Environmental and physico-chemical factors induce VBNC state in Listeria monocytogenes

Abstract

-Investigations of bacterial survival in natural environments have indicated that some organisms lose culturability on appropriate media under certain conditions and yet still exhibit signs of metabolic activity and thus viability. This reproducible loss of culturability in many bacterial species led to the description of a "Viable But Non Culturable" (VBNC) state. The purpose of this article is to determine environmental and physico-chemical factors which induce the VBNC state in a food-borne pathogen that has become a public concern: Listeria monocytogenes. The factors, i.e. inoculum size, natural sunlight, temperature (4 o C or 20 o C), NaCl concentration (0% or 7%) and pH (5 or 6) were studied on 4 strains (LO28, ATCC 19115, Scott A, CNL 895807). The culturability of the starved cell suspension was determined in each condition tested by the spread plate count, and the cell activity was determined by the Direct Viable Count technique and CTC-DAPI double staining. A strain effect was found in different test conditions. For the LO 28 and ATCC 19115 strains, the VBNC state was very transient in certain conditions. For the other strains tested (Scott A, CNL 895807), the VBNC state was maintained throughout the observation period. In the dark, the incubation temperature was the main factor in the production of VBNC forms in L. monocytogenes. However, natural sunlight rapidly produced the VBNC state in L. monocytogenes cells in microcosm water. We conclude that because of its ubiquity and the factors studied which are met in the food industry, the presence of VBNC L. monocytogenes cells could pose a major public health problem since they cannot be detected by traditional culturing methods. Further investigations are needed to establish virulence before and after resuscitation of VBNC L. monocytogenes cells. bactéries perdent leur capacité à former des colonies sur des milieux de culture, tout en conservant une activité métabolique. L'objectif de ce travail est d'étudier l'influence de facteurs physico-chimiques et environnementaux, intervenant dans l'entrée à l'état VNC de Listeria monocytogenes. Les facteurs : taille de l'inoculum, exposition à la lumière naturelle, température (4-20 o C), concentration en NaCl (0-7%), pH (5-6) ont été étudiés chez 4 souches de L. monocytogenes : LO28, ATCC19115, Scott A, CNL 895807. Les cellules ont été placées dans des conditions de privation nutritionnelle (eau distillée filtrée). La capacité à former des colonies a été déterminée par étalement sur boîte de gélose, alors que l'activité métabolique des bactéries a été établie par 2 techniques : le Direct Viable Count et la double coloration CTC-DAPI. Un effet souche a été constaté: pour les souches LO28 et ATCC 19115, l'état VNC semblait très transitoire, alors que pour les souches Scott A et CNL 895807, l'état VNC s'est maintenu pendant toute la durée de l'expérimentation. A l'obscurité, la température d'incubation est apparue comme le facteur primordial d'entrée à l'état VNC, mais l'exposition à la lumière naturelle a...

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

“…the ability to colonize the mouse spleens and the ability to form plaques within an enterocyte cell line monolayer. Listeria monocytogenes has been reported to enter the VBNC state and to remain as such for extended periods in microcosms, depending on a variety of factors such as temperature and salinity [4,6]. In this study, LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in a microcosm water system at 20 °C, and for the Scott A strain at 4 °C.…”

“…The predominant factors of VBNC state induction have been previously determined [6]. After two 24 h subcultures in PCA at 37 °C, the cells were collected, washed and suspended in bottles containing 500 mL of filtered sterilized distilled water (Nalgene 0.2 µm, Bioblock, Strasbourg, France) adjusted to pH 5.0 ± 0.1 (HCl 0.1 mol/L) to obtain a final concentration of about 10 6 bacteria/mL.…”

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

“…These strains were chosen because of their origin (clinical and food isolates) and because they were previously used in VBNC studies in which optimal conditions to induce the VBNC state were described [3,5,12]. Two subcultures were performed under similar conditions and then the cells were placed in filtered sterilised distilled water (microcosm water) in order to induce the VBNC state.…”

“…A number of animal models have been tested by various authors with different efficiencies: mice [33], chicks [45], embryonated eggs [11,14] and human volunteers [17]. In Listeria monocytogenes the VBNC condition has been recently described and is well documented [3,5]. This major pathogen was implicated in the last two decades in several food borne disease outbreaks with high death rates [6,18].…”

Detection and enumeration of viable Listeria monocytogenes cells from ready-to-eat and processed vegetable foods by culture and DVC-FISH

“…Listeria monocytogenes can enter the VBNC state under environmental stress conditions such as low nutrient source, extreme temperatures or high osmotic concentration of the medium (Besnard, Federighi, Declerq, Jugiau & Cappelier, 2002). A recent study tested the ability of the bacteria to recover its culturability after entering the VBNC state.…”