Discrimination of Viable and Dead Fecal
            <i>Bacteroidales</i>
            Bacteria by Quantitative PCR with Propidium Monoazide

Bae, Sung Woo; Wuertz, Stefan

doi:10.1128/aem.01333-08

Cited by 183 publications

(174 citation statements)

References 17 publications

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“…Intact cell membranes are impermeable to PMA and this molecule can thus be used to differentiate naked DNA and dead cells from bacteria with an intact cell membrane (Nocker et al, 2007). To test whether the DNA extracted from the 2-m permafrost and the active layer soil was from viable cells, the samples were treated with PMA following the concentrations and conditions given for soils in previous studies (Pisz et al, 2007;Bae and Wuertz, 2009). The material (10 g) was mixed with 10 ml sterile ultrapure water and 50 ml of 20 mM PMA.…”

Section: Pma Treatmentsmentioning

confidence: 99%

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

et al. 2010

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The fate of the carbon stocked in permafrost following global warming and permafrost thaw is of major concern in view of the potential for increased CH 4 and CO 2 emissions from these soils. Complex carbon compound degradation and greenhouse gas emissions are due to soil microbial communities, but no comprehensive study has yet addressed their composition and functional potential in permafrost. Here, a 2-m deep permafrost sample and its overlying active layer soil were subjected to metagenomic sequencing, quantitative PCR (qPCR) and microarray analyses. The active layer soil and the 2-m permafrost microbial community structures were very similar, with Actinobacteria being the dominant phylum. The two samples also possessed a highly similar spectrum of functional genes, especially when compared with other already published metagenomes. Key genes related to methane generation, methane oxidation and organic matter degradation were highly diverse for both samples in the metagenomic libraries and some (for example, pmoA) showed relatively high abundance in qPCR assays. Genes related to nitrogen fixation and ammonia oxidation, which could have important roles following climatic change in these nitrogen-limited environments, showed low diversity but high abundance. The 2-m permafrost showed lower abundance and diversity for all the assessed genes and taxa. Experimental biases were also evaluated using qPCR and showed that the whole-community genome amplification technique used caused representational biases in the metagenomic libraries by increasing the abundance of Bacteroidetes and decreasing the abundance of Actinobacteria. This study describes for the first time the detailed functional potential of permafrost-affected soils.

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Section: Pma Treatmentsmentioning

confidence: 99%

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

et al. 2010

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“…97 The main disadvantage of DNA-based PCR techniques is the inability to distinguish viable DNA sequences from inactive or dead organisms, 98-100 unless supplementary methods are used. 101,102 This small substudy examined the identification of pathogens present in suspected infected DFUs using both conventional plating and culture-based techniques and PCR. This enabled us to investigate the agreement between the two analysis techniques (i.e.…”

Section: Appropriateness Of Empirical Antimicrobial Therapy Based On mentioning

confidence: 99%

Concordance in diabetic foot ulceration: a cross-sectional study of agreement between wound swabbing and tissue sampling in infected ulcers

Nelson

Wright-Hughes

Brown

et al. 2016

Health Technol Assess

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BackgroundThere is inadequate evidence to advise clinicians on the relative merits of swabbing versus tissue sampling of infected diabetic foot ulcers (DFUs).ObjectivesTo determine (1) concordance between culture results from wound swabs and tissue samples from the same ulcer; (2) whether or not differences in bacterial profiles from swabs and tissue samples are clinically relevant; (3) concordance between results from conventional culture versus polymerase chain reaction (PCR); and (4) prognosis for patients with an infected DFU at 12 months’ follow-up.MethodsThis was a cross-sectional, multicentre study involving patients with diabetes and a foot ulcer that was deemed to be infected by their clinician. Microbiology specimens for culture were taken contemporaneously by swab and by tissue sampling from the same wound. In a substudy, specimens were also processed by PCR. A virtual ‘blinded’ clinical review compared the appropriateness of patients’ initial antibiotic regimens based on the results of swab and tissue specimens. Patients’ case notes were reviewed at 12 months to assess prognosis.ResultsThe main study recruited 400 patients, with 247 patients in the clinical review. There were 12 patients in the PCR study and 299 patients in the prognosis study. Patients’ median age was 63 years (range 26–99 years), their diabetes duration was 15 years (range 2 weeks–57 years), and their index ulcer duration was 1.8 months (range 3 days–12 years). Half of the ulcers were neuropathic and the remainder were ischaemic/neuroischaemic. Tissue results reported more than one pathogen in significantly more specimens than swabs {86.1% vs. 70.1% of patients, 15.9% difference [95% confidence interval (CI) 11.8% to 20.1%], McNemar’sp-value < 0.0001}. The two sampling techniques reported a difference in the identity of pathogens for 58% of patients. The number of pathogens differed in 50.4% of patients. In the clinical review study, clinicians agreed on the need for a change in therapy for 73.3% of patients (considering swab and tissue results separately), but significantly more tissue than swab samples required a change in therapy. Compared with traditional culture, the PCR technique reported additional pathogens for both swab and tissue samples in six (50%) patients and reported the same pathogens in four (33.3%) patients and different pathogens in two (16.7%) patients. The estimated healing rate was 44.5% (95% CI 38.9% to 50.1%). At 12 months post sampling, 45 (15.1%) patients had died, 52 (17.4%) patients had a lower-extremity ipsilateral amputation and 18 (6.0%) patients had revascularisation surgery.LimitationsWe did not investigate the potential impact of microbiological information on care. We cannot determine if the improved information yield from tissue sampling is attributable to sample collection, sample handling, processing or reporting.ConclusionsTissue sampling reported both more pathogens and more organisms overall than swabbing. Both techniques missed some organisms, with tissue sampling missing fewer than swabbing. Results from tissue sampling more frequently led to a (virtual) recommended change in therapy. Long-term prognosis for patients with an infected foot ulcer was poor.Future workResearch is needed to determine the effect of sampling/processing techniques on clinical outcomes and antibiotic stewardship.FundingThe National Institute for Health Research Health Technology Assessment programme.

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“…PMA treatment for this purpose has been shown to be effective in a range of contexts (Nocker et al, 2007a;Bae and Wuertz, 2009;Kralik et al, 2010;Nam et al, 2011;Taskin et al, 2011), including the assessment of microbiota present in CF airway samples (Rogers et al, 2008(Rogers et al, , 2010. Further, this strategy has been used previously in conjunction with bacterial pyrosequencing in waste-water treatment systems .…”

Section: Introductionmentioning

confidence: 99%

Reducing bias in bacterial community analysis of lower respiratory infections

et al. 2012

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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.

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Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide

Cited by 183 publications

References 17 publications

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

Concordance in diabetic foot ulceration: a cross-sectional study of agreement between wound swabbing and tissue sampling in infected ulcers

Reducing bias in bacterial community analysis of lower respiratory infections

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