Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB101

Poster Communications

Investigation of diabetes-induced changes in lung microbiome using next-generation sequencing.

S. Vasiljevs1, D. L. Baines1

1. Institute for Infection and Immunity, St George's University of London, London, United Kingdom.

High blood glucose (hyperglycaemia) has been linked with increased glucose concentration in the airway surface liquid (ASL). This has been shown to be associated with increased risk of developing pulmonary infection in patients in the intensive care unit and in patients who have diabetes and chronic lung disease1. Contrary to the popular belief that the lungs are sterile, it was recently discovered that the lung is inhabited by resident bacteria and that it possesses its own unique microbiome. Several chronic lung conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), have been shown to induce changes to the resident lung microbiota. We are investigating the effect of high glucose concentration in the ASL on the resident lung microbiome. Preliminary studies were performed in db/db and GK+/- diabetic mice which represent models of Type 2 and Type 1 diabetes. As the environment is a key factor in determining the microbiota in other tissues, diabetic and non-diabetic littermates were housed under similar conditions. Animals were subjected to Schedule 1 terminal anaesthesia (i.p. injection of 0.2 mL pentobarbital (60 mg/ml) diluted 1:1 with saline) and samples were collected using bronchoalveolar lavage (BAL) technique. Cannulas were inserted via the trachea and lungs were washed using 1ml of sterile PBS. Negative control samples were created by repeating all the steps without BAL in order to account for environmental contaminants. The 16S ribosomal RNA amplification approach has become the standard for identifying bacterial species in the microbiota. 16S is present in all bacteria and allows precise species identification from a small amount of starting material, making it a strong choice for analysis of the samples with low bacterial yield, such as samples of lung microbiome. The lung microbiome of diabetic and non-diabetic mice was analysed using next-generation sequencing technique Illumina MiSeq and NanoPore. Analysis was performed using bacterial 16S rRNA gene (1500bp). Primers amplifying V3-V4 regions of 16S gene were used for the MiSeq analysis. Pure bacterial DNA was amplified using Touchdown PCR approach with annealing temperatures from 64°C to 54°C with a 1°C decrease per step. Cycle was repeated 38 times for sufficient amplification. Preliminary studies suggest a shift in diversity in the resident lung microbiome with diabetes. Abundance of several types of Actinobacteria was notably increased in the diabetic mice in comparison to the wild-type mice. Both Staphylococcaceae and Streptococcaceae abundance was also increased in the lungs of diabetic mice. This data supports previous findings that diabetes mellitus is a predisposing factor for nasal colonisation of S. aureus2.

Where applicable, experiments conform with Society ethical requirements