Changes in the lung microbiome following lung transplantation include the emergence of two distinct Pseudomonas species with distinct clinical associations

Abstract

Background: Multiple independent culture-based studies have identified the presence of Pseudomonas aeruginosa in respiratory samples as a positive risk factor for bronchiolitis obliterans syndrome (BOS). Yet, culture-independent microbiological techniques have identified a negative association between Pseudomonas species and BOS. Our objective was to investigate whether there may be a unifying explanation for these apparently dichotomous results.

Methods: We performed bronchoscopies with bronchoalveolar lavage (BAL) on lung transplant recipients (46 procedures in 33 patients) and 26 non-transplant control subjects. We analyzed bacterial communities in the BAL fluid using qPCR and pyrosequencing of 16S rRNA gene amplicons and compared the culture-independent data with the clinical metadata and culture results from these subjects.

Findings: Route of bronchoscopy (via nose or via mouth) was not associated with changes in BAL microbiota (p = 0.90). Among the subjects with positive Pseudomonas bacterial culture, P. aeruginosa was also identified by culture-independent methods. In contrast, a distinct Pseudomonas species, P. fluorescens, was often identified in asymptomatic transplant subjects by pyrosequencing but not detected via standard bacterial culture. The subject populations harboring these two distinct pseudomonads differed significantly with respect to associated symptoms, BAL neutrophilia, bacterial DNA burden and microbial diversity. Despite notable differences in culturability, a global database search of UM Hospital Clinical Microbiology Laboratory records indicated that P. fluorescens is commonly isolated from respiratory specimens.

Interpretation: We have reported for the first time that two prominent and distinct Pseudomonas species (P. fluorescens and P. aeruginosa) exist within the post-transplant lung microbiome, each with unique genomic and microbiologic features and widely divergent clinical associations, including presence during acute infection.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. 16S rRNA gene qPCR of DNA prepared from unfractionated BAL samples.

The number of copies of bacterial 16S rRNA genes per 5's multiple comparisons test.

Figure 2. Comparison of transplant BAL specimens obtained via nasal and oral routes of bronchoscopy.

Unsupervised principal component analysis (PCA) labeled by route of bronchoscope insertion. Group dissimilarity tested using ANOVA permutation test of ordination constrained by route of insertion.

Figure 3. Ordination analyses of bacterial communities detected in BAL samples.

Unsupervised principal component analysis (PCA): labeled by specimen group (A) and with specimen group centroids labeled (B) C: Ordination constrained by specimen group (RDA). Group dissimilarity tested using ANOVA permutation test. D: Biplot analysis of PCA plot with prominent OTUs labeled.

Figure 4. Shannon Diversity Indices of bacterial communities in BAL samples from lung transplant recipients.

A-C: Comparison of bacterial community diversity by transplant status (A), specimen group (symptomatic/transplant status) (B) and antibiotic exposure (C). D-E: Bacterial community diversity correlated with BAL neutrophilia (D) and 16S DNA (E). F: Comparison of bacterial community diversity among transplant subjects by culture results. Comparison of group means performed with unpaired t-test and ANOVA with Tukey's multiple comparisons test. Continuous variables assessed for correlation with linear regression.

Figure 5. Taxonomic classification of the bacterial OTUs detected in BAL samples.

Families and OTUs are ranked in descending order of mean relative abundance among all subjects. Box plots are colored according to phylum (see Phyla legend). Outliers are plotted as circles. A: Relative abundance of the 10 most abundant bacterial families. B: Relative abundance of 20 most abundant bacterial OTUs.

Figure 6. Clinical comparison among lung transplant recipients in relationship to two distinct Pseudomonas OTUs in BAL fluid.

P. aeruginosa refers to OTU 1065, and P. fluorescens refers to OTU 1025 (see text). A: Relative abundance of each OTU in transplant recipients with greater than 10% of either. B-E: Comparison of P. aeruginosa-prominent and P. fluorescens-prominent BAL specimens by BAL neutrophilia (B), bacterial DNA burden (C), bacterial community diversity (D) and culture results (E). F: Relative abundance of P. fluorescens among pre-defined subject groups. Comparison of group means performed using unpaired t-test and ANOVA with Tukey's multiple comparisons test. Contingency testing performed using Fisher's exact test.

Figure 7. In vivo associations between P. fluorescens (OTU 1025) and other prominent bacteria: Prevotella (A) and Veillonella (B).

Group means compared using unpaired t-test.