Friday, November 30, 2012
Reducing bias in bacterial community analysis of lower respiratory infections.
Reducing bias in bacterial community analysis of lower respiratory infections.
Nov 2012
Rogers GB, Cuthbertson L, Hoffman LR, Wing PA, Pope C, Hooftman DA, Lilley AK, Oliver A, Carroll MP, Bruce KD, van der Gast CJ.
Source
Molecular Microbiology Research Laboratory, Institute of Pharmaceutical Science, King's College London, London, UK.
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.
Labels: bacterial community analyses, cystic fibrosis, DNA, lower respiratory infections, NVCE, PCR, Pseudomonas aeruginosa
# posted by Pat O'Connor @ 8:34 AM
Saturday, November 17, 2012
Real-time PCR as a diagnostic tool for bacterial diseases.
Real-time PCR as a diagnostic tool for bacterial diseases.
2012
Source
Laboratoire de Bactériologie, Département des Agents Infectieux, Institut de Biologie et Pathologie, CHU de Grenoble, Université Joseph Fourier Grenoble 1, France and LAPM, CNRS UMR 5163, Institut Jean Roget, Campus Santé, Université Joseph Fourier-Grenoble 1, France. mmaurin@chu-grenoble.fr.
Abstract
In recent years, quantitative real-time PCR tests have been extensively developed in clinical microbiology laboratories for routine diagnosis of infectious diseases, particularly bacterial diseases. This molecular tool is well-suited for the rapid detection of bacteria directly in clinical specimens, allowing early, sensitive and specific laboratory confirmation of related diseases. It is particularly suitable for the diagnosis of infections caused by fastidious growth species, and the number of these pathogens has increased recently. This method also allows a rapid assessment of the presence of antibiotic resistance genes or gene mutations.
Although this genetic approach is not always predictive of phenotypic resistances, in specific situations it may help to optimize the therapeutic management of patients. Finally, an approach combining the detection of pathogens, their mechanisms of antibiotic resistance, their virulence factors and bacterial load in clinical samples could lead to profound changes in the care of these infected patients.
Labels: bacterial diseases, diagnostic tool, infectious diseases, PCR, routine diagnosis
# posted by Pat O'Connor @ 7:28 AM
