讨了该方法在囊性纤维化患者随访中的潜在应用价值。未来需开展更多采用PMA预处理的研究，以深化我们对囊性纤维化患者气道的组学认知。