UBC Theses and Dissertations
The lung microbiome in chronic obstructive pulmonary disease Sze, Marc Alexander
Until recently the normal human lung was thought to be sterile below the larynx, but recent reports from other laboratories indicate that a diverse microbiome exists and becomes less diverse in smokers. These reports led naturally to the hypothesis that pathogens emerging from the abnormal microbiome in smokers could drive the innate and adaptive immune response that has been associated with the pathology of peripheral lung abnormalities observed in Chronic Obstructive Pulmonary Disease (COPD). The purpose of the present study was to examine this hypothesis in human lung tissue. This began with a preliminary experiment in which DNA isolated from 2 samples from a control lung were compared to DNA isolated from 5 different samples of a severe COPD lung, using 75 based pair-end tag sequencing (metagenomic sequencing). For bacteria, a weighted average genome size representing bacterial species identified was applied and the results validated using PCR and qPCR assays. This preliminary experiment was followed by a qPCR, T-RFLP, and targeted sequencing analysis of the bacterial 16S rRNA gene in DNA isolated from single samples of frozen lung tissue obtained from 8 non-smoking and 8 smoking controls, 8 COPD (GOLD 4), and 8 cystic fibrosis patients. The metagenomic sequencing conducted in the preliminary study showed that the 5 samples from a single COPD patient had an average of 2.4 ± 0.7 bacteria/1000 human genomes while the smoking control had 1.6 ± 0.8 bacteria/1000 human genomes. The qPCR results obtained from a single sample from 32 different subjects showed that on average the 8 samples/group of non-smokers, smokers, and COPD (GOLD 4) patients had 34.5 ± 21.8, 44.3 ± 47.0, and 24.1 ± 36.9 bacteria/1000 human cells, respectively, while cystic fibrosis patients had (20 ± 54) x 10 4 bacteria /1000 human cells. T-RFLP analysis showed three distinct community compositions: smokers and non-smokers, cystic fibrosis, and COPD (GOLD 4) patients. These results confirm the presence of a small number of bacteria within the human lung of non-smoker and smoker controls and in COPD patients with a shift in bacterial composition in lungs of those with COPD (GOLD 4).
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