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

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 $^{\circ}$C or 20 $^{\circ}$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.Les facteurs environnementaux et physico-chimiques induisent l'état VNC chez Listeria monocytogenes . Des études sur la viabilité bactérienne dans l'environnement ont démontré l'existence d'un état particulier qualifié d'état Viable non Cultivable (VNC), dans lequel certaines 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 $^{\circ}$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 entraîné une perte rapide du caractère cultivable. Compte tenu d'une part de l'ubiquité de L. monocytogenes, d'autre part du fait que les facteurs étudiés peuvent être rencontrés dans l'environnement des industries agro-alimentaires, les auteurs pensent que l'état VNC chez L. monocytogenes pourrait représenter un réel problème de santé publique. Des travaux complémentaires concernant la virulence et le retour à l'état cultivable de ces formes sont en cours afin de préciser le risque lié à l'état VNC chez L. monocytogenes

Detection of Listeria monocytogenes in raw and pasteurized liquid whole eggs and characterization by PFGE.

International audienceListeria monocytogenes has been recognized as a human pathogen for decades and is known to be an important foodborne pathogen. There have been no documented foodborne L. monocytogenes illnesses due to the consumption of eggs or egg products, even though the bacterium has been isolated from faeces, body fluid, and oviducts of asymptomatic laying hens. In order to describe L. monocytogenes contamination of egg products, 144 liquid whole egg samples were collected from 3 different egg-breaking plants during 3 sampling periods. L. monocytogenes detection was performed on raw samples stored at 2 degrees C for two days (D+2) and on pasteurized samples stored at 2 degrees C at D+2 and at shelf-life date (SLD). L. monocytogenes was detected in 25 of the 144 raw egg samples collected, in 4 of the 144 pasteurized egg samples at D+2 and in 2 of the 144 ones analysed at SLD. Contamination of raw egg products appeared to be season dependant and was higher during summer and winter than during autumn. One hundred and ninety-six L. monocytogenes isolates were collected and serotyped; 3 serovars were demonstrated. The dominant serovar was L. monocytogenes 1/2a which was presented by 94.4% of the isolates. Typing of 196 L. monocytogenes isolates was carried out by macrorestriction of the genomic DNA with ApaI and AscI enzymes followed by pulsed field gel electrophoresis (PFGE). A large diversity was observed with 21 genotypes of L. monocytogenes, even for a given manufacturer. Nevertheless, most of the egg product samples were contaminated by one genotype, except for five samples which were contaminated by two or three distinct genotypes. The genotypes seem to be specific to each manufacturer. No cluster of L. monocytogenes was found to recur in the different plants over successive seasons

Characterization of Campylobacter spp. transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board

International audienceCampylobacter represents the leading cause of gastroenteritis in Europe. Campylobacteriosis is mainly due to C. jejuni and C. coli. Poultry meat is the main source of contamination, and cross-contaminations in the consumer's kitchen appear to be the important route for exposure. The aim of this study was to examine the transfer of Campylobacter from naturally contaminated raw poultry products to a cooked chicken product via the cutting board and to determine the characteristics of the involved isolates. This study showed that transfer occurred in nearly 30% of the assays and that both the C. jejuni and C. coli species were able to transfer. Transfer seems to be linked to specific isolates: some were able to transfer during separate trials while others were not. No correlation was found between transfer and adhesion to inert surfaces, but more than 90% of the isolates presented moderate or high adhesion ability. All tested isolates had the ability to adhere and invade Caco-2 cells, but presented high variability between isolates. Our results highlighted the occurrence of Campylobacter cross-contamination via the cutting board in the kitchen. Moreover, they provided new interesting data to be considered in risk assessment studies. (C) 2013 Elsevier B.V. All rights reserved