Conceptualizing Stress as a Mediator of Lung Microbiota’s Influence on Respiratory Health

Purpose: Stress responses have been shown to alter the microbial ecosystem along the respiratory tract affecting the elimination and migration of microbes. However, to date little is known about the mechanisms through which bacteria function as a resident flora within the respiratory system, especially when confronted to stress. Using an experimental murine model, the purpose of the current study was to assess whether bacteria could be recovered from the lower respiratory tract (e.g. lung tissue), as well as assess the quality and quantity of bacterial species given stress exposure. Methods: C57 B6 mice strain were assigned to either a control group or a stress group and were subjected to a restraint stress paradigm reported to elicit a neuroendocrine stress response. Mice weight was recorded daily as an indicator of stress sensitivity over the course of the study. Following the stress paradigm, mice lungs were harvested, homogenized, and plated onto prepared Brain Heart Infusion agar plates. Eighteen (18) hours later, bacterial numbers were quantified by colony forming unit (CFU) techniques. Gram staining methods were also performed on lung bacterial isolates to access Gram (+) and Gram (-) species. In addition, 16S DNA amplification of lung bacterial isolates was performed for future bacterial sequence analysis. Results: The weights recorded throughout the stress paradigm showed stressed mice had greater variations in weight fluctuations than non-stressed mice. Quantification of bacteria isolated from the lungs demonstrated that while bacteria were recovered from both stressed and non-stressed mice; 8 of 9 stressed mice demonstrated quantifiable colonies where only 3 of 9 non-stressed mice had bacterial counts above the limit of detection. Qualitatively differences in the proportion of Gram (+) and Gram (-) bacteria were observed in the lungs of stressed mice compared to non-stressed mice. Amplification of 16S Illumina V4 primer and gel electrophoresis confirmed the presence of bacterial DNA necessary for downstream sequence analysis to identify species-specific differences present in the lungs of stressed and non-stressed mice. Conclusions: Initial data indicates that stress could be a factor that regulate lung microbiota. Future research will investigate microbiota diversity in the lung and how changes due to stress impact asthma disease pathogenesis.