BAL samples analysis
STERILE
For each lavage sample of the upper and lower airway, 20 ml of sterile isotonic saline solution were instilled with subsequent gentle suctioning. As soon as the upper and lower airway lavage samples were collected, they were centrifuged (11,000 g for 10 min). Subsequently, the pellet was collected and stored at − 80 °C until analysis. Defrosted samples were centrifuged a second time (22,500 g for 30 min), and the resulting pellet was used for DNA isolation by the DNeasy PowerSoil Kit (QIAGEN) according to manufacturer’s instructions. Of the 62 BAL samples, one sample belonging to the standard nutrition group (ID #16 at T7) was excluded by the study because it resulted damaged at the opening. An aliquot of the sterile saline used in sample collection and processing was included in the set of analyzed samples as a negative extraction control. It was processed and sequenced along with all the collected samples, and the identified features were evaluated in order to exclude potential sources of contamination. Extracted DNA was quantified using a high-sensitivity Qubit™ fluorometer.