Deuterium isotope probing (DIP) on Listeria innocua: Optimisation of labelling and impact on viability state

Abstract

An innovative approach, Raman microspectroscopy coupled with deuterium isotope probing (Raman-DIP), can be used to evaluate the metabolism of deuterated carbon source in bacteria and also to presume different anabolic pathways. This method requires the treatment of cells with heavy water that could affect the bacterial viability state at higher concentration. In this study, we evaluated the effect of heavy water incorporation on the viability state of Listeria innocua cells. We exposed the L. innocua suspensions to different heavy water concentrations (0%, 25%, 50% and 75%) from 30 minutes to 72 h of incubation times at 37°C. The total, viable and viable culturable populations were quantified by qPCR, PMA-qPCR and plate count agar respectively. We analyzed heavy water incorporation by Raman-DIP. The exposure of L. innocua cells to different concentrations of heavy water did not alter their cell viability to 24 h incubation time. In addition, the maximum intensity for C-D band, specific for the incorporation of heavy water, was reached after 2 h of exposure in a media containing 75% v/v D2O but an early detection of the labelling was possible at t = 1 h 30 min. In conclusion, the use of D2O as a metabolic marker was validated and can be developed for the detection of L. innocua cell viability state.

Fig 1

Quantification of total L. innocua ATCC 33090 population (Log (GE/mL)), viable population (Log (GE/mL)), and viable culturable population (Log (CFU/mL)) with A) 0%, B) 25%, C) 50% and D) 75% of D₂O in nutrient media. The error bars represented the standard deviation (n = 6). Statistical significant differences were evaluated at p< 0.05 and represented by “*”.

Fig 2. Raman spectra (1800–2500 cm^-1) of single L. innocua cells grown in TSBYe with heavy water (0%, 25%, 50% and 75% D₂O of nutrient medium water).

Fig 3. Quantification of the C-D band (between 2,040 and 2,300 cm⁻¹) in non-deuterated (0% of D₂O (curve blue) and deuterated (25% (curve orange), 50% (curve green), 75% (curve red) of D₂O) bacterial suspension of L. innocua ATCC 33090.

Each points represents the mean height of the C-D band (n > 60). The coloured halo represents one standard deviation (n > 60). Statistical significant differences were noticed when blue halo is separated to other colored halo.