Composition and dynamics of the respiratory tract microbiome in intubated patients

Abstract

Background: Lower respiratory tract infection (LRTI) is a major contributor to respiratory failure requiring intubation and mechanical ventilation. LRTI also occurs during mechanical ventilation, increasing the morbidity and mortality of intubated patients. We sought to understand the dynamics of respiratory tract microbiota following intubation and the relationship between microbial community structure and infection.

Results: We enrolled a cohort of 15 subjects with respiratory failure requiring intubation and mechanical ventilation from the medical intensive care unit at an academic medical center. Oropharyngeal (OP) and deep endotracheal (ET) secretions were sampled within 24 h of intubation and every 48-72 h thereafter. Bacterial community profiling was carried out by purifying DNA, PCR amplification of 16S ribosomal RNA (rRNA) gene sequences, deep sequencing, and bioinformatic community analysis. We compared enrolled subjects to a cohort of healthy subjects who had lower respiratory tract sampling by bronchoscopy. In contrast to the diverse upper respiratory tract and lower respiratory tract microbiota found in healthy controls, critically ill subjects had lower initial diversity at both sites. Diversity further diminished over time on the ventilator. In several subjects, the bacterial community was dominated by a single taxon over multiple time points. The clinical diagnosis of LRTI ascertained by chart review correlated with low community diversity and dominance of a single taxon. Dominant taxa matched clinical bacterial cultures where cultures were obtained and positive. In several cases, dominant taxa included bacteria not detected by culture, including Ureaplasma parvum and Enterococcus faecalis.

Conclusions: Longitudinal analysis of respiratory tract microbiota in critically ill patients provides insight into the pathogenesis and diagnosis of LRTI. 16S rRNA gene sequencing of endotracheal aspirate samples holds promise for expanded pathogen identification.

Fig. 1

Subject enrollment and antibiotic exposure. Fifteen recently intubated subjects were enrolled in the study. The horizontal, gray bars depict the duration of each subject’s enrollment: longitudinal data was collected for ten of the 15 enrolled subjects. Thinner horizontal lines indicate the intravenous or oral antibiotic exposure of each subject, with line color representing antibiotic identity, from 2 days prior to enrollment through the end of sample collection. The color corresponding to each antibiotic is summarized to the right. Asterisks indicate subjects who died during the ICU admission

Fig. 2

Upper and lower respiratory tract bacterial communities of intubated subjects and healthy controls. Heatmaps for a upper and b lower respiratory samples are depicted. Each column represents a single sample, and each row represents family-level taxonomic assignment of the sample’s 16S rRNA gene sequences. Vertical lines separate the healthy controls and each intubated subjects. The color indicates proportional abundance of the sequences assigned to each bacterial family within the sample

Fig. 3

Proportional abundance of bacterial community members in intubated subjects and healthy controls. Each point represents an OTU; the samples from which the OTUs were identified are arrayed along the horizontal axis, including both upper and lower respiratory tract sites; the proportional abundance of each OTU in the sample from which it was identified is indicated by its position along the vertical axis. All OTUs that accounted for >200 reads across all samples are included; OTUs from healthy controls are colored red; OTUs from intubated subjects are colored blue. Asterisks indicate samples with concurrent documentation of suspected pneumonia by the critical care attending physician

Fig. 4

Alpha diversity of intubated subjects over time. Within-sample (alpha) diversity, measured by the Shannon index, is shown for upper (OP) and lower (ET for intubated subjects, BAL for healthy controls) respiratory tract sites. Comparison is made between the diversity of communities in healthy controls, versus the communities of intubated subjects within 24 h of intubation or more than 24 h post intubation