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

PLoS One. 2016 Dec 28;11(12):e0168860. doi: 10.1371/journal.pone.0168860. eCollection 2016.

Abstract

Introduction and purpose: Propidium 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 discussion: We 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.

MeSH terms

  • Adolescent
  • Adult
  • Anti-Bacterial Agents / pharmacology
  • Azides / pharmacology*
  • Biodiversity
  • Cystic Fibrosis / drug therapy
  • Cystic Fibrosis / genetics
  • Cystic Fibrosis / microbiology*
  • DNA, Bacterial / genetics
  • Disease Progression
  • Female
  • Forced Expiratory Volume
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lung / drug effects
  • Lung / microbiology*
  • Lung / physiopathology
  • Male
  • Metagenome
  • Microbiota / drug effects
  • Microbiota / genetics*
  • Middle Aged
  • Mycobiome / drug effects
  • Mycobiome / genetics*
  • Propidium / analogs & derivatives*
  • Propidium / pharmacology
  • Prospective Studies
  • Respiratory System / drug effects
  • Respiratory System / metabolism
  • Respiratory System / microbiology*
  • Sputum / microbiology
  • Young Adult

Substances

  • Anti-Bacterial Agents
  • Azides
  • DNA, Bacterial
  • propidium monoazide
  • Propidium

Grant support

Laurence Delhaes has received research grants from the French Ministry of Health and Research (PHRC no. 2006/1902), Lille Hospital, Lille 2 University (Lille and Ghent Universities partnership: R7000), and Pasteur Institute of Lille. These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. GenesDiffusion’s company provided support in the form of salaries for authors (SM, AL, AC) of GenesDiffusion’s company, but did not have any additional role in the study design, data collection and analysis, or decision to publish. The specific roles of these authors are articulated in the ‘author contributions’ section.