The characterisation of polymicrobial bacterial communities in the lower respiratory tract of individuals with chronic pulmonary disease

Purcell, Paul (2013) The characterisation of polymicrobial bacterial communities in the lower respiratory tract of individuals with chronic pulmonary disease. Doctoral thesis, Northumbria University.

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Abstract

Microbial diversity encompasses the whole of the Earth’s biosphere and is incredibly vast. The microbial diversity of three disparate micro-environments using two culture-independent techniques (denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing) were revealed. Five commercially available DNA polymerase (pol) enzymes were assessed in determining the bacterial community generated in sandy soil. The V3 region of the 16S rRNA gene was targeted for amplification by polymerase chain reaction (PCR). Using a PCR-DGGE approach, different DNA pols exhibited differences in the DGGE profiles produced. Both high-fidelity DNA pols Ex Taq™ Hot Start (HS) and Platinum® Pfx detected greater microbial diversity present within sandy soil than the other DNA polymerase enzymes.

We employed Ex Taq™ HS to characterise the microbial communities present in two chronic respiratory tract diseases, non-cystic fibrosis bronchiectasis (nCFBR) and chronic obstructive pulmonary disease (COPD). Seventy individuals expectorated sputum, and using 16S and 28S rRNA PCR-DGGE polymicrobial communities were revealed. From the 70 patients investigated, 20 presented with symptoms consistent with an exacerbation, the remainder being clinically stable. Demographic and culture data were used in constrained ordination analyses to identify any significant associations between these data and changes in the sputum microbiota. The data presented indicates that bacterial lung communities in adult nCFBR patients have distinct differences between exacerbating and clinically stable episodes. Persistent colonisation by Pseudomonas aeruginosa is significantly associated with reduced lung function, and is negatively correlated with Haemophilus influenzae carriage. Bacterial communities seem to be predominantly assembled by stochastic processes. Fungal taxa present were scarce.

Stable COPD populations have been previously investigated using culture-dependent techniques. Eleven clinically stable COPD patients had a bronchoalveolar lavage (BAL) fluid taken from the right lower lobe. Both 16S and 28S rRNA PCR-DGGE was performed on all clinical samples from extracted DNA. Co-migration of bands was then compared to a 16S and 28S standard ladder consisting of pure cultivars. Additionally, execution of 454-pyrosequencing and interrogation of the V3-V5 region of 16S rRNA genes resulted in 1799 unique OTUs being identified. Dominant bacterial genera identified were Streptococcus, Arthrobacter, and Staphylococcus respectively. Bacterial taxa identified were then subjected to multivariate statistical analysis to identify relationships between the microbial communities and patient phenotypes. Metagenomic analysis demonstrated that heterogeneous bacterial populations exist in all eleven individuals. This preliminary study shows that the lungs of COPD sufferers are colonised with multiple species of bacteria and demonstrate that a complex microbial community is present. Furthermore, bacterial phylotypes resolved to class-level indicated three potential drivers of community structure within the COPD lung microbiome: lung function, moderate and severe COPD progression, and smoking status in cohort. The identification of a greater number of bacterial taxa was also apparent in culture-negative patients using both PCR-DGGE and 454-pyrosequencing approaches.

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