In vivo Lung Microbiome Alterations from Burn Pit Emissions and/or Sand Inhalation Exposures

Jeanette S. Frey^1,2Matthew W. Grogg³Andrew Hoisington^4,5,6Brian A. Wong⁷Camilla A Mauzy^8*

• ¹Air Force Research Laboratory Aerospace Systems Directorate, Wright-Patterson Air Force Base, United States
• ²Henry M. Jackson Foundation for the Advancement of Military Medicine, Dayton, OH, United States
• ³Air Force Reseach Laboratory, Human Effectiveness Directorate, Air & Space Biosciences Div, Dayton, OH, United States
• ⁴Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Rocky Mountain Regional VA Medical Center (RMRVAMC),, Aurora, CO, United States
• ⁵Department of Physical Medicine & Rehabilitation, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
• ⁶Military and Veteran Microbiome: Consortium for Research and Education, Aurora, CO, United States
• ⁷US Naval Medical Research Unit Dayton, Wright-Patterson Air Force Base, United States
• ⁸Human Effectiveness Directorate, Air & Space Biosciences Div, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, United States

In-theater inhalation exposures to burn pit emissions (BPEs) and sand, and their possible linkage to respiratory issues, led to a study to identify molecular alterations and potential biomarkers of exposure and outcome. Using a complex in vivo exposure scenario to mimic in-theatre inhalation exposures, Sprague Dawley rats were exposed to clean air (Control), BPEs, Sand, or a combination of BPE+Sand via whole body exposure chambers. After euthanasia, bronchoalveolar lavage fluid was collected at 4 and 90 days post-exposure and bacterial amplicon sequence variants identified using genomic DNA extraction and 16S rRNA gene sequencing. Both BPE and BPE+Sand exposures significantly altered the lung microbiome, demonstrating increased mean alpha diversity and highest unique ASVs. Both BPE and BPE+Sand exposures initiated changes to the lung microbiome at 4 days post-exposure that endured throughout the 90 day post-exposure assessment. BRE and BRE+Sand groups had increased levels of Bradyrhizobium and Methylobacterium and decreased levels of Pseudomonas compared to either Control or Sand groups. Genera most associated with the differences at 4 days post-exposure seen between BPE vs. Control and BPE+Sand vs. Control groups were Corynebacterium, Geobacillus, Sphingomonas, and Streptococcus. Interestingly, the lung microbiome from the Sand or Control groups were not significantly altered based on alpha or beta diversity and shared the most abundant genera. These data indicate that BPE exposures significantly alter the lung microbiome, whereas sand inhalation exposures alone did not seem to implement significant changes nor, when combined with BPE, provide an additive effect. While the sub-chronic exposure study design initiated more subtle molecular alterations in the lung tissue than expected, BPE exposures initiated distinct and significant microbiome compositional changes in the lung. The observed population shifts provide a signature specific to the type of inhalation exposure. Further efforts could lead to an understanding of the role of individual lung microbiomes play in inhalation exposure risks and outcome.