Dissimilarity of the gut-lung axis and dysbiosis of the lower airways in ventilated preterm infants

Abstract

Background: Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth, but the role of the microbiome in its development remains unclear. Therefore, we assessed the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways, and assessed the gut-lung axis by comparing bacterial communities in the upper and lower airways with stool findings. Finally, we assessed whether the bacterial communities were associated with lung inflammation to suggest dysbiosis.

Methods: We serially sampled multiple anatomical sites including the upper airway (nasopharyngeal aspirates), lower airways (tracheal aspirate fluid and bronchoalveolar lavage fluid) and the gut (stool) of ventilated preterm-born infants. Bacterial DNA load was measured in all samples and sequenced using the V3-V4 region of the 16S rRNA gene.

Results: From 1102 (539 nasopharyngeal aspirates, 276 tracheal aspirate fluid, 89 bronchoalveolar lavage, 198 stool) samples from 55 preterm infants, 352 (32%) amplified suitably for 16S RNA gene sequencing. Bacterial load was low at birth and quickly increased with time, but was associated with predominant operational taxonomic units (OTUs) in all sample types. There was dissimilarity in bacterial communities between the upper and lower airways and the gut, with a separate dysbiotic inflammatory process occurring in the lower airways of infants. Individual OTUs were associated with increased inflammatory markers.

Conclusions: Taken together, these findings suggest that targeted treatment of the predominant organisms, including those not routinely treated, such as Ureaplasma spp., may decrease the development of CLD in preterm-born infants.

FIGURE 1

Total bacterial load in the gut and airways of preterm infants. a) Change in total bacterial load over time shown for the four anatomical sites: bronchoalveolar lavage (BAL), nasopharyngeal aspirate (NPA), tracheal aspirate fluid (TAF) and stool samples. BAL, TAF and NPA samples were measured in copies of the 16S RNA gene per mL of supernatant. Stool samples were measured in copies of the 16S DNA gene per mg of stool. Data are presented as mean±se. b) Table showing the number of samples available at each time point for each sample type, and the percentage of the samples from which the 16S rRNA gene could be successfully amplified at each time point of each sample type. Data are presented as n/N (%).

FIGURE 2

Microbiome analyses for each sample site shown by phylum: a) tracheal aspirate fluid; b) bronchoalveolar lavage; c) nasopharyngeal aspirate; and d) stool. The relative abundance of the predominant phyla of the preterm infant is shown for each time period. Each bar represents an individual sample with relative abundance shown after all operational taxonomic units from each phylum were combined and averaged.

FIGURE 3

Microbiome analyses for each sample site shown at genus level: a) tracheal aspirate fluid; b) bronchoalveolar lavage; c) nasopharyngeal aspirate; and d) stool. Each bar represents an individual sample with relative abundance shown after all operational taxonomic units from each genus were combined.

FIGURE 4

Microbiome community structure between sample sites. a) Nonmetric multidimensional scaling (NMDS) plots for individual babies for each sample site: i) tracheal aspirate fluid (TAF); ii) bronchoalveolar lavage (BAL); iii) nasopharyngeal aspirate (NPA); and iv) stool. Each coloured polygon joins all samples from an individual infant. b) α-diversity of observed genera/operational taxonomic units (OTUs) and Chao1 shown at i) genus and ii) operational taxonomic unit level. c) NMDS plot showing the relationship between the bacterial community between the anatomical sites. PERMANOVA showed significant differences between BAL versus TAF, BAL versus NPA, BAL versus stool, TAF versus NPA, TAF versus stool and NPA versus stool using Bonferroni correction (all p<0.01).

FIGURE 5

Association of the lower airway microbiome with markers of inflammation. a) Log₁₀ scale for interleukin (IL)-6 and -8 in tracheal aspirate fluid (TAF) and bronchoalveolar lavage (BAL) fluid samples for samples where bacterial sequencing was positive or negative. b) The relationship between IL-6, IL-8, antibiotic treatment (blue) in upper graph and bacterial load (black) and bars showing genus constituents in three individual babies. Generally, parallel increases and decreases were observed for all markers despite antibiotic treatment. c) Averaged IL-6 and IL-8 in TAF samples shown for each time point regardless of bacterial presence showing a peak of both cytokines at day 7 as previously shown [2, 3, 19] Data are presented as mean±sem. d) Association between the presence of IL-6 and IL-8 in BAL samples with specific operational taxonomic units (OTUs) showing increased IL-6 and IL-8 if an OTU was identified when compared to the negative for sequencing group. E. coli: Escherichia coli.