ANSES, Laboratory for Food Safety, Boulogne-Sur-Mer, France.
Department of Microtechnologies for Biology and Health, LETI, CEA, University Grenoble Alpes, Grenoble, France.
PLoS One. 2023 Mar 9;18(3):e0280885. doi: 10.1371/journal.pone.0280885. eCollection 2023.
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.
一种创新的方法，即拉曼微光谱学与氘同位素探测（Raman-DIP）相结合，可以用于评估细菌中氘源的代谢情况，也可以推测不同的合成途径。该方法需要用重水来处理细胞，这可能会在更高的浓度下影响细菌的存活状态。在本研究中，我们评估了重水掺入对无害李斯特菌细胞存活状态的影响。我们将无害李斯特菌悬浮液暴露于不同的重水浓度（0%、25%、50%和 75%）下，在 37°C 下孵育 30 分钟至 72 小时。通过 qPCR、PMA-qPCR 和琼脂平板计数法分别定量总细胞数、活细胞数和可培养活细胞数。我们通过 Raman-DIP 分析重水的掺入情况。将无害李斯特菌细胞暴露于不同浓度的重水中，在 24 小时的孵育时间内不会改变其细胞活力。此外，在含有 75%v/v D2O 的培养基中暴露 2 小时后，C-D 带的最大强度（专门用于重水的掺入）达到峰值，但在 t = 1 小时 30 分钟时就可以早期检测到标记。总之，验证了 D2O 作为代谢标记物的使用，并可用于检测无害李斯特菌细胞的存活状态。
