Next-generation sequencing and cytokine analysis of bronchoalveolar lavage samples from mechanically ventilated trauma patients

Purpose: Mechanically ventilated trauma patients are at high risk for ventilator-associated pneumonia (VAP). VAP diagnosis relies on several clinical factors including the identification of a specific pathogen by culture-dependent techniques. Often, patients exhibit signs of VAP, yet the hospital lab is unable to culture a pathogen from a bronchoalveolar lavage (BAL) sample. We hypothesize that these culture-negative, presumptive positive patients are infected with potentially pathogenic bacteria that are not detected by traditional culture techniques. Methods: Culture-positive and -negative hospital results were tested again by Sanger and next-generation sequencing (NGS) on the Ion Torrent PGM. Sample cytokine levels were determined using a Bio-Plex Pro Human Th17 cytokine panel. Results: No significant difference was seen between the identification methods in culture-positive BAL. However, NGS analysis of culture-negative BAL identified hundreds of bacterial genera, including a group of patients that exhibited similar bacterial composition, diversity, and abundance. This group was significantly different from other culture-negative BAL that were dominated by one or two suspected pathogens. Culture-negative BAL contained significantly less IL-1β, IL-6 and TNF-α than culture-positive BAL. Conclusions: NGS is a valuable method for pathogen identification, particularly for difficult to culture BAL. Culture-positive BAL exhibit less bacterial diversity and increased cytokine production than culture-negative BAL. The grouping of culture-negative BAL with similar characteristics may denote a core lung microbiome.