Lung-enriched organisms and aberrant bacterial and fungal respiratory microbiota after lung transplant

Abstract

Rationale: Long-term survival after lung transplantation is limited by infectious complications and by bronchiolitis obliterans syndrome (BOS), a form of chronic rejection linked in part to microbial triggers.

Objectives: To define microbial populations in the respiratory tract of transplant patients comprehensively using unbiased high-density sequencing.

Methods: Lung was sampled by bronchoalveolar lavage (BAL) and upper respiratory tract by oropharyngeal wash (OW). Bacterial 16S rDNA and fungal internal transcribed spacer sequencing was used to profile organisms present. Outlier analysis plots defining taxa enriched in lung relative to OW were used to identify bacteria enriched in lung against a background of oropharyngeal carryover.

Measurements and main results: Lung transplant recipients had higher bacterial burden in BAL than control subjects, frequent appearance of dominant organisms, greater distance between communities in BAL and OW indicating more distinct populations, and decreased respiratory tract microbial richness and diversity. Fungal populations were typically dominated by Candida in both sites or by Aspergillus in BAL but not OW. 16S outlier analysis identified lung-enriched taxa indicating bacteria replicating in the lower respiratory tract. In some cases this confirmed respiratory cultures but in others revealed enrichment by anaerobic organisms or mixed outgrowth of upper respiratory flora and provided quantitative data on relative abundances of bacteria found by culture.

Conclusions: Respiratory tract microbial communities in lung transplant recipients differ in structure and composition from healthy subjects. Outlier analysis can identify specific bacteria replicating in lung. These findings provide novel approaches to address the relationship between microbial communities and transplant outcome and aid in assessing lung infections.

Figure 1.

Quantification of bacterial 16S rDNA gene copies in oropharyngeal wash (OW) and bronchoalveolar lavage (BAL) from control and lung transplant subjects. 16S copy number in oropharyngeal (A) and BAL (B) samples of control and transplant subjects. Copy number was also examined in BAL samples of major transplant subgroups (C, D) as described in Table 2. The y axis indicates the 16S rRNA gene copy number by quantitative PCR. Each sample was analyzed in triplicate. *P < 0.05; **P < 0.01 (Mann-Whitney test).

Figure 2.

Relative abundance of bacterial taxa derived from pyrosequencing data. Each column represents an individual sample. Sample type and subject group is indicated at the bottom of each group of columns. Each row corresponds to specific bacterial taxa, with their proportional representation in the given sample indicated by the color code at right. Bacterial operational taxonomic units (OTUs) were collected into genera, so some rows represent multiple OTUs. OTUs not assigned at the genera level and genera with less than 1,000 total reads are omitted. Hierarchical clustering groups the rows to emphasize lineages with similar abundance patterns.

Figure 3.

Relative abundance of fungal taxa derived from pyrosequencing data. Fungal taxa identified in bronchoalveolar lavage and oropharyngeal wash samples are shown as described for bacterial lineages in Fig. 2. Fungal operational taxonomic units (OTUs) were collected into classes, so some rows represent multiple OTUs. OTUs not assigned at the class level and classes with less than 250 total reads are omitted. Along the top is the concentration of ITS DNA post-PCR for each sample, keyed by gray-scale at top right.

Figure 4.

Rarefaction analysis of bacterial and fungal operational taxonomic units (OTUs) in the oropharynx of healthy and lung transplant subjects. The y axis denotes the number of OTUs detected by pyrosequencing of bacterial 16S genes (A) or fungal internal transcribed spacer genes (B) at the corresponding sequencing depths shown along the x axis. The subject group is indicated by the color key on the right. Error bars denote the standard deviation within subject groups.

Figure 5.

Alpha diversity of bacterial communities in healthy and transplant subjects. Diversity was calculated using the Shannon Index for oropharyngeal wash (A) and bronchoalveolar lavage (B). *P < 0.05; **P < 0.01 (Wilcoxon rank sum).

Figure 6.

Relationship between bronchoalveolar lavage (BAL) and oropharyngeal bacterial communities within individuals. Weighted UniFrac distances were calculated between all pairs of samples within BAL or oropharyngeal wash (OW), and then each sample type was plotted separately in 3D space by principal coordinate analysis. The two plots (BAL and OW) were then transformed by Procrustes analysis to achieve maximum alignment. Each point corresponds to a bacterial community, with transplant subjects’ communities shown in blue, healthy subjects’ communities shown in red, and the two communities from each subject connected by a bar. The orange end of each bar connects to the OW sample data; the black end connects to the BAL sample data from the same individual. If BAL and OW plots are similar, then the relative distance between connected points (residuals) will be small. The overall similarity is summarized by the M² value, and statistical goodness of fit is measured by a Monte Carlo label permutation approach (10,000 iterations).

Figure 7.

Identification of lung-enriched bacteria using single-sided outlier plots to compare taxa abundances in bronchoalveolar lavage (BAL) and oropharyngeal wash (OW) samples. Six representative outlier plots are shown (A–F). Each dot represents an operational taxonomic unit (OTU), with its abundance in OW plotted on the x axis and in BAL on the y axis. Taxa found in equal proportions in lung and oropharynx are close to the diagonal line of identity from lower left to upper right, whereas those enriched in BAL are above the diagonal and to the left. Taxa enriched in BAL compared with OW at a level that reaches statistical significance (P < 0.05 after correction for false discovery rate) are highlighted in red.