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


CONCLUSIONS
PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.
IRSTEA, 17 avenue de Cucillé, 35044, Rennes, France. Jeremy.desneux@irstea.fr.|Université Européenne de Bretagne, Rennes, France. Jeremy.desneux@irstea.fr.
Jérémy
J
Desneux
Université Européenne de Bretagne, Rennes, France. Marianne.chemaly@anses.fr.|French Agency for Food Environmental and Occupational Health Safety, Anses, Laboratory of Ploufragan-Plouzané, F-22440, Ploufragan, France. Marianne.chemaly@anses.fr.
Marianne
M
Chemaly
IRSTEA, 17 avenue de Cucillé, 35044, Rennes, France. anne-marie.pourcher@irstea.fr.|Université Européenne de Bretagne, Rennes, France. anne-marie.pourcher@irstea.fr.
Anne-Marie
AM
Pourcher
D000818:Animals; D001386:Azides / Q000378:metabolism*; D058491:Bacterial Load / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008372:Manure / Q000382:microbiology*; D050296:Microbial Viability; D008962:Models, Theoretical; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D013997:Time Factors
D001386:Azides; D004791:Enzyme Inhibitors; D008372:Manure; C533957:propidium monoazide; D011419:Propidium