title: Reducing bias in bacterial community analysis of lower respiratory infections.
abstract: High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing 'core' and 'satellite' taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.
authors.affiliation: Molecular Microbiology Research Laboratory, Institute of Pharmaceutical Science, King's College London, London, UK.
authors.forename: Geraint B
authors.initials: GB
authors.lastname: Rogers
authors.forename: Leah
authors.initials: L
authors.lastname: Cuthbertson
authors.forename: Lucas R
authors.initials: LR
authors.lastname: Hoffman
authors.forename: Peter A C
authors.initials: PA
authors.lastname: Wing
authors.forename: Christopher
authors.initials: C
authors.lastname: Pope
authors.forename: Danny A P
authors.initials: DA
authors.lastname: Hooftman
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Lilley
authors.forename: Anna
authors.initials: A
authors.lastname: Oliver
authors.forename: Mary P
authors.initials: MP
authors.lastname: Carroll
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Bruce
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: van der Gast
mesh_terms: D000328:Adult; D001386:Azides / Q000627:therapeutic use*; D001419:Bacteria / Q000145:classification* / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D003550:Cystic Fibrosis / Q000150:complications / Q000188:drug therapy* / Q000382:microbiology*; D006801:Humans; D011419:Propidium / Q000031:analogs & derivatives* / Q000627:therapeutic use; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D012141:Respiratory Tract Infections / Q000188:drug therapy / Q000382:microbiology*; D013183:Sputum / Q000382:microbiology
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium