Towards unbiased bacterial community analysis in lower respiratory infections

High-throughput pyrosequencing and quantitative 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 is an important factor, 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 PMA treatment on community characteristics (abundance, diversity, composition and structure) is not known. Here, we use adult cystic fibrosis (CF) sputum samples as a paradigm. The effects of PMA treatment on CF bacterial community characteristics, as analysed by pyrosequencing, species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR enumeration, 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 significant differences in diversity, however, PMA treatment resulted in a significant alteration in local community membership in all cases. 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 certain recognised and emerging CF pathogens, significantly influencing ‘core’ and ‘satellite’ taxa group membership. Our findings suggest failure to implement NVCE will result in skewed bacterial community analyses.