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

Jérémy Desneux,Marianne Chemaly,Anne-Marie Pourcher

doi:10.1186/s12866-015-0505-6

Abstract

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

Highlights

Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge
Use of the desirability approach to determine the optimal conditions for propidium monoazide (PMA) pretreatment We studied the concentration of PMA, incubation time and photoactivation time in a raw manure and in a lagoon effluent using an experimental design matrix based on a Doehlert design (Table 1)
The values of Δviable ranged from 0.2 to 1.3 log10 cfu-eq in manure and from 0.1 to 1.0 log10 cfu-eq in lagoon effluent, demonstrating that the combination of the three methodological factors affected the efficiency of the PMA-quantitative PCR (qPCR)

Summary

Introduction

Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. 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). Accurate and reliable detection of viable pathogenic bacteria in complex matrices like manure and biosolids is a major challenge. The Live/Dead BacLight viability assay can detect viable bacteria but this microscopic method is not suitable for environmental matrices due to Desneux et al BMC Microbiology (2015) 15:164 interfere with the photoactivation process. Questions still remain about the applicability of PMA in such matrices

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