Airway Mycobiota-Microbiota During Pulmonary Exacerbation of Cystic Fibrosis Patients: A Culture and Targeted Sequencing Study

Abstract

Background: The airways of patients with cystic fibrosis (pwCF) harbour complex fungal and bacterial microbiota involved in pulmonary exacerbations (PEx) and requiring antimicrobial treatment. Descriptive studies analysing bacterial and fungal microbiota concomitantly are scarce, especially using both culture and high-throughput-sequencing (HTS).

Objectives: We analysed bacterial-fungal microbiota and inter-kingdom correlations in two French CF centres according to clinical parameters and antimicrobial choices.

Methods: Forty-eight pwCF with PEx from Creteil (n = 24) and Lille (n = 24) CF centres were included over 2 years. Sputa were collected for culture and targeted-HTS (ITS2 and V3-V4 targets). Sequencing and culture data, along with clinical, radiological and treatment data, were analysed. Two-level stratified analysis was performed to study potential confounding factors (age, CF mutation, FEV1 and antibiotics) on the centre factor. Inter-kingdom correlations were analysed.

Results: Significant differences in the bacterial microbiota profile were found between centres (p-value = 0.03). For mycobiota, the taxonomic distribution and diversity were comparable. HTS provided concordant but more detailed information than culture and increased detection of main CF fungi (> 25% more positive samples for Aspergillus or Scedosporium). FEV1 and systemic antibiotic before PEx influenced bacterial microbiota, but no clinical association was found with the mycobiota. No inter-kingdom correlation between Pseudomonas and fungi was found.

Conclusions: Describing concomitant bacterial and fungal communities of pwCF at the beginning of PEx using culture and HTS shows greater diversity in HTS and better detection in case of low microbial load. Interesting inter-kingdom correlations were observed, requiring further research on larger cohorts to understand the potential microbial interactions.

Keywords: Aspergillus; Candida; Scedosporium apiospermum; colonization; cystic fibrosis; internal transcribed spacer; pneumonia.

FIGURE 1

Combined heat map of the bacterial (A) and fungal (B) relative abundances of the predominant taxa detected through culture and targeted‐amplicon high‐throughput‐sequencing (TA‐HTS). §Missing data for the bacterial culture; *detection of Pneumocystis jirovecii by qPCR. Patients from Creteil are marked in green, those from Lille in purple. For bacterial culture, semi‐quantification is distributed as follows: rare: < 10³ UCF/mL; light: 10³–10⁴ UFC/mL; moderate: 10⁵–10⁶ UFC/mL; numerous: ≥ 10⁷ UFC/mL. For fungal culture, semi‐quantification is distributed as follows: rare: 1–10 colonies; light: 10–50 colonies; moderate: 50–100 colonies; numerous: ≥ 100 colonies. For 16S‐HTS and ITS2‐HTS, the relative abundances of the 16 most predominant taxa are represented in the heat map. The concordance between the culture and TA‐HTS for the main CF microorganisms is detailed in Figure S1.

FIGURE 2

Diversity of bacterial and fungal microbiota in sputa of CF patients at time of pulmonary exacerbation (PEx) according to their centre (Creteil or Lille). Thirty sputa were collected from Creteil's CF patients and 26 from Lille's. (a) Beta‐diversity of bacterial (A) or fungal (B) microbiota assessed via PCoA analysis, statistical significance was assessed using Permanova test. (b) Taxa plots summarising the relative abundances of most predominant bacterial (A) or fungal (B) genera identified by centre. The diversity indexes (alpha‐diversity measurements) for bacterial and fungal metagenomic analyses are available in Table S3.

FIGURE 3

Correlation analysis based on interspecies correlation network between bacterial and fungal taxa detected in sputa (n = 56) of CF patients with pulmonary exacerbation (PEx). Only taxa harbouring strong intra or inter‐kingdom correlations (> 0.70) on the ReBoot method were plotted on this chart. Blue circles represent bacterial taxa, red circles the fungal taxa; circle size reflects the relative abundance of microorganisms in the dataset. Green lines connecting circles represent positive correlations (< 0.70), the thicker the line, the stronger the correlation. Taxa with < 50 reads were excluded from this analysis.

FIGURE 4

Boxplot of log2 abundances of bacterial (A) and fungal (B) taxa detected in Creteil and Lille samples. Taxa with significantly different relative abundance in Creteil versus Lille are marked with an asterisk (*). No significant differences in relative abundances of main bacterial taxa were detected. For fungi, Aspergillus appeared to be slightly more abundant in Lille versus Creteil (p‐value = 0.05).