Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

Agustí, Gemma; Fittipaldi, Mariana; Morató, Jordi; Codony, Francesc

doi:10.1007/s00253-012-4524-z

Cited by 14 publications

(6 citation statements)

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“…These two taxonomic groups appeared to be very sensitive to PMA treatment, confirming that PMA-pretreatment did in fact affect the detectability of community members other than bacteria. PMA chemistry has previously been used to discern viable from dead fungi [ 28 , 29 ] and viruses [ 30 – 33 ]. The authors are aware, however, that PMA-based viability assays are limited in their ability to accurately distinguish viable spores and archaea from their expired counterparts.…”

Section: Resultsmentioning

confidence: 99%

A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses

et al. 2015

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BackgroundRecent studies posit a reciprocal dependency between the microbiomes associated with humans and indoor environments. However, none of these metagenome surveys has considered the viability of constituent microorganisms when inferring impact on human health.ResultsReported here are the results of a viability-linked metagenomics assay, which (1) unveil a remarkably complex community profile for bacteria, fungi, and viruses and (2) bolster the detection of underrepresented taxa by eliminating biases resulting from extraneous DNA. This approach enabled, for the first time ever, the elucidation of viral genomes from a cleanroom environment. Upon comparing the viable biomes and distribution of phylotypes within a cleanroom and adjoining (uncontrolled) gowning enclosure, the rigorous cleaning and stringent control countermeasures of the former were observed to select for a greater presence of anaerobes and spore-forming microflora. Sequence abundance and correlation analyses suggest that the viable indoor microbiome is influenced by both the human microbiome and the surrounding ecosystem(s).ConclusionsThe findings of this investigation constitute the literature’s first ever account of the indoor metagenome derived from DNA originating solely from the potential viable microbial population. Results presented in this study should prove valuable to the conceptualization and experimental design of future studies on indoor microbiomes aimed at inferring impact on human health.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0129-y) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses

et al. 2015

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“…While PMA-pretreatment has been combined with various molecular techniques and successfully applied for discriminating between living and dead cells in microbiology [6,8,10–17,35], it has been combined to pyrosequencing in only few studies to analyze the bacterial community of environmental and human samples [6,11–15]. Given the importance of detecting the whole viable members at metacommunity level, we studied both respiratory mycobiome and bacteriome of CF patients colonized with P .…”

Section: Discussionmentioning

confidence: 99%

Effects of Propidium Monoazide (PMA) Treatment on Mycobiome and Bacteriome Analysis of Cystic Fibrosis Airways during Exacerbation

et al. 2016

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Introduction and PurposePropidium monoazide (PMA)-pretreatment has increasingly been applied to remove the bias from dead or damaged cell artefacts, which could impact the microbiota analysis by high-throughput sequencing. Our study aimed to determine whether a PMA-pretreatment coupled with high-throughput sequencing analysis provides a different picture of the airway mycobiome and bacteriome.Results and DiscussionWe compared deep-sequencing data of mycobiota and microbiota of 15 sputum samples from 5 cystic fibrosis (CF) patients with and without prior PMA-treatment of the DNA-extracts. PMA-pretreatment had no significant effect on the entire and abundant bacterial community (genera expressed as operational taxonomic units (OTUs) with a relative abundance greater than or equal to 1%), but caused a significant difference in the intermediate community (less than 1%) when analyzing the alpha biodiversity Simpson index (p = 0.03). Regarding PMA impact on the airway mycobiota evaluated for the first time here; no significant differences in alpha diversity indexes between PMA-treated and untreated samples were observed. Regarding beta diversity analysis, the intermediate communities also differed more dramatically than the total and abundant ones when studying both mycobiome and bacteriome. Our results showed that only the intermediate (or low abundance) population diversity is impacted by PMA-treatment, and therefore that abundant taxa are mostly viable during acute exacerbation in CF. Given such a cumbersome protocol (PMA-pretreatment coupled with high-throughput sequencing), we discuss its potential interest within the follow-up of CF patients. Further studies using PMA-pretreatment are warranted to improve our “omic” knowledge of the CF airways.

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“…In addition to vegetative cells of commonly studied bacteria, it has been applied to fastidious bacteria (41,42), bacterial spores (35,43), protozoa (44,45), fungi (46,47), and some viruses (48)(49)(50)(51). Moreover, the method can be applied to any genetic target that can be amplified by PCR or by other DNA amplification procedures (52).…”

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Dead or Alive: Molecular Assessment of Microbial Viability

Cangelosi

Meschke

2014

Appl Environ Microbiol

264

207

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Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.Yet it hath happened that the veritable body without the spirit hath walked.-Ambrose Bierce, The Death of Halpin Frayser M icrobiologists, like characters in zombie fiction, quickly learn the critical importance of distinguishing the living from the dead. In addition to characterizing the numbers and species of microorganisms in samples, it is important to collect data on their physiological states. The most fundamental physiological state of microbial cells is their viability, defined here as the capacity to form progeny. For ecologists, pathobiologists, metagenomicists, food or water safety analysts, infectious-disease clinicians, and virtually every other stripe of microbiologist, the observation of a viable microorganism in a sample means something entirely different from the observation of a dead one. Despite its importance, this distinction remains extremely challenging by current microbiological methods.Microbiological culture meets this requirement, as it selectively detects viable organisms. However, because only a small percentage of species can be cultured, this strategy underestimates microbial diversity (1-6). In contrast, nucleic acid-based methods, ranging from species-specific PCR to metagenomic methods, have greatly advanced our ability to detect diverse microorganisms independently of microbiological culture (7,8). However, these methods provide only limited information on the physiology of microorganisms in samples. They can assess microbial viability retrospectively, by measuring quantitative changes over time, but they cannot discern viability cross-sectionally (in single measurements). Traditional PCR is notoriously poor at differentiating DNA associated with a viable bacterial cell from DNA associated with an inactivated one or from a free DNA fragment. All of these analytes register as "hits" in PCR, despite their very distinct meanings.In order to address this limitation, alternative PCR-based strategies have been developed. This article reviews two complementary strategies. One strategy, term...

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Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

Abstract: Postprint (published version

Cited by 14 publications

References 55 publications

A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses

A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses

Effects of Propidium Monoazide (PMA) Treatment on Mycobiome and Bacteriome Analysis of Cystic Fibrosis Airways during Exacerbation

Dead or Alive: Molecular Assessment of Microbial Viability

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