Survival and virulence changes in the VNC state of Salmonella Typhimurium in relation to simultaneous UV radiation, salinity and nutrient deprivationexposure.

The release of enteric pathogenic bacteria in aquatic environments poses th e 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 medi a. However, the use of direct detection methods (DAPI stained cells) allows the visualization of these Viable but Non Culturable cells (VNC). But, bey ond 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 Typ himurium. The virulence of this strain, which is the agent of the murine ty phoid, was evaluated on a mouse model. Experimentally, the effects of some environmental factors on the survival and on the maintenance of virulence o f Salmonella Typhimurium were measured in microcosms exposed to UV radiatio n (four germicidal lamps 8 mW s(-1) cm(-2), wave length: 254 nm), salt conc entration (Sea Salt Sigma, 37), nutrient starvation. The microcosms were si multaneously 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 meas ured through different physiological states notable in the cells, after usi ng different fluorescent dyes. The stained cells were observed by epifluore scence microscopy and analysed by image cytometry. So, the cellular populat ions are characterised by enumeration of respiring bacteria (CTC, [39]), me tabolising bacteria (YEK, [22] modified), bacteria owning an undamaged cyto plasmic membrane (LD, Live/Dead BacLight Viability Kit. Molecular Probes In c.); 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 th e plate count cell density rapidly decreased from #10(7) cells mL(-1) to < 0.1 cell mL(-1), physiological states of these viable but non-culturable ce lls are similar to those of nonexposed cells. On the other hand, after expo sure for three hours, only 10 % of the cells deposit a CTC formazan-crystal and 20 % are substrate responsive cells (enlarged cells in presence of Yea st Extract and Cephalexin: YEC). Half of the cellular population presents a n undamaged cytoplasmic membrane and the level of fluorecence of DAPI stain ed cells is close to 85 %, which shows that the DNA of these cells is weakl y damaged. After exposure to the three experimental factors for 24 hours (3 15 J cm(-2)), weak replies to the physiological tests used in this study to characterize the viability of the non-culturable cellular population are o bserved (CTC: 4 %; YEC: 2 %; LD: 11.8 %) while the fluorescence level of DA PI stained cells remains firm at 80 %. At the same time, the virulence expr ession of VNC cells of Salmonella Typhimurium, evaluated by intraperitoneal injection to the mouse (route which excludes uncontrolled parameters, unli ke the per os route) does not seem to be correlated with the cellular viabi lity 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-culturabili ty 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 th e one of non-exposed cells. The injection of 1000 of those cells (<0.001 cu lturable cells in 100 mu L inoculated) into the mouse (a group of ten mice) does not cause any mortality four weeks post-inoculation, whereas the inje ction of the same dose of nonexposed cells leads to the death of all mice i n the group one week post-inoculation. According to our preliminary experim ents on Salmonella Typhimurium, the loss of the state of culturability and the loss of virulence towards mice by intraperitoneal route, because of the exposure to associated effects of UV irradiation (254 nm), salinity (37) a nd nutrient starvation, seem to be concomitant. (C) Elsevier, Paris.