Reducing bias in bacterial community analysis of lower respiratory infections

Geraint B. Rogers, Leah Cuthbertson, Lucas R. Hoffman, Peter A C Wing, Christopher Pope, Danny A P Hooftman, Andrew K. Lilley, Anna Oliver, Mary P. Carroll, Kenneth D. Bruce, Christopher J. Van Der Gast

Research output: Contribution to journalArticle

53 Citations (Scopus)

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.

LanguageEnglish
Pages697-706
Number of pages10
JournalISME Journal
Volume7
Issue number4
DOIs
Publication statusPublished - 1 Apr 2013

Keywords

  • propidium monoazide; pyrosequencing; metacommunity; quantitative PCR; cystic fibrosis; commonness and rarity

ASJC Scopus subject areas

  • Microbiology
  • Ecology, Evolution, Behavior and Systematics

Cite this

Rogers, G. B., Cuthbertson, L., Hoffman, L. R., Wing, P. A. C., Pope, C., Hooftman, D. A. P., ... Van Der Gast, C. J. (2013). Reducing bias in bacterial community analysis of lower respiratory infections. ISME Journal, 7(4), 697-706. https://doi.org/10.1038/ismej.2012.145