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|Title:||MOLECULAR AND CULTURE-BASED PROFILING OF SPUTUM MICROBIOTA FROM CYSTIC FIBROSIS PATIENTS|
|Abstract:||Background/Objectives: Chronic airway infections, characterized by pulmonary exacerbations, are responsible for >90% of morbidity and mortality in CF. Although conventional CF pathogens are targets of antibiotic therapy, colonization by complex polymicrobial communities is now recognized. Our previous research has observed novel pathogens in CF, and polymicrobial interactions, where increased P. aeruginosa virulence may contribute to airway infections. However, these culture-based findings are not recapitulated in culture-independent microbiome profiling studies. Because expectorated sputum produces highly heterogeneous mucous plugs, only a percentage is representative of the active subpopulation driving the disease. To distinguish populations, culture will be compared with DNA and RNA based molecular profiling. Methods: Spontaneous sputum produced by adult patients attending regular CF clinic visits were obtained within 30 min and processed. Aliquots were treated for quantitative culture and processed for DNA and RNA extraction. Bacterial composition was determined by profiling of the v3 variable region of the 16S rRNA gene (DNA) and 16S rRNA (RNA), and sequenced on an Illumina MiSeq and processed using DADA2. Results: Quantitative culture allowed for recovery of majority of the CF airway microbiome and resulted in distinct cultured organisms at each fractionation conditions for each patient. This individual specific composition was recapitulated in molecular profiles; however, DNA and RNA profiles were dissimilar at each fractionation step. Conclusions: Each bacterial profiling method resulted in distinct bacterial composition. Processing allowed for varying areas of sputum to be isolated resulting in high microbial diversity within the sample, highlighting high sputum microbiome heterogeneity.Incongruent DNA and RNA profiles across sputum suggest viable bacterial populations, represented by the RNA profile, may be more representative of disease state. Overall, the microbiology of sputum is highly patient specific and very difficult to identify a unifying pattern.|
|Appears in Collections:||Open Access Dissertations and Theses|
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|Nair_Gayatri_2019September_MSc.pdf||29.3 MB||Adobe PDF||View/Open|
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