Lung Microbiota of Critically Ill Patients with COVID-19 Are Associated with Nonresolving Acute Respiratory Distress Syndrome

Abstract

Rationale: Bacterial lung microbiota are correlated with lung inflammation and acute respiratory distress syndrome (ARDS) and altered in severe coronavirus disease (COVID-19). However, the association between lung microbiota (including fungi) and resolution of ARDS in COVID-19 remains unclear. We hypothesized that increased lung bacterial and fungal burdens are related to nonresolving ARDS and mortality in COVID-19. Objectives: To determine the relation between lung microbiota and clinical outcomes of COVID-19-related ARDS. Methods: This observational cohort study enrolled mechanically ventilated patients with COVID-19. All patients had ARDS and underwent bronchoscopy with BAL. Lung microbiota were profiled using 16S rRNA gene sequencing and quantitative PCR targeting the 16S and 18S rRNA genes. Key features of lung microbiota (bacterial and fungal burden, α-diversity, and community composition) served as predictors. Our primary outcome was successful extubation adjudicated 60 days after intubation, analyzed using a competing risk regression model with mortality as competing risk. Measurements and Main Results: BAL samples of 114 unique patients with COVID-19 were analyzed. Patients with increased lung bacterial and fungal burden were less likely to be extubated (subdistribution hazard ratio, 0.64 [95% confidence interval, 0.42-0.97]; P = 0.034 and 0.59 [95% confidence interval, 0.42-0.83]; P = 0.0027 per log₁₀ increase in bacterial and fungal burden, respectively) and had higher mortality (bacterial burden, P = 0.012; fungal burden, P = 0.0498). Lung microbiota composition was associated with successful extubation (P = 0.0045). Proinflammatory cytokines (e.g., tumor necrosis factor-α) were associated with the microbial burdens. Conclusions: Bacterial and fungal lung microbiota are related to nonresolving ARDS in COVID-19 and represent an important contributor to heterogeneity in COVID-19-related ARDS.

Keywords: artificial respiration; critical illness; host–microbial interactions; lung microbiome.

Figure 1.

Lung bacterial and fungal burden are correlated with clinical outcomes of coronavirus disease (COVID-19)-related acute respiratory distress syndrome. (A) Lung bacterial DNA burden (quantified using a 16S rRNA gene quantitative PCR assay) was lower in patients with COVID-19 successfully extubated at Day 60 after initiation of mechanical ventilation (n = 44 of 114 unique patients with COVID-19). Patients with the lowest bacterial burden had a higher incidence of successful extubation than those with high bacterial burdens. (B) Likewise, lung fungal DNA burden (quantified by quantitative PCR assay targeting the 18S rRNA gene) was lower in patients who were successfully extubated, and patients with high lung burdens of fungal DNA were less likely to be extubated. (C) Shannon α-diversity did not correlate with the liberation of mechanical ventilation. At Day 60 after initiation of mechanical ventilation, 10 patients were still intubated (and thus did not achieve extubation or death). Hypothesis testing was performed using a Wilcoxon rank sum test (left panels) and Fine and Gray competing risk regression models, treating extubation as outcome and mortality as competing risk. *P < 0.05 and **P < 0.01.

Figure 2.

Lung microbiota community composition is associated with successful extubation in coronavirus disease (COVID-19)-related acute respiratory distress syndrome but not driven by specific bacterial genera. (A) Community composition of lung bacterial microbiota differed between patients who were extubated (n = 44) and those who were deceased or still intubated (n = 70) at 60 days after initiation of mechanical ventilation. Significance of differences in community composition are determined using permutational multivariate ANOVA with the weighted UniFrac distance. Principal coordinates analysis focusing on the center of the figure (leaving out outlying samples with distinct microbiota) is depicted in Figure E6 in the online supplement. (B and C) Biplot analysis (B) and rank abundance analysis (C) revealed no earlier reported bacterial taxa (family and genus level, respectively) driving the differences in community composition. Contaminant taxa (e.g., Massilia and Ralstonia spp.) were abundant in both groups. Rank abundance analysis included the 15 most abundant genera in decreasing order of relative abundance (mean ± SD), and no genera breached Benjamini-Hochberg adjusted significance (α < 0.05; Wilcoxon rank sum test).

Figure 3.

Lung microbiota alterations correspond with secondary pulmonary infections in severe coronavirus disease (COVID-19). (A) Increased lung bacterial DNA burden in patients with a positive microbiologic culture (n = 39 BAL samples) and in those for whom oral bacteria were cultured (n = 95), compared with those with negative cultures (n = 29). (B) The lung fungal DNA burden was higher in patients with proven or probable COVID-19–associated pulmonary aspergillosis (CAPA) (n = 35 BAL samples of 30 unique patients with COVID-19), although considerable overlap did occur. (C–E) High relative abundances (proportion of total 16S rRNA reads) of Staphylococcus species (C), Klebsiella spp. (D), and Serratia spp. (E) in BAL samples of patients with microbiological diagnosis of these pathogens as obtained via nondirected culture. In the boxplots, the rectangle spans the interquartile range with a line at the median. P values are calculated using a Wilcoxon rank sum test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4.

Lung bacterial and fungal burden are correlated with alveolar inflammatory cytokine responses in coronavirus disease (COVID-19)-related acute respiratory distress syndrome. (A) The lung bacterial DNA burden of patients with COVID-19 (n = 163 BAL samples of 114 unique patients) was positively correlated with alveolar concentrations of the proinflammatory cytokines TNF-α (tumor necrosis factor-α), IL-6, IL-1β and TGF-α (tumor growth factor-α. (B) Fungal DNA burden was positively correlated with the alveolar concentrations of proinflammatory cytokines (TNF-α and TGF-α) and ILs involved in the antifungal response (IL-12p70 and IL-17A). Statistical significance was determined using Spearman correlations between cytokine measurements and DNA burdens.