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

Linh Do Ngoc Nguyen,Sophie Merlin,Eric Viscogliosi,Mario Vaneechoutte,Pieter Deschaght,Laurence Delhaes,Christophe Audebert,Sabine Van Daele,Alexandre Loywick,Sanjay Haresh Chotirmall

doi:10.1371/journal.pone.0168860

Abstract

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.

Highlights

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 results showed that only the intermediate population diversity is impacted by PMA-treatment, and that abundant taxa are mostly viable during acute exacerbation in Cystic fibrosis (CF)
Overall effect of PMA on the polymicrobial community in CF sputum samples The mean abundance of P. aeruginosa cells in PMA-untreated samples using qPCR was significantly higher than that of PMA-treated samples (5.17 ± 6.50 x 107 viable bacteria/ml; p = 0,00006 using pairwise t-test) and that of cultures (2.91 ± 3.98 x 107 cells/ml), whereas P. aeruginosa abundances estimated by qPCR in PMAtreated and culture samples were not statistically different (Fig 1)

Summary

Introduction

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. Strategies to manage CF lung disease principally consist of routine microbiology (including microbiological culture of sputum samples) and appropriate antibiotic treatment [1,2]. While these conventional methods identify viable and abundant pathogens, they could not detect uncultivable or difficult-to-cultivate microorganisms. The recent use of cultureindependent methods based on high-throughput sequencing (HTS) has provided a more complete view of the CF lung bacterial microbiome and its evolution during respiratory alteration [3,4,5,6,7]. Sample pretreatment with propidium monoazide (PMA) might facilitate viable microorganism detection, as recently proposed [6]

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