Alterations in airway microbiota in patients with PaO₂/FiO₂ ratio ≤ 300 after burn and inhalation injury

<div><p>Background</p><p>Injury to the airways after smoke inhalation is a major mortality risk factor in victims of burn injuries, resulting in a 15–45% increase in patient deaths. Damage to the airways by smoke may induce acute respiratory distress syndrome (ARDS), which is partly characterized by hypoxemia in the airways. While ARDS has been associated with bacterial infection, the impact of hypoxemia on airway microbiota is unknown. Our objective was to identify differences in microbiota within the airways of burn patients who develop hypoxemia early after inhalation injury and those that do not using next-generation sequencing of bacterial 16S rRNA genes.</p><p>Results</p><p>DNA was extracted from therapeutic bronchial washings of 48 patients performed within 72 hours of hospitalization for burn and inhalation injury at the North Carolina Jaycee Burn Center. DNA was prepared for sequencing using a novel molecule tagging method and sequenced on the Illumina MiSeq platform. Bacterial species were identified using the MTToolbox pipeline. Patients with hypoxemia, as indicated by a PaO₂/FiO₂ ratio ≤ 300, had a 30% increase in abundance of <i>Streptococcaceae</i> and <i>Enterobacteriaceae</i> and 84% increase in <i>Staphylococcaceae</i> as compared to patients with a PaO₂/FiO₂ ratio > 300. Wilcoxon rank-sum test identified significant enrichment in abundance of OTUs identified as <i>Prevotella melaninogenica (p</i> = 0.042), <i>Corynebacterium</i> (<i>p</i> = 0.037) and <i>Mogibacterium</i> (<i>p</i> = 0.048). Linear discriminant effect size analysis (LefSe) confirmed significant enrichment of <i>Prevotella melaninognica</i> among patients with a PaO₂/FiO₂ ratio ≤ 300 (<i>p</i><0.05). These results could not be explained by differences in antibiotic treatment.</p><p>Conclusions</p><p>The airway microbiota following burn and inhalation injury is altered in patients with a PaO₂/FiO₂ ratio ≤ 300 early after injury. Enrichment of specific taxa in patients with a PaO₂/FiO₂ ratio ≤ 300 may indicate airway environment and patient changes that favor these microbes. Longitudinal studies are necessary to identify stably colonizing taxa that play roles in hypoxemia and ARDS pathogenesis.</p></div

Increased detection of unique facultative anaerobic taxa.

<p>(A) Unique facultative anaerobic OTUs were detected significantly more frequently than obligate aerobes or anaerobes among all patients. (B) No significant difference was found between number of unique facultative anaerobic OTUs when the data was split by patient PaO₂/FiO₂ ratio. OTUs were identified as facultative anaerobes, obligate anaerobes, or obligate aerobes among all patients. OTUs were quantified and normalized to the molecule tag count and averaged by bacterial aerobic or anaerobic capability. One-way ANOVA detected a significant difference among the mean taxa of facultative anaerobes (<i>p</i> = 0.029). (n = 48)</p

Patient clinical characteristics.

<p>Patient clinical characteristics.</p

Percent abundance OTU level differences.

<p>Percent abundance OTU level differences.</p

Average abundance of taxa detected among 80% of patients.

<p>Average abundance of taxa detected among 80% of patients.</p

Patient airway microbiota composition.

<p>OTUs identified as the families Streptococcaceae and Enterobacteriaceae dominate the airway microbiota within 72 hours following burn and inhalation injury. (A) The microbiota composition among patients with hypoxemia. (B) The microbiota composition among patients without hypoxemia. Each horizontal bar represents an individual patient microbiota normalized to 100%. Different colored sections within each bar indicate abundance of specific families. The category ‘Other’ includes bacterial taxa present at less than 1% of the total community. (n = 24)</p

OTU level detection differences.

<p>OTU level detection differences.</p

Percent abundance of OTUs with significant enrichment detected by LEfSe.

<p>Of the three taxa LEfSe identified as significantly enriched in patients with hypoxemia, <i>Staphylococcus</i> had the highest percent increase in abundance. However, <i>Prevotella melaninogenica</i> was more consistently present among patients with hypoxemia, resulting in its higher ranking over <i>Staphylococcus spp</i>. (A) Bacterial abundances for taxa identified as significantly enriched by LEfSe are displayed per patient. The X axis is labeled with each patient’s PaO₂/FiO₂ ratio and the Y axis displays relative abundance of the taxa. (B) The range of abundances of the three significantly enriched taxa are split by patient hypoxemia status along the X axis. (n = 48).</p

Clustering by principle components analysis suggests microbiota similarity in the presence of <i>P</i>. <i>melaninogenica</i>.

<p>The R package phyloseq was used to generate a PCA plot of patient microbiota abundance. Patient samples containing <i>P</i>. <i>melaninogenica</i> cluster to the left side of the graph and are split based on PaO₂/FiO₂ ratio. Manhattan distance was used to perform the ordination prior to plotting the PCA graph. Data points are colored by patient PaO₂/FiO₂ ratio (Red: P/F < 300; Green: P/F > 300) and data point shape indicates presence (square) or absence (triangle) of <i>P</i>. <i>melaninogenica</i>. Control samples are included for comparison (blue circles). (n = 48).</p