-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...
. C A P P E L I E R . 2000. A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only ef®cient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in ®ltered, sterilized distilled water. We used different concentrations of cipro¯oxacin, ef®cient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and cipro¯oxacin. The mixture was incubated at different incubation times at 37 C. After different incubation times, cells were ®ltered through an isopore polycarbonate black membrane ®lter and covered with a DAPI solution or orange acridine. The ®lters were prepared and examined by epi¯uorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of cipro¯oxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.
Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (107 cells ml−1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride–4′,6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 μl mg of protein−1 for the culturable form and 10.96 μl mg of protein−1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.
Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Campylobacter species involved in this infection usually include the thermotolerant species Campylobacter jejuni. The major reservoir for C. jejuni leading to human infections is commercial broiler chickens. Poultry flocks are frequently colonized by C. jejuni without any apparent symptoms. Risk assessment analyses have identified the handling and consumption of poultry meat as one of the most important sources of human campylobacteriosis, so elimination of Campylobacter in the poultry reservoir is a crucial step in the control of this foodborne infection. To date, the use of probiotics has demonstrated promising results to reduce Campylobacter colonization. This review provides recent insights into methods used for probiotic screening to reduce the prevalence and colonization of Campylobacter at the farm level. Different eukaryotic epithelial cell lines are employed to screen probiotics with an anti-Campylobacter activity and yield useful information about the inhibition mechanism involved. These in vitro virulence models involve only human intestinal or cervical cell lines whereas the use of avian cell lines could be a preliminary step to investigate mechanisms of C. jejuni colonization in poultry in the presence of probiotics. In addition, in vivo trials to evaluate the effect of probiotics on Campylobacter colonization are conducted, taking into account the complexity introduced by the host, the feed, and the microbiota. However, the heterogeneity of the protocols used and the short time duration of the experiments lead to results that are difficult to compare and draw conclusions at the slaughter-age of broilers. Nevertheless, the combined approach using complementary in vitro and in vivo tools (cell cultures and animal experiments) leads to a better characterization of probiotic strains and could be employed to assess reduced Campylobacter spp. colonization in chickens if some parameters are optimized.
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