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

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.

The microbiome of translocated bacterial populations in patients with and without inflammatory bowel disease

“…mRNA undergoes rapid metabolism by intracellular and extracellular RNase 15 and has an overall lifetime of only a few minutes to hours. 26,27 The aims of our study were: to investigate the existence of viable organisms in the lymph nodes of subjects with and without IBD using molecular techniques and to compare the bacteria present in the lymph nodes with the same patient's gut microbiome. 17 While 16S rRNA is generally stable, degradation occurs under stressful conditions.…”

“…However, as this has been associated with a moderate false positive rate, 25 RNA detection is preferred in tissues with low bacterial abundances. 26,27 The aims of our study were: to investigate the existence of viable organisms in the lymph nodes of subjects with and without IBD using molecular techniques and to compare the bacteria present in the lymph nodes with the same patient's gut microbiome.…”

The Pulmonary Microbiome in Cystic Fibrosis

“…One obvious inconsistency is the use of propidium monoazide (PMA), a chemical compound that binds DNA in cells with damaged membranes and hence allows exclusion of non-viable DNA from sequencing. Excluding non-viable DNA has been suggested to be important for accurately identifying which members of the community are active at times of exacerbation and helps to avoid overestimation of viable microorganisms following treatment with antibiotics, but it is not utilised by all groups [51,52]. There are concerns that PMA may incompletely penetrate sputum, hence only identifying a portion of non-viable cells.…”

The Human Lung Mycobiome in Chronic Respiratory Disease: Limitations of Methods and Our Current Understanding

“…Therefore, it is vital that such analyses choose an appropriate and consistent DNA extraction method which will provide efficient extraction from all cell types and comparable extraction efficiency from different sample types. Nyugen et al performed 16S and internal transcribed spacer (ITS) sequencing and identified Candida as the most predominant fungal genera in CF sputum samples [67]. However, the enzyme-based DNA extraction protocol they used may have favoured the extraction of yeast compared to fungi.…”

“…A high person-to-person variation in mycobiome composition has been reported. Few studies to date have analysed samples in replicates and most only include technical replicates at the PCR stage [37,67]. Bittinger et al [20] performed repeat extractions from samples and found poor reproducibility between replicates, with instances of high abundance of an operational taxonomic unit (OTU) in one replicate and absence in another.…”