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Applied and Environmental Microbiology, October 2006, p. 6508-6513, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00798-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Effects of Temperature and Starvation on Induction of the Viable-but-Nonculturable State in the Coral Pathogens Vibrio shiloi and Vibrio tasmaniensis

Thomas Vattakaven,1 Peter Bond,2 Graham Bradley,1 and Colin B. Munn1*

School of Biological Sciences,1 Plymouth Electron Microscopy Centre, University of Plymouth, Plymouth PL4 8AA, United Kingdom2

Received 5 April 2006/ Accepted 24 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20°C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17°C), the VBNC state was not induced by incubation at 4°C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30°C), was induced into the VBNC state by incubation at 4°C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20°C, and V. shiloi became VBNC after incubation for 126 days at 20°C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The viable-but-nonculturable (VBNC) state in bacteria has been defined as "a cell that can be demonstrated to be metabolically active, while being incapable of undergoing a sustained cellular division required for growth in or on a medium normally supporting growth of that cell" (20). So far, the VBNC state has been reported for about 60 different species (22) and has been demonstrated both in the laboratory and in situ (23). The conditions inducing the VBNC state are as diverse as the species themselves; however, all inducing factors generally are some form of environmental stress, such as starvation or changes in temperature or salinity, suggesting that the VBNC state may be an adaptive or dormant state allowing survival under adverse conditions (4, 20-22, 29).

The VBNC state was first described for Vibrio cholerae (36) and has since been characterized for at least 11 Vibrio species. The VBNC state in vibrios is generally induced by incubation at low temperatures (4 to 6°C), while elevated temperatures (>25°C) induce the same state in non-Vibrio species (10). As a part of our study of the effects of environmental temperatures on the symbiotic and pathogenic associations of bacteria with corals, we compared the effects of prolonged incubation at different temperatures on the survival of two Vibrio spp. that have been associated with coral disease in very different habitats.

Vibrio shiloi has been confirmed to cause bleaching in the coral Oculina patagonica (16). At a high temperature (25°C), V. shiloi adheres to and penetrates into coral tissue (2, 3, 34), where it is thought to divide and transform into an intracellular VBNC state (11); however, induction of the VBNC state in vitro has not been shown previously. The studies mentioned above showed that successful infection by V. shiloi is highly dependent upon elevated temperatures, although Bourne and Munn (7) identified clones with high levels of sequence identity to V. shiloi in healthy Pocillopora damicornis on the Great Barrier Reef at temperatures of 28 to 30°C.

The second species, Vibrio tasmaniensis, has recently been characterized as part of the biota associated with marine organisms (33). In our study, a strain isolated from a diseased gorgonian coral from cool temperate waters with a temperature range of 7.5 to 17°C was used.

It was hypothesized that variations in the in vitro culture temperature might have different effects on induction of the VBNC state in these coral-pathogenic vibrios isolated from cold- and warm-water habitats.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacterial strains and preparation of microcosms.
V. tasmaniensis strain CC056 used in this study was isolated from necrotic tissue of the gorgonian octocoral Eunicella verrucosa during a disease outbreak in the waters off Lundy Island, Devon, England, in June 2003. It was identified as V. tasmaniensis by F. Thompson (Universiteit Gent; personal communication) using fluorescent amplified fragment length polymorphism patterns, which were generated and analyzed as described by Thompson et al. (32). LMG 19703 is the type strain of V. shiloi, and it was obtained from the BCCM/LMG Culture Collection, Laboratorium voor Microbiologie, Universiteit Gent, Belgium; it was originally isolated from bleached Oculina patagonica scleractinian coral in the Mediterranean Sea (16).

The inoculum for each species was prepared by culturing the bacteria overnight in tryptone soy broth (Oxoid) supplemented with NaCl at a final concentration of 1.5% (TSBNa). The bacterial cells were harvested by centrifugation at 4,000 x g for 10 min. The resulting pellet was resuspended in autoclaved artificial seawater (ASW) (Instant Ocean Aquarium Systems) and dispensed into conical flasks. Triplicate flasks were incubated at 4°C and 20°C. Sampling was done at regular intervals to determine the numbers of CFU, live cells, and dead cells and the total counts.

Colony counts.
The culturability of the samples was monitored by using a modified Miles-Misra method (18). Tenfold serial dilutions of the sample were prepared in ASW, and 20-µl aliquots were placed on triplicate plates containing dried tryptone soy agar (Oxoid) supplemented with 1.5% (final concentration) NaCl (TSANa) and incubated at 21°C.

Measurement of viability.
The viability of cells was determined using a LIVE/DEAD BacLight bacterial viability kit (L13152; Molecular Probes) according to the manufacturer's instructions. A bacterial sample (1 ml) was centrifuged (4,000 x g, 5 min). The pellet was resuspended in 50 µl of 0.85% (wt/vol) NaCl. To this suspension, an equal amount of the dye (6 µM [final concentration] SYTO9 and 30 µM [final concentration] propidium iodide) was added, mixed well, and incubated for 15 min. Finally, 3 µl of the stained sample was placed on a microscope slide, covered with a coverslip, and observed immediately using an Olympus Vanox fluorescence microscope with a blue filter (excitation wavelength, 380 to 490 nm; emission wavelength, >590 nm). Digital images of five random fields for each replicate sample were acquired with an Olympus CAMEDIA C-2020 zoom camera, and the live, dead, and total bacteria were counted.

Scanning electron microscopy.
One milliliter of a sample was filtered through 0.2-µm Nuclepore polycarbonate filters, and the filtrate was collected for observation by transmission electron microscopy (TEM) with negative staining. The filters were then fixed with 2.5% glutaraldehyde in ASW for 1 h, rinsed, dehydrated using an ethanol series, and critical point dried (Emitech K550). The filters were mounted, sputter coated with gold (Emitech K850), and observed with a JEOL 6100 or JEOL 5600LV scanning electron microscope (SEM). Overnight cultures of the bacteria grown in TSBNa were used as controls.

Transmission electron microscopy.
One milliliter of a sample was fixed with 2.5% glutaraldehyde for 30 min in an Eppendorf tube and centrifuged (1,400 x g, 2 min). The pellet was washed with ASW and then resuspended in 0.1 M cacodylate buffer to which 2% molten agarose was added and concentrated by centrifugation. The agarose was solidified, trimmed, postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 30 min, rinsed, dehydrated using a graded ethanol series, infiltrated with Spurr's resin, and polymerized at 70°C for 8 h. Ultrathin sections were stained with lead citrate and uranyl acetate. Overnight cultures grown in TSBNa were used as controls. Images were acquired with a JEOL 1200EXII TEM with a Mega view digital camera (SIS).

Resuscitation of V. shiloi from the VBNC state.
Aliquots (1.0 ml) were transferred from the microcosm samples after 125 days into a sterile container, 0.1 ml of TSBNa was added, and then the preparations were incubated at 20°C for 4 h. Culturable colony counts were obtained by the spread plate technique on TSANa after incubation for 18 h at 20°C. The sensitivity of the VBNC cells of V. shiloi to reactive oxygen intermediates was checked using filter-sterilized catalase (bovine liver; Sigma); 0.1 ml of an enzyme solution containing 2,000 to 5,000 U was aseptically spread on the surface of TSANa plates. After drying, the samples were counted using the spread plate technique (18 h, 20°C).


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Effects of long-term incubation in ASW on V. tasmaniensis.
After a decline after transfer of the cells into the microcosm with ASW at 4°C, the culturable colony counts rose to ca. 109 CFU ml–1 (Fig. 1A). This level was maintained throughout the sampling period (157 days), except that there was a very slight decline at the end of the experiment. This species can grow at 4°C (33); thus, it should not be stressed at this temperature. Although the samples incubated at 20°C exhibited a similar trend in the initial 30 days, the decline in the culturable counts was very pronounced after 30 days (Fig. 1B), and the values decreased to ca. 106 CFU ml–1 by day 100 and to 1.3 x 103 CFU ml–1 on the last day of sampling (day 157); i.e., there was a difference of almost 6 orders of magnitude from the start of the experiment to the end. Both temperatures resulted in an initial decrease and a subsequent peak in the culturable counts by days 11 to 14. The recovery in the colony counts that occurred after the decrease following transfer of the cells into ASW could have occurred as a result of nutrients provided by leaching from damaged and dead cells or as a result of the transient osmotic effects of transfer from growth medium (containing 1.5% NaCl) to ASW (containing 3.5% total salts). Another factor which could account for the initial rise in cell numbers is reductive division, which has been observed in the early stages of starved vibrios known to enter the VBNC state (20, 24).


Figure 1
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  		FIG. 1. Comparison of culturable colony counts (•) (CFU ml–1) and live ({blacksquare}) and total ({circ}) counts (cells ml–1) for V. tasmaniensis incubated at 4°C (A) and 20°C (B) in seawater. The values are means ± standard errors for 6 replicates for colony counts and for 10 replicates for live/dead counts.

 
Contrary to the trends observed for the culturable counts, the live, dead, and total counts remained almost constant at ca. 1010 cells ml–1. This pattern was maintained even when the culturable counts fell in the cultures incubated at 20°C, indicating that ca. 4.8 x 109 cells per ml were VBNC after 157 days. This is the first description of the VBNC state in V. tasmaniensis. Although 20°C is above the temperature range in the natural habitat of V. tasmaniensis and could therefore be considered stressful, causing induction of the VBNC state, this organism grows in the laboratory at temperatures up to at least 30°C.

SEM of 55-day-old samples obtained at both temperatures showed that the control cells (grown in TSBNa) were mostly rods that were 1 to 1.5 µm long (Fig. 2A), while mesocosm samples obtained at both temperatures contained numerous coccoid cells displaying blebs thought to be membrane vesicles or extrusions (Fig. 2B). At day 147, cultures incubated at both temperatures showed almost complete transformation of cells to the coccoid shape. Cells grown at 20°C showed a greater degree of blebbing and clumping, apparently due to a polymer-like substance. TEM images of the cells at day 147 indicated that the cells were characterized by the absence of the clear three-layer envelope (Fig. 2D) seen in the control cells (Fig. 2C).


Figure 2
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  		FIG. 2. Representative electron micrographs of V. tasmaniensis. (A) SEM micrograph of control (overnight culture in TSBNa), showing rod-shaped cells. (B) SEM micrograph showing blebbing in coccoid cells after 55 days of incubation at 4°C. (C) TEM micrograph of a control cell, showing clear periplasmic space and cytoplasmic organization. (D) TEM micrograph showing a lack of periplasmic space in cells after 147 days of incubation at 4°C.

 
Effects of long-term incubation in ASW on V. shiloi.
The culturable counts for V. shiloi declined shortly after transfer into ASW (Fig. 3). As observed with V. tasmaniensis, the counts in both samples not only recovered to the original levels but increased to more than 1010 CFU ml–1 by day 11. Subsequent sampling showed that there was a steady decrease in the culturable colony counts at both 4°C and 20°C. Sampling on day 60 revealed that the culturable counts were 3.2 x 108 and 3.3 x 108 CFU ml–1 for cells incubated at 4 and 20°C, respectively. While sampling was not done for the intervening period, no colony counts were obtained on day 126 at either temperature. There were always more than 109 live cells ml–1 in both samples, indicating entry into the VBNC state. The data for both samples showed a peak value for live cells on day 11, which correlated with the peaks in culturable counts. The total counts exhibited comparable trends throughout sampling.


Figure 3
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  		FIG. 3. Comparison of culturable colony counts (•) (CFU ml–1) and live ({blacksquare}) and total ({circ}) counts (cells ml–1) for V. shiloi incubated at 4°C (A) and 20°C (B) in seawater. The values are means ± standard errors for 9 replicates for colony counts and for 15 replicates for live/dead counts. The arrow at 126 days indicates that no colonies were observed (i.e., <50 CFU ml–1).

 
Resuscitation of the VBNC population of V. shiloi from microcosms incubated at both temperatures was achieved by addition of nutrients after 147 days. The culturable colony counts rose to 3.7 x 10 4 and 2.0 x 10 4 CFU ml–1 for cultures incubated at 4 and 20°C, respectively, after addition of 0.1 ml of TSBNa and incubation for 4 h. This suggests that starvation conditions play a major role in induction of the VBNC state in V. shiloi, but it is important to note that this result could have been due to the presence of one or more undetected culturable cells in the VBNC population (5, 6, 28, 35). Previous studies have shown that the starvation response in bacteria is different from the VBNC response. For example, in Vibrio vulnificus, the VBNC state can be induced by low temperature under nutrient-rich conditions as well as under nutrient-deficient conditions (25), while Vibrio cholerae displays the VBNC response at 5°C and a starvation response at 18°C (26). Addition of catalase resulted in significant revival of colony formation in the VBNC population of V. shiloi, and the level increased from zero to 1.8 x 10 4 CFU ml–1 for the culture incubated at 4°C and to 8.8 x 10 3 CFU ml–1 for the culture incubated at 20°C. The inability of cells in the VBNC state to detoxify peroxides and other free radicals commonly present in culture media or induced within the cells themselves during exposure to rich media has been shown to be a key explanation for the phenomenon of nonculturability (15). In V. vulnificus, this has recently been shown to be due to repression of the gene encoding periplasmic catalase (30).

To our knowledge, our results are the first report of in vitro induction of the VBNC state and resuscitation for V. shiloi, and this induction appears to be linked more strongly with starvation than with temperature. The intracellular VBNC state in the infection of corals by V. shiloi described by Israely et al. (11) differs markedly from the conventionally defined VBNC state, and the reported manifestation of nonculturability probably reflects a shift in metabolic requirements rather than an adaptation to stress. Sussman et al. (31) have suggested that the marine fireworm Hermodice caranculata acts as a winter reservoir and vector of the bacterium between successive summer infection of corals, with V. shiloi differentiating into the VBNC state inside the worm. Our results suggest that viable V. shiloi could successfully persist in seawater for significant periods during the lower temperatures that may be experienced in winter conditions. This raises the possibility that V. shiloi could also cause infection of corals directly from seawater; however, the inability of the bacterium to adhere to and penetrate into corals at temperatures below 16°C (34) could explain why it does not cause infection in the winter months despite its presence in the surrounding seawater.

It is not clear whether the in vitro VBNC state of V. shiloi observed here is an adaptive response or a transient debilitative response, although several features observed by electron microscopy indicate that there are modifications that are directed at enhanced survival. TEM revealed that V. shiloi cells had significant convolutions of the outer membrane and that there was an increase in the periplasmic space after 41 days of incubation at both temperatures (Fig. 4C). Such observations have been reported for other vibrios (1, 8, 13) and may be due to condensation of the cytoplasm resulting from dehydration of the cell (8). Cell lysis and curling of membranes into whorl-like structures were also observed with starved V. shiloi cells (Fig. 4D). The membrane changes in V. shiloi were very different from those observed in V. tasmaniensis. SEM showed that prolonged incubation in the mesocosms was associated with rounding and clumping of the cells (Fig. 4A and B). The transition to coccoid cells observed for both V. tasmaniensis and V. shiloi has been reported for starved cells of several species (1, 8, 9, 12, 14, 17, 24). A change in morphology and a reduction in size have been proposed to be starvation survival strategies that allow the cell to minimize cell maintenance requirements (19, 29).


Figure 4
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  		FIG. 4. Representative electron micrographs of V. shiloi. (A) SEM micrograph of control (overnight culture in TSBNa), showing rod-shaped cells. (B to D) Micrographs of cells after 41 days of incubation at 4°C. (B) SEM micrograph showing clumping of coccoid cells. (C) TEM micrograph showing enlarged periplasmic space. (D) TEM micrograph showing release of cellular contents and curling of the membrane.

 
In conclusion, our results revealed differential effects of starvation and incubation at 4°C on the VBNC state in two coral pathogens, which may be important in future studies of the ecology of the necrotic and bleaching diseases with which these bacteria are associated. Our hypothesis that the cold-water-adapted organism V. tasmaniensis does not enter the VBNC state at 4°C while the warm-water-adapted organism V. shiloi does was confirmed. However, induction of the VBNC state for both species at 20°C was unexpected, since this temperature is well within their optimal growth ranges. Vibrios are considered more likely to enter the VBNC state at 4 to 6°C (10), although it should be noted that Vibio harveyi and Vibrio fischeri became VBNC at 22°C in nutrient-limited ASW (27). The observation of a number of morphological changes in both species at both temperatures suggests that the cells underwent general adaptation to nutrient starvation rather than adaptation to temperature. Although our preliminary observations suggest that the morphological changes in the two species differ, further studies of membrane composition are required. Field studies are necessary to confirm whether the pathogens occur in a VBNC state in their habitats during the winter, where they could remain as a source for summer infections of their coral hosts.


   ACKNOWLEDGMENTS
 
We thank Paul Waines for technical support and Fabiano Thompson (Laboratorium voor Microbiologie, Universiteit Gent, Belgium) for identification of V. tasmaniensis used in this study.


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Biological Sciences, Portland Square, University of Plymouth, Drake Circus, Plymouth PL4 8AA, United Kingdom. Phone: 441752233549. Fax: 441752232970. E-mail: colin.munn{at}plymouth.ac.uk. Back


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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, October 2006, p. 6508-6513, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00798-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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