Document Detail


Reducing bias in bacterial community analysis of lower respiratory infections.
MedLine Citation:
PMID:  23190732     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
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
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-11-29
Journal Detail:
Title:  The ISME journal     Volume:  7     ISSN:  1751-7370     ISO Abbreviation:  ISME J     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-19     Completed Date:  2013-09-30     Revised Date:  2014-04-01    
Medline Journal Info:
Nlm Unique ID:  101301086     Medline TA:  ISME J     Country:  England    
Other Details:
Languages:  eng     Pagination:  697-706     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Adult
Azides / therapeutic use*
Bacteria / classification*,  drug effects*,  genetics,  isolation & purification
Cystic Fibrosis / complications,  drug therapy*,  microbiology*
Humans
Propidium / analogs & derivatives*,  therapeutic use
Pseudomonas aeruginosa / drug effects,  genetics,  isolation & purification
Real-Time Polymerase Chain Reaction
Respiratory Tract Infections / drug therapy,  microbiology*
Sputum / microbiology
Grant Support
ID/Acronym/Agency:
K02 HL105543/HL/NHLBI NIH HHS; UL1 TR000423/TR/NCATS NIH HHS
Chemical
Reg. No./Substance:
0/Azides; 0/propidium monoazide; 36015-30-2/Propidium
Comments/Corrections

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