Microbiología, Departamento de Biología Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, Lagoas-Marcosende s/n, 36200 Vigo, Spain
Correspondence
Teresa P. Nieto
mtperez{at}uvigo.es
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ABSTRACT |
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INTRODUCTION |
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The role of the VBNC state on the ecology and life-cycle of different bacterial species is being evaluated (Lebaron et al., 1999; Grimes et al., 2000
; Huq et al., 2000
). Current research is strongly guided by evidence of the existence of different cellular physiological states that marine bacteria display under variable environmental conditions (Kaprelyants et al., 1996
; Comas & Vives-Rego, 1998
; Fegatella & Cavicchioli, 2000
).
The aim of the present work was to study the effect of temperature, salinity and nutrient concentration on the survival responses of V. splendidus, using both natural freshwater and defined medium laboratory microcosms. Also resuscitation experiments were conducted, to establish the growth-limiting natural conditions of this species.
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METHODS |
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The freshwater was collected from a public fountain in Vigo (chemically untreated; pH 6·16), sterilized through 0·22 µm filters and placed into aseptic flasks.
The defined media were based on those described by Weichart & Kjelleberg (1996), although three different nutrient and salt concentrations were prepared. Nutrients were aseptically added to the microcosms at final concentrations of 17 or 1 g l-1, or 5 mg l-1, each one containing 49·8 % peptone (Cultimed), 24·9 % yeast extract (Cultimed), 24·9 % glucose (Panreac) and 0·4 % Na2HPO4 (Panreac). Iron (FeSO4 . 7H2O; Panreac), prepared in 0·4 M Tricine (Sigma), was added from a filter-sterilized solution at a final concentration of 0·01 mM. A 3·3 % salinity stock solution was prepared with (g l-1) NaCl (29·4), Na2SO4 (2·4), NaHCO3 (0·1), KCl (0·4), KBr (0·07), MgCl2 . 6H2O (3·1), CaCl2 . 2H2O (0·7), SrCl2 (0·013) and H3BO3 (0·013) (all purchased from Panreac). Two additional 1·6 and 0·9 % salinity working solutions were obtained by 5x10-1 and 2·5x10-1 dilutions of the initial stock. The salinities were measured with a portable salinometer (Orion 130). The survival experiments were performed at 4, 10 or 22 °C.
Nalidixic acid direct counts (NADC) and acridine orange total counts (AOTC).
Duplicate samples from the microcosms were mixed with both nalidixic acid (NA; Sigma) and yeast extract (Oxoid) at final concentrations of 0·02 % (w/v) and incubated overnight at 22 °C in darkness with shaking at 100 r.p.m. (modified from Kogure et al., 1979). NA-treated and untreated (control) samples were then stained with acridine orange (Sigma) at a 0·01 % (w/v) final concentration for 2 min. Samples were filtered through 0·22 µm pore-size black polycarbonate filters (Millipore). Counts were performed in an Olympus BH2-RFC fluorescence microscope (blue-light excitation; 1250x times; final magnification) with a micrometer eyepiece. The length of 250400 control cells was measured and the mean value was calculated. NA-active cells were recorded as those which were elongated at least 1·5-fold in length with respect to the control mean length (Barcina et al., 1995
). AOTC and NADC were counted in 40 fields using a 10x10 ocular grid and the mean number of cells per field was obtained.
Tetrazolium salt direct counts (TSDC).
Aliquots from the microcosms were incubated with 0·01 % (w/v) of the tetrazolium salt (TS) p-iodonitrophenyltetrazolium chloride (Sigma) at 22 °C for 1 h, in darkness and with shaking at 100 r.p.m. (Zimmermann et al., 1978). The samples were fixed with 2 % (w/v) formaldehyde and placed in a counting chamber (Neubauer Improved). Counts were performed in duplicate using an Olympus BH2 microscope (1250x times; final magnification). Those cells containing an intracellular reddish-brown formazan spot were recorded as TS-responsive.
Plate counts (PC).
The number of c.f.u. was determined by duplicate plating on tryptone soy agar (TSA) and 5x10-2 and 2·5x10-4 dilutions of this medium. These media were amended with NaCl to mimic the salinity of the microcosms. After 23 days at 22 °C, the plates were inspected for c.f.u. determination. The experiments in which culturable cells could still be detected after a minimum of 3 months were stopped.
In vitro resuscitation assays.
The resuscitation experiments were started once the culturable cell numbers were fixed at a level below 0·1 cells ml-1. To attain this value, once PC reached zero by 0·1 ml seeding, cells from 10 ml samples were collected onto sterile 0·2 µm cellulose nitrate filters (Albet). These filters were then placed on TSA, 5x10-2 TSA and 2·5x10-4 TSA (with the appropriate NaCl concentration) and plates were incubated for 1 week at 22 °C. Afterwards, samples of 1 and 2 ml were added in duplicate to tubes containing 9 or 2 ml (double-strength), respectively, fresh liquid medium. TSB and 5x10-2 and 2·5x10-4 dilutions of this medium with the appropriate NaCl concentration were used. After 1 week at 22 °C with shaking, the presence of turbidity was recorded as a positive result.
Statistical analysis.
The statistical analysis was performed using the SPSS 10.0 program. The survival times of the strains and the counting procedures were compared two by two using one-way variance analysis (ANOVA), including temperature, salinity and nutrient content as co-variables. The influence of these variables on the survival time of the strains was evaluated by regression analysis. Probability values lower than or equal to 0·05 were considered statistically significant. The mean values of duplicate samples and triplicate experiments for all the counting procedures were used for graphic representation.
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RESULTS |
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Strains 16N and 43N maintained under controlled laboratory conditions led to the development of at least four subpopulations, which differed in their culturability and activity. These subpopulations were composed of (i) NA- and TS-responsive (active) culturable cells, (ii) active non-culturable cells; (iii) only TS-active non-culturable cells and (iv) non-active non-culturable cells. Subpopulations (ii) and (iii) are referred to as VBNC.
When strains 16N and 43N were maintained in the defined medium of 3·3 % salinity and nutrient concentrations of 1 g l-1 or 5 mg l-1, the populations remained homogeneous and only subpopulation (i) could be detected (Fig. 1). During the time in which population assessment was carried out, AOTC remained constant, except for an initial increase due to nutrient availability, and cell populations remained both active and culturable. After a minimum of 96 and a maximum of 130 days of maintained culturability the experiments were terminated (Table 1
). In some of these long-term culturable microcosms, NADC dropped below the limits of detection before both PC and TSDC (Fig. 1a
). The differences in the limits of detection of viable counting procedures and NADC are responsible for these results.
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In vitro resuscitation of non-culturable cells
The trials for resuscitation in the 14 microcosms in which a VBNC response was observed (Table 1) were not successful.
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DISCUSSION |
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Also, high nutrient content has been found to induce a VBNC state in V. vulnificus (Oliver, 1995). The studied temperatures did not affect the survival of V. splendidus, showing the efficiency of the species to maintain itself as culturable at low temperatures.
V. splendidus biotype I displayed several survival strategies in laboratory microcosms when challenged with different environmental conditions. Joux et al. (1997) described the death kinetics of starved Salmonella typhimurium as a process beginning with cells losing both the ability to grow on solid medium and to respond to the NA assay, at the same time. Later cells lost the respiratory activity measured by a TS reduction and finally, cellular integrity. Although the compounds used to measure the respiratory activity were not the same, this sequence in the decay of cell activities was only observed when strain 43N was cultured in freshwater at 22 °C. In this study we have found that stressful conditions produce different responses in V. splendidus.
According to Villarino et al. (2000) cells in the VBNC state could be metabolically reactivated. In our case, resuscitation of VBNC populations was not achieved. Although only variations in the temperature and nutrient concentrations were assayed, some authors have pointed out that reversal of the inducing factor is not always enough to produce a return to culturability (Ghezzi & Steck, 1999
).
With respect to direct viable counting procedures, TSDC and NADC have been widely used for assessing cell activities in several environmental studies (Huq et al., 2000) and different authors have found a good correlation between values obtained by both procedures (Wolf & Oliver, 1992
). In our study, the statistical comparison of TSDC and NADC has revealed higher yields of the former, indicating that these assays measure different cellular activities. As pointed out by Joux et al. (1997)
, NADC and PC measure a potential growth ability, as a result of nutrient addition. NA blocks the activity of the DNA gyrase, thus inhibiting cell division. However, TSDC measure a real respiratory activity.
In conclusion, although the results stated above are not directly extrapolable to natural habitats, salinities of marine environments produce long-term survival responses, whereas those of freshwater or estuarine areas and/or high organic matter concentrations lead to the formation of a VBNC state in V. splendidus. In all but one of the experiments heterogeneity within populations was found, instead of a programmed sequence leading to cell death. However, the recovery of non-culturable populations was not attained by in vitro procedures. These results suggest that the VBNC state is an adaptation strategy in V. splendidus and the isolation of this species from its natural habitat can be unsuccessful.
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ACKNOWLEDGEMENTS |
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Received 12 March 2002;
revised 18 October 2002;
accepted 23 October 2002.
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