Pneumocystis jirovecii is a potential pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly

Abstract

Early life gut microbiota is being increasingly recognized as a major contributor to short and/or long-term human health and diseases. However, little is known about these early-life events in the human microbiome of the lower respiratory tract. This study aims to investigate fungal and bacterial colonization in the lower airways over the first year of life by analyzing lung tissue from autopsied infants. The fungal and bacterial communities of lung tissue samples from 53 autopsied infants were characterized by Next-Generation Sequencing (NGS), based on universal PCR amplification of the ITS region and the 16S rRNA gene, respectively. Our study highlights a high degree of inter-individual variability in both fungal and bacterial communities inhabiting the infant lung. The lower respiratory tract microbiota is mainly composed of transient microorganisms that likely travel from the upper respiratory tract and do not establish permanent residence. However, it could also contain some genera identified as long-term inhabitants of the lung, which could potentially play a role in lung physiology or disease. At 3-4 months of age, important dynamic changes to the microbial community were observed, which might correspond to a transitional time period in the maturation of the lung microbiome. This timeframe represents a susceptibility period for the colonization of pathogens such as Pneumocystis. The asymptomatic colonization of Pneumocystis was associated with changes in the fungal and bacterial communities. These findings suggest that the period of 2-4 months of age is a "critical window" early in life. Pneumocystis jirovecii could be a potential pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly, and to the future health of children.

Fig. 1. Blank controls cluster separately from the lung tissue samples.

CCA plots of bacterial (A) and fungal (B) microbiomes according to sample type. Red characters represent lung tissue samples. Blue characters are blank controls.

Fig. 2. Early-life lung fungal communities.

A Relative abundance of the most abundant fungal genera identified in lung tissue samples (present in almost 37% of samples). Sequencing of the ITS region was carried out on 53 lung tissue samples using the Illumina MiSeq platform. A complete list of taxa is provided in the Supplementary Table S2. B Plot of fungal genus prevalence versus relative abundance across samples. Each point corresponds to a different or unique taxon. Red dotted line represents the 30% of prevalence.

Fig. 3. Early-life lung bacterial communities.

A Relative abundance of the most abundant bacterial genera identified in lung tissue samples (present in almost 50% of samples). Sequencing of the 16S rRNA gene region was carried out on 53 lung tissue samples using the Illumina MiSeq platform. A complete list of taxa is provided in Supplementary Table S3. B Plot of bacterial genus prevalence versus relative abundance across samples. Each point corresponds to a different or unique taxon. Red dotted line represents the 50% of prevalence.

Fig. 4. Major changes in the abundance of fungal and bacterial communities at 3–4 months of age.

Dynamic changes of the most abundant fungal (>37% of prevalence) (A) and bacterial (>50% of prevalence) (B) genera during the six months of life. Samples are clustered by age and samples over 6 months of age were ranged in the same age class. The changes of the most prevalent taxa were modeled using the edgeR package in R.

Fig. 5. Early-life fungal community clusters into four distinct microbiota profiles.

A Composition of fungal profiles (MPs) identified by PAM clustering in the total cohort (n = 53), based on the most abundant fungal genera (present in almost 37% of samples). B Cumulative distribution of samples over the first year of life, stratified by MPs. C Prevalence of Pneumocystis at [0–2], [2–4] and [>4] months life. D Relative abundance of Pneumocystis at [0–2], [2–4] and [>4] months life. Pneumocystis detection was confirmed by qPCR.

Fig. 6. Early-life bacterial community clusters into two distinct microbiota profiles.

A Composition of bacterial profiles (MPs) identified by PAM clustering in the total cohort (n = 53), based on the most abundant bacterial genera (present in almost 50% of samples). B Cumulative distribution of samples over the first year of life, stratified by MPs.

Fig. 7. Level of Pneumocystis colonization affects the early-life fungal community.

A Canonical correspondence analysis (CCA) plots of lung mycobiome according to the load of Pneumocystis. B Canonical correspondence analysis (CCA) plots of lung mycobiome in the three groups after removing the Pneumocystis sequencing reads. C LEfSe analysis of lung mycobiome composition (after release of Pneumocystis sequencing reads). Histogram of the LDA scores reveals the most differentially abundant taxa among different level of Pneumocystis colonization.

Fig. 8. Level of Pneumocystis colonization moderately affects the early-life bacterial community.

A Canonical correspondence analysis (CCA) plots of lung microbiome. B LEfSe analysis of lung microbiome composition. Histogram of the LDA scores reveals the most differentially abundant taxa among different level of Pneumocystis colonization.