Survie et maintien de la virulence de Salmonella Typhimurium VNC exposée simultanément à trois facteurs stressants expérimentaux

Abstract

The release of enteric pathogenic bacteria in aquatic environments poses the problem of the fate of these bacteria under the effects of environmental factors (solar radiation, salt concentration, temperature, nutrient level, pH, competition). Frequently, these bacterial cells, potentially pathogenic, enter into a non-culturable state on routine bacteriological plating media. However, the use of direct detection methods DAPI stained cells allows the visualization of these Viable but Non Culturable cells VNC. But, beyond the characterization of the viability of the cells electron transport activity, metabolic activity, membrane integrity, structure and/or quantity of DNA, what happens with the virulence of these cells? This problem was experimentally investigated according to the bacterial model Salmonella Typhimurium. The virulence of this strain, which is the agent of the murine typhoid, was evaluated on a mouse model. Experimentally, the effects of some environmental factors on the survival and on the maintenance of virulence of Salmonella Typhimurium were measured in microcosms exposed to UV radiation (four germicidal lamps 8 mW s −1 cm −2, wave length: 254 nm, salt concentration (Sea Salt Sigma, 37, nutrient starvation. The microcosms were simultaneously submitted to these three factors, with variable exposure times. For each of those times, the viability of the nonculturable cells which became nonculturable because of the exposure to the three factors was measured through different physiological states notable in the cells, after using different fluorescent dyes. The stained cells were observed by epifluorescence microscopy and analysed by image cytometry. So, the cellular populations are characterised by enumeration of respiring bacteria CTC, [39], metabolising bacteria YEK, [22] modified, bacteria owning an undamaged cytoplasmic membrane LD, Live/Dead BacLight Viability Kit. Molecular Probes Inc.; we also determined the quantity and/or structure of DNA of the cells fluorescence level of DAPI stained cells), After exposure to the three factors for one hour 13.56 J cm −2, while the plate count cell density rapidly decreased from #10 7 cells mL −1 to0.1 cell mL −1, physiological states of these viable but non-culturable cells are similar to those of non-exposed cells. On the other hand, after exposure for three hours, only 10 % of the cells deposit a CTC formazan-crystal and 20 % are substrate responsive cells enlarged cells in presence of Yeast Extract and Cephalexin: YEC. Half of the cellular population presents an undamaged cytoplasmic membrane and the level of fluorecence of DAPI stained cells is close to 85 %, which shows that the DNA of these cells is weakly damaged. After exposure to the three experimental factors for24 hours 315 J cm −2, weak replies to the physiological tests used in this study to characterize the viability of the non-culturable cellular population are observed CTC: 4 %; YEC: 2 %; LD: 11.8 % while the fluorescence level of DAPI stained cells remains firm at 80 %. At the same time, the virulence expression of VNC cells of Salmonella Typhimurium, evaluated by intraperitoneal injection to the mouse route which excludes uncontrolled parameters, unlike the per os route does not seem to be correlated with the cellular viability such as it has been evaluated in this study. A 30 min exposure (6.73 J cm −2) to the three environmental factors, leading to the non-culturability of almost the entire exposed cell population 0.08 culturable cell mL −1 whereas the level of viability of those culturable cells is closed to the one of non-exposed cells. The injection of 1000 of those cells 0.001 culturable cells in 100 μL inoculated into the mouse a group of ten mice does not cause any mortality four weeks post-inoculation, whereas the injection of the same dose of non-exposed cells leads to the death of all mice in the group one week post-inoculation. According to our preliminary experiments on Salmonella Typhimurium, the loss of the state of culturability and the loss of virulence towards mice by intra-peritoneal route, because of the exposure to associated effects of UV irradiation 254 nm, salinity 37 and nutrient starvation, seem to be concomitant.