Vibrio parahaemolyticus is a gram-negative bacterium ubiquitous in seawater or estuarine water throughout the world. It is a major cause of seafood gastroenteritis complication. In this study, the presence of V. parahaemolyticus was investigated in 66 seawater samples collected during 2018 from 15 stations spread along the Tunisian coast using selective media including CHROMagar Vibrio media. The results show that only eight samples contained V. parahaemolyticus. However, while Vibrio alginolyticus was detected in all samples; both Vibrio cholerae and Vibrio vulnificus were not found. Nine of the presumed V. parahaemolyticus colonies were purified on tryptic soy agar from eight positive samples then identified by the API 20E biochemical test and confirmed by the presence of a specific target toxR gene. The detection of virulence genes, thermostable direct haemolysin (tdh) and thermostable-related haemolysin (trh), by the polymerase chain reaction (PCR) showed the presence of only two trh-positive isolates. The assessment of antibiotic susceptibility of the V. parahaemolyticus isolated revealed a complete resistance to colistin, amikacin, penicillin and cefotaxime and a total sensitivity to chloramphenicol, nitrofurantoin and sulfamethoxazole-trimethoprim with a multiple antibiotic resistance index (MAR) ranged from 0.4 to 0.5.
The effect of sunlight exposure on Salmonella typhimurium isogenic strains harboring an rpoS gene functional (rpoS+) or not functional (rpoS-) was investigated in microcosms of sterile sea water at 20 degrees C. The two strains rapidly lost their ability to produce colonies on solid culture media. The detrimental action of sunlight was more important when the salinity of sea water increased. The survival of stationary phase cells was influenced by RpoS. Bacteria grown in media with high salinity or osmolarity and transferred to sea water in stationary phase were more resistant to irradiation than those grown in media with low salinity. Prior growth under oxidative (0.2 mmol/L of H2O2) or amino acid starved (minimal medium) conditions did not modify the survival of either strain when they were exposed to sunlight. Bacteria were more resistant when cells were incubated in sea water in the dark prior to being exposed to sunlight. The resistance to sunlight irradiation was also greater in clones of both strains isolated from microcosms exposed to sunlight for 90 min, then further inoculated into sea water and reexposed to sunlight.
The effect of sunlight and salinities (10, 20, 39, and 60 psu) on the survival of Vibrio parahaemolyticus strains carrying either (thermostable direct hemolysin) tdh, the (thermostable related hemolysin) trh, and both or none of them were studied in water microcosms stabilized at 20 C using plate count agar and acridine orange direct viable count. All V. parahaemolyticus strains exposed to sunlight rapidly lose their culturability and evolve into a viable but non-culturable state (VBNC). However, the tdh positive strains remain more culturable than the non-virulent or trh positive strain but statically insignificant. At tested salinities, the survival time was higher at 10, 20, and 60 psu compared with that observed in seawater (39 psu). In seawater under dark condition, Vibrio strains remain culturable for up to 200 days with a significant difference between strains (p < 0.05). Furthermore, the non-pathogenic strain survives longer than the virulent ones. At different salinities, a better adaptation is observed at 10 and 20 psu compared with 39 and 60 psu. Resuscitations essays performed on VBNC bacteria in a nutrient broth at 20 C and 37 C does not show any revivification.
Practitioner Points• Effect of sunlight and salinities on the survival of V. parahaemolyticus in the marine environment.• Resuscitation essay performed on viable but no cultivable bacteria.• Microscope motility examines show that all strains exposed to sunlight remain motile after the loss of cultivability.
The survival of Salmonella enterica serovar Typhimurium rpoS+ and rpoS− exposed to sunlight or incubated in the dark was studied in a seawater microcosm and a diffusion chamber. The total number of bacteria and physiologically active ones were estimated by direct counting using epifluorescence microscopy, while cultivable bacteria were enumerated by plating onto an agar culture medium. The results obtained showed that the bacteria exposed to sunlight, either in the microcosm or in the diffusion chamber, exhibited a fast decline of cultivable cells, whereas the epifluorescence enumeration revealed a conservation of physiological activity after a total loss of cultivability. In a transparent diffusion chamber, the evolution of cultivable and physiological active bacteria was characterized by a diurnal decrease and a nocturnal stabilization in the count levels, reflecting the effect of phototoxicity on the survival of Salmonella. The transformation and persistence of a physiological active state and cellular integrity in the diffusion chamber or the microcosm showed a clear dependence on the presence of the rpoS gene. Survival in the dark was also characterized by a decrease of cultivability; however, this was significantly inferior to that observed in bacteria exposed to sunlight. This loss of cultivability was accompanied by the preservation of a physiologically active state. The study of seasonal survival carried out in the transparent diffusion chamber was characterized by an enhanced survival of the rpoS+ strain in comparison with the rpoS– mutant. The highest survival rates were obtained in the autumn, thus highlighting the relevant effect of temperature and exposure to sunlight on the persistence of bacteria in seawater.
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