Experimental design for the optimization of propidium monoazide treatment to quantify viable and non-viable bacteria in piggery effluents

Desneux, Jérémy; Chemaly, Marianne; Pourcher, Anne‐Marie

doi:10.1186/s12866-015-0505-6

Cited by 31 publications

(25 citation statements)

References 53 publications

(84 reference statements)

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“…But only a few studies have demonstrated the presence of indicator or pathogenic bacteria in the VBNC state in sludge or in manured soil using PMA or RNA based techniques ( Jiang et al, 2013 ; Fu et al, 2014 ). Although the use of qPCR PMA to detect viable bacteria in sludge is limited by problems with turbid matrices ( Wagner et al, 2008 ), we previously demonstrated that qPCR PMA can be applied to manures after optimisation of the conditions of exposure ( Desneux et al, 2015 ). In the present study, analysis of cell viability in the microcosms, conducted using PMA, showed that L. monocytogenes entered the VBNC state in piggery eﬄuents.…”

Section: Discussionmentioning

confidence: 99%

“…Propidium monoazide (Biotium, Inc., Hayward, CA, USA) was dissolved in water to prepare a stock solution (1 mg mL -1 ) stored in the dark at -20°C. PMA was used as described in Desneux et al (2015) . Briefly, 400 μL of manure or 600 μL of lagoon eﬄuent were transferred in transparent polypropylene reaction tubes (Greiner Bio-One, France).…”

Section: Methodsmentioning

confidence: 99%

“…However, it is worth noting that qPCR PMA remains difficult to apply in turbid matrices such as wastewater or manure containing high rates of suspended matter ( Wagner et al, 2008 ; Varma et al, 2009 ). To overcome this drawback, our team has recently compared different experimental conditions to achieve optimal qPCR PMA quantification of L. monocytogenes in pig manures ( Desneux et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

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Fate of Viable but Non-culturable Listeria monocytogenes in Pig Manure Microcosms

et al. 2016

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The fate of two strains of Listeria monocytogenes and their ability to become viable but non-culturable (VBNC) was investigated in microcosms containing piggery eﬄuents (two raw manures and two biologically treated manures) stored for 2 months at 8 and 20°C. Levels of L. monocytogenes were estimated using the culture method, qPCR, and propidium monoazide treatment combined with qPCR (qPCRPMA). The chemical composition and the microbial community structure of the manures were also analyzed. The strains showed similar decline rates and persisted up to 63 days. At day zero, the percentage of VBNC cells among viable cells was higher in raw manures (81.5–94.8%) than in treated manures (67.8–79.2%). The changes in their proportion over time depended on the temperature and on the type of eﬄuent: the biggest increase was observed in treated manures at 20°C and the smallest increase in raw manures at 8°C. The chemical parameters had no influence on the behavior of the strains, but decrease of the persistence of viable cells was associated with an increase in the microbial richness of the manures. This study demonstrated that storing manure altered the culturability of L. monocytogenes, which rapidly entered the VBNC state, and underlines the importance of including VBNC cells when estimating the persistence of the pathogens in farm eﬄuents.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Fate of Viable but Non-culturable Listeria monocytogenes in Pig Manure Microcosms

et al. 2016

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“…Factors directly interfere with PMA's chemical reactions also affect its e cacy [12,21], such as turbidity, which in uences optical density, thus affecting photoactivation [21,22]. The presence of organic content provides cation exchange sites that inactivate PMA [13,22,23]. These parameters all varied PMA performance in environmental samples, which are particle-rich and chemically heterogeneous.…”

Section: Discussionmentioning

confidence: 99%

Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach

Wang

Yan

Thompson

et al. 2020

Preprint

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Background: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g. qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting “viable” communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark “PMA-seq” (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. Results: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis . However, in realistically complex communities (computer screens, computer mice, soil and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability, with its performance largely affected by community properties such as initial biomass, sample types and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the “PMA-responsive” microbes, we compared our results with previous PMA-based studies and found that PMA-responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. Conclusions: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative accuracy in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities, but provided only qualitative assessments in realistically complex community samples.

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“…Pero también, se ha demostrado que el estado VBNC puede ser parte de la patogenia normal de la infección por Lm. Estudios recientes, ponen de manifiesto que Lm puede presentar una etapa de persistencia en las vacuolas en estado VBNC que podría explicar el largo periodo de incubación de la enfermedad y la mala respuesta a los antibióticos (28), (29) .…”

Section: Escenarios Donde El Estado Vbnc De Lm Es Importanteunclassified

Retos actuales en la detección e identificación de Listeria monocytogenes

Balsalobre

Lepe

2020

REMASP

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Desde el punto de vista microbiológico la identificación y detección de Listeria monocytogenes (Lm) está bien establecida, pero no bien resuelta en algunas circunstancias. En la actualidad, los mayores retos en la identificación de Lm son: la identificación segura de los aislamientos de Lm con características hemolíticas atípicas y la detección de Lm en un estado fisiológicamente viable pero no cultivable (VBNC) en respuesta al estrés. Estos aspectos, no están bien establecidos o no están contemplados en la normativa UNE-EN ISO 11290. Desde el punto de vista de la identificación segura, los mayores problemas ocurren con los aislados de Lm con débil o ausente capacidad hemolítica, los aislados con fenotipo de hemólisis atípico y en la discriminación de los aislados de Listeria innocua con características hemolíticas. Respecto a la detección Lm con fenotipo de no cultivabilidad, los problemas radican en la puesta en evidencia de los aislados en las biopelículas que se forman en las instalaciones de procesamiento de alimentos o en las infecciones que involucran dispositivos protésicos. Otro hecho importante, es la detección en las hojas de los vegetales sometidos a procesos de conservación y en las infecciones que producen abscesos.

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Experimental design for the optimization of propidium monoazide treatment to quantify viable and non-viable bacteria in piggery effluents

Cited by 31 publications

References 53 publications

Fate of Viable but Non-culturable Listeria monocytogenes in Pig Manure Microcosms

Fate of Viable but Non-culturable Listeria monocytogenes in Pig Manure Microcosms

Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach

Retos actuales en la detección e identificación de Listeria monocytogenes

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