Pulmonary microbiome and cytokine analysis of Bronchoalveloar lavage in mechanically ventilated trauma patients
Ryan Huebinger, UT Southwestern Medical Center; Deborah Carlson; Ashley Smith, University of North Texas Health Science Center; Sara Ireland, UT Southwestern Medical Center; Ding Chen, UT Southwestern Medical Center; Christian Minshall, UT Southwestern Medical Center; Joseph Minei, UT Southwestern Medical Center; Robert Barber, University of North Texas Health Science Center; Michael Allen, University of North Texas Health Science Center; Steven Wolf, UT Southwestern Medical Center; Nancy Monson, UT Southwestern Medical Center
Mechanically ventilated patients have a variety of bacterial lung flora that are often undetectable by traditional culture methods. The source of infection in many of these patients remains unclear. To better address this problem clinically, we have used next generation sequencing to query the lung microbiome of such patients as a means to identify the source of infection. Expanding on ourprevious immunophenotyping and microbiome work we examined the levels of cytokine production in BAL fluid within this patient cohortHypothesis:
Levels of cytokine response will differ in BAL samples based upon the presence of a predominant bacterial pathogen identified by traditional culture and microbiome sequencing.Methods:
Bronchoalveolar lavage samples were collected in the SICU as a part of standard of care for intubated individuals that had a CPIS>6 or would be intubated >48 hours (screening). Raw BAL was frozen at -80 for microbiome analysis. The remaining BAL fluid was filtered through a 70 micron filter and centrifuged to pellet cells. BAL supernatant was stored at -80 for cytokine analysis. Cytokine analysis was conducted with the Bioplex Pro Human Th17 cytokine panel (15-plex). The microbiome of BAL samples were sequenced for the 16S rRNA region using the ION Torrent PGM. Bacterial species with a relative abundance of less than 1% were excluded from the microbiome profile.Results:
A total of 8 culture positive and 6 culture negative BAL samples were analyzed. Five of the 6 culture negative BAL samples were screening BALs. Microbiomes of the culture positive BAL were dominated by one or two pathogens. The average number of species in the culture positive BAL (with relative abundance >1%) was 3.4 species, whereas the culture negative BAL had an average of 17.3 species (with relative abundance >1%) and were not dominated by a single bacterial species. Comparison of cytokine expression (represented as pg/ml) between the culture positive and culture negative BAL showed an increased level of cytokines in culture positive samples: Interferon-gamma (15.4:0.6), IL-17F (20.7:2.0), IL-1 beta (1189.9:9.3), and IL-6 (417.0:114.8) (Culture Positive:Culture Negative).Conclusions:
Culture positive BAL samples exhibited a more robust immune response and reduced diversity of bacterial species. Lower cytokine levels in culture negative samples, despite a greater number of bacterial species, suggests a diverse resident non-pathogenic bacterial community may be indicative of a healthy environment.