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Survival of the turbot-virulent strain 7P of Listonella pelagia was evaluated at 3 incuba- tion temperatures (4, 10 and 22°C) in microcosms of natural freshwater and defined media of differ- ent salinities (9, 16 and 33‰) and nutrient concentrations (0.005, 1 and 17 g l -1 ). During the experi- mental period, different counting procedures were used to assess the population state. These were: (1) acridine orange total counts (AOTC); (2) nalidixic acid direct counts (NADC); (3) 2-(p-iodophenyl)- 3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) direct counts (INTDC); and (4) plate counts (PC). Resuscitation of the viable but nonculturable (VBNC) populations was performed both in vitro and in vivo. Electron microscopy and membrane protein profiling of the cells was conducted. Irre- spective of the temperatures assayed, L. pelagia remained culturable for long periods in the defined- medium microcosms of 33‰ and 0.005 or 1 g l -1 nutrient content, but entered a VBNC state in fresh- water, at lower salinities (9 or 16 ‰) and/or at the highest nutrient content (17 g l -1 ). Although, in these cases, cells displayed a spheroid morphology, a significant reduction in size (p < 0.05) was only observed in long-term surviving cells. No changes were found in membrane protein profiles. VBNC cells were recovered in vitro in samples that contained less than 0.0001 culturable cells ml -1 , or that had been treated with ampicilline at bacteriolytic concentrations, and from the internal organs of infected sea bream. The overall results suggest long-term survival of L. pelagia, and that the VBNC state is part of its life cycle.

Long-term survival and the viable but nonculturable state as part of the life cycle of Listonella pelagia