# Identification and viability state determination of Listeria monocytogenes and Vibrio parahaemolyticus at a single-cell level by Raman microspectroscopy

Darius L. Abedini, Jonathan Dikec, Célia Afgoun, Mélanie Gay, Graziella Midelet, Pierre R Marcoux, Thomas Brauge

► To cite this version:

Darius L. Abedini, Jonathan Dikec, Célia Afgoun, Mélanie Gay, Graziella Midelet, et al., Identification and viability state determination of Listeria monocytogenes and Vibrio parahaemolyticus at a single-cell level by Raman microspectroscopy. JSDA, Jun 2025, Maisons-Alfort, France. 2025. hal-05421626

HAL Id: hal-05421626

https://hal.science/hal-05421626v1

Submitted on 17 Dec 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

Public Domain
<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

# Identification and viability state determination of *Listeria monocytogenes* and *Vibrio parahaemolyticus* at a single-cell level by Raman microspectroscopy

ABEDINI Darius¹, ², DIKEC Jonathan¹, AFGOUN Célia¹, GAY Mélanie¹, MIDELET Graziella¹, MARCOUX Pierre R.², BRAUGE Thomas¹

¹Anses Boulogne-sur-Mer, Unité Sécurité sanitaire des aliments d'origine aquatique F-62200, ²CEA Grenoble, Laboratoire d'électronique et de technologie de l'information, Département Technologies et Innovation pour la Santé F-38000

## INTRODUCTION

*Vibrio parahaemolyticus* and *Listeria monocytogenes* are two major human pathogenic bacteria commonly found in seafood and/or in their processing environments. When exposed to environmental stress —such as biocide treatments used during cleaning procedures— these bacteria can enter the **Viable But Non-Culturable (VBNC) state**. In this reversible state, the cells remain metabolically active, but they lose the ability to grow on standard culture media. As a result, current detection methods described in ISO 11290-1:2017 for *L. monocytogenes* and ISO 21872-1:2017 for *V. parahaemolyticus*, which rely on selective solid media, fail to detect cells in the VBNC state.

To circumvent this failure, the objective of the presented thesis is to develop Raman microspectroscopy coupled with Deuterium Isotope Probing (DIP) for the identification and viability state determination at the single-cell level of *L. monocytogenes* and *V. parahaemolyticus*. Raman-DIP spectroscopy allows the measurement of deuterium incorporation at the single-cell level, indicating the metabolic activity of the bacterium. This method also enables species identification using the 400–1800 cm⁻¹ region of the spectrum, known as the bacterial fingerprint.

## MATERIALS AND METHODS

<figure><img src="image_3.png" /></figure>

## POPULATION QUANTIFICATION UNDER BIOCIDE STRESS CONDITIONS

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Quantification of **total population (qPCR)**, **viable population (PMA-qPCR)** and **viable culturable population (Agar plate counting)** of *L. monocytogenes* (A) and *V. parahaemolyticus* (B) after a short or long treatment with two biocides. * Genom Unit (GU) **Colony-Forming Unit (CFU)**</figcaption></figure>

## CLASSIFICATION OF RAMAN SPECTRA

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Preprocessed mean Raman spectra of *Listeria monocytogenes* strain L6202V1 in control condition in short exposition. The spectra are preprocessed with a baseline removal using airPLS algorithm and L2 normalization.</figcaption></figure>

(A) Classification according to growth conditions using spectra from two *L. monocytogenes* strains, with an accuracy of 94% (± 0.09). (B) Classification according to genus (*L. monocytogenes* vs *V. parahaemolyticus*) under identical growth conditions, using spectra from two strains of each species, with an accuracy of 95% (± 0.05). Both models were evaluated using stratified 10-fold cross-validation.

## CONCLUSION AND PERSPECTIVES

The results demonstrated that Raman-DIP spectroscopy not only differentiates *V. parahaemolyticus* from *L. monocytogenes* but also distinguishes VBNC state from the VC state. This distinction is made possible by metabolic labelling through DIP. VBNC occurs more especially after biocide stress.

The next step is to expand our spectral database by incorporating Raman fingerprints from a wider range of bacterial species. This is essential to enable the tool to perform both accurate bacterial identification and reliable viability assessment in a single analysis.