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

Desneux J, Chemaly M, Pourcher AM - BMC Microbiol. (2015)

Bottom Line: An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results.Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells.PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.

View Article: PubMed Central - PubMed

Affiliation: IRSTEA, 17 avenue de Cucillé, 35044, Rennes, France. Jeremy.desneux@irstea.fr.

ABSTRACT

Background: Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Among existing methods, propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. However, its efficiency depends on the composition of the effluent, particularly on total suspended solids (TSS)) and on methodological parameters. The aim of this study was evaluate the influence of three methodological factors (concentration of PMA, incubation time and photoactivation time) on the efficiency of PMA-qPCR to quantify viable and dead cells of Listeria monocytogenes used as a microorganism model, in two piggery effluents (manure and lagoon effluent containing 20 and 0.4 TSS g.kg(-1), respectively). An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results.

Results: The quantification of viable cells of L. monocytogenes was mainly influenced by the concentration of PMA in the manure and by the duration of photoactivation in the lagoon effluent. Optimal values differed with the matrix: 55 μM PMA, 5 min incubation and 56 min photoactivation for manure and 20 μM PMA, 20 min incubation and 30 min photoactivation for lagoon effluent. Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells.

Conclusion: PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.

No MeSH data available.


Pareto chart of the individual, quadratic and interactive effect of PMA concentration (PMA), incubation time (Inc) and photoactivation time (Phot) on the Δviable (a and b) and Δdead (c and d) values in samples of manure and lagoon effluents
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4537567&req=5

Fig1: Pareto chart of the individual, quadratic and interactive effect of PMA concentration (PMA), incubation time (Inc) and photoactivation time (Phot) on the Δviable (a and b) and Δdead (c and d) values in samples of manure and lagoon effluents

Mentions: The estimated effects of the three independent factors with their second-order interactions on the Δviable and Δdead responses are presented on the standardized Pareto charts in Fig. 1, which graphically displays the magnitude of the effects according to the statistical analysis of the experimental data. The effects are ranked in descending order. Analysis of the data showed that the significance of the variables depends on the nature of the matrix. When viable L. monocytogenes was inoculated in manure, the PMA concentration were the most important factor affecting the Δviable values (Fig. 1a). The two other factors had little or no significant effect on the Δviable values. On the other hand, when viable bacteria were inoculated in the lagoon, only the length of the photoactivation period had a positive effect on the Δviable values (Fig. 1b). The interpretation of the significance of the factors obtained with the dead cells was more difficult given the high number of similar responses corresponding to runs in which the limit of quantification of the qPCR was reached. This may explain the low significance of the three factors observed in the lagoon effluent (Fig. 1d) and the high number of factors and interactions influencing the Δdead values in manure (Fig. 1c). Nevertheless, overall, the reduction in Δviable and the increase in Δdead values were mainly affected by the concentration of PMA and /or the length of the photoactivation period, whereas incubation time had less impact.Fig. 1


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

Desneux J, Chemaly M, Pourcher AM - BMC Microbiol. (2015)

Pareto chart of the individual, quadratic and interactive effect of PMA concentration (PMA), incubation time (Inc) and photoactivation time (Phot) on the Δviable (a and b) and Δdead (c and d) values in samples of manure and lagoon effluents
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4537567&req=5

Fig1: Pareto chart of the individual, quadratic and interactive effect of PMA concentration (PMA), incubation time (Inc) and photoactivation time (Phot) on the Δviable (a and b) and Δdead (c and d) values in samples of manure and lagoon effluents
Mentions: The estimated effects of the three independent factors with their second-order interactions on the Δviable and Δdead responses are presented on the standardized Pareto charts in Fig. 1, which graphically displays the magnitude of the effects according to the statistical analysis of the experimental data. The effects are ranked in descending order. Analysis of the data showed that the significance of the variables depends on the nature of the matrix. When viable L. monocytogenes was inoculated in manure, the PMA concentration were the most important factor affecting the Δviable values (Fig. 1a). The two other factors had little or no significant effect on the Δviable values. On the other hand, when viable bacteria were inoculated in the lagoon, only the length of the photoactivation period had a positive effect on the Δviable values (Fig. 1b). The interpretation of the significance of the factors obtained with the dead cells was more difficult given the high number of similar responses corresponding to runs in which the limit of quantification of the qPCR was reached. This may explain the low significance of the three factors observed in the lagoon effluent (Fig. 1d) and the high number of factors and interactions influencing the Δdead values in manure (Fig. 1c). Nevertheless, overall, the reduction in Δviable and the increase in Δdead values were mainly affected by the concentration of PMA and /or the length of the photoactivation period, whereas incubation time had less impact.Fig. 1

Bottom Line: An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results.Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells.PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.

View Article: PubMed Central - PubMed

Affiliation: IRSTEA, 17 avenue de Cucillé, 35044, Rennes, France. Jeremy.desneux@irstea.fr.

ABSTRACT

Background: Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Among existing methods, propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. However, its efficiency depends on the composition of the effluent, particularly on total suspended solids (TSS)) and on methodological parameters. The aim of this study was evaluate the influence of three methodological factors (concentration of PMA, incubation time and photoactivation time) on the efficiency of PMA-qPCR to quantify viable and dead cells of Listeria monocytogenes used as a microorganism model, in two piggery effluents (manure and lagoon effluent containing 20 and 0.4 TSS g.kg(-1), respectively). An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results.

Results: The quantification of viable cells of L. monocytogenes was mainly influenced by the concentration of PMA in the manure and by the duration of photoactivation in the lagoon effluent. Optimal values differed with the matrix: 55 μM PMA, 5 min incubation and 56 min photoactivation for manure and 20 μM PMA, 20 min incubation and 30 min photoactivation for lagoon effluent. Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells.

Conclusion: PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.

No MeSH data available.