Culture-Independent Analysis of Pediatric Bronchoalveolar Lavage Specimens

Abstract

Rationale: The clinical utility of culture-independent testing of pediatric BAL specimens is unknown. In addition, the variability of the pediatric pulmonary microbiome with patient characteristics is not well understood.

Objectives: To compare testing with 16S rRNA gene-based sequencing to conventional cultures of BAL specimens in children Methods: Study subjects were not more than 22 years old and underwent BAL from May 2013 to August 2015 as part of clinical care. DNA extracted from BAL specimens was used for 16S rRNA gene-based analysis, and results were compared with routine cultures from the same samples. Indices of microbial diversity and relative taxon abundances were compared on the basis of subject characteristics (diagnosis and antibiotic use).

Results: From 81 participants (male, 51%; median age, 9 yr), 89 samples were collected. The 16S rRNA genes of 77 samples (86.5%) from 70 subjects were successfully analyzed. These 70 subjects included 23 with cystic fibrosis, 19 who were immunocompromised, and 28 who were nonimmunocompromised. Of 68 organisms identified in culture, 16S rRNA gene-based analyses detected corresponding taxa in 66 (97.1%) and also identified potentially clinically significant organisms missed by cultures (e.g., Staphylococcus, Legionella, and Pseudomonas). Taxa that varied significantly with diagnosis and antibiotic use included Veillonella, Corynebacterium, Haemophilus, and Streptococcus. The microbiota of cystic fibrosis samples was less diverse. A "core" group of 15 taxa present in all three diagnosis groups was identified.

Conclusions: Culture-independent analysis was concordant with routine cultures and showed the potential to detect noncultured pathogens. Although culture-independent testing identified relative changes in organism abundance associated with clinical characteristics, distinct microbiome profiles associated with disease states were not identified.

Keywords: bronchoalveolar lavage; children; microbiome.

Figure 1.

(A) Relative abundance, in operational taxonomic unit (OTU) read counts, of the most abundant taxon in each sample. The relative abundance of predominant cystic fibrosis (CF) OTUs was significantly higher than those of immunocompromised (IC) and nonimmunocompromised (nIC) samples following Kruskal-Wallis testing with Dunn’s multiple comparisons test. (B) Relative abundance of taxa that were isolated in the clinical laboratory. Solid points correspond to the most abundant taxa. Shaded points are nonpredominant taxa that were cultured. All samples were rarefied to 4,025 counts. **P = 0.0023, ****P < 0.0001.

Figure 2.

(A) α diversity (Shannon-Weiner [SW]) within groups was compared using the Kruskal-Wallis test with Dunn’s multiple comparisons test. Cystic fibrosis (CF) samples were significantly less diverse than immunocompromised (IC) and nonimmunocompromised (nIC) samples. (B) Influence of concurrent treatment antibiotics on diversity (Shannon-Weiner) within diagnosis groups by two-way ANOVA with Tukey’s multiple comparisons test. CF samples, with and without treatment antibiotics, were less diverse than IC samples. CF samples without treatment antibiotics were also less diverse than nIC samples without antibiotics. There was no significant reduction in diversity within diagnosis groups due to concurrent treatment antibiotic use. Abx = antibiotics.*0.05 > P > 0.01, **0.01 > P > 0.001, ****P < 0.0001.

Figure 3.

β diversity. Top: Dendrogram clustered by Bray-Curtis dissimilarity. Middle: Diagnosis class and concurrent antibiotic use of each sample. Bottom: Percent abundance of the top 10 most prevalent genera is shown in the corresponding stacked bar chart, with any remaining genera included in “other.” CF = cystic fibrosis; IC = immunocompromised; nIC = nonimmunocompromised.

Figure 4.

Correlation of α diversity (Shannon-Weiner [SW]) with relative abundance of the most prevalent genera. Genera shown are significantly correlated with P values less than 0.0001. Corynebacterium (r = 0.37) and Haemophilus (r = 0.35) were also significantly correlated with P = 0.0011 and 0.0016, respectively. r = Pearson correlation statistic.

Figure 5.

BAL core taxa identified as nonrandomly distributed. Taxa listed were present in at least 30% (gray) or at least 50% (bold) of samples. There are two [Prevotella] operational taxonomic units (OTUs) (belonging to the family Paraprevotellaceae) included in group A and two Stenotrophomonas OTUs in group E. CF = cystic fibrosis; IC = immunocompromised; nIC = nonimmunocompromised.