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Accurate and informative microbiologic testing is essential for guiding diagnosis and management of pneumonia in critically ill patients. Sampling of tracheal aspirate (TA) is less invasive compared to mini-bronchoalveolar lavage (mBAL) and is now recommended as a frontline diagnostic approach in mechanically ventilated patients, despite the historical belief that TA was suboptimal due to contamination from oral microbes. Advancements in metagenomic next generation sequencing (mNGS) now permit assessment of airway microbiota without a need for culture, and as such provide an opportunity to examine differences between mBAL and TA at a resolution previously unachievable. Here, we engaged shotgun mNGS to quantitatively assess the airway microbiome in matched mBAL and TA specimens from a prospective cohort of critically ill adults. We observed moderate differences betweensampletypes across all patients(Pearson correlation of 0.72, 95% CI: 0.68 - 0.76), however we found significant compositional similarity in patients with bacterial pneumonia, whose microbial communities were characterized by a dominant pathogen (Pearson correlation of 0.92, 95% CI: 0.88 - 0.95). In addition, we found that both mBAL and TA were similar in terms of microbial burden, abundance of oropharyngeal taxa, and microbial diversity. Our findings suggest that TA sampling provides a similar assessment of airway microbiota as more invasive testing by mBAL, and that this similarity is most significant in the setting of bacterial pneumonia.

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