FEMS MicrobiologyLetters 100 (1992) 205-210 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
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Low temperature induced non-culturability and killing of Vibrio vulnificus D i e t e r W e i c h a r t a, J a m e s D. O l i v e r b a n d S t a f f a n K j e l l e b e r g a o Department of General and Marine Microbiology, University of Gdteborg, G6teborg, Sweden, and b Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina, USA
Received 16 June 1992 Accepted 19 June 1992 Key words: Vibrio vulnificus; Viable but non-culturable; Survival; Resuscitation
1. S U M M A R Y Vibrio vulnificus ceils progressively lose culturability during incubation at 5°C. This process is accelerated by the addition of supernatants from non-culturable cells obtained by incubation at 5°C for 17 days. Thus the organism apparently produces a factor upon cold incubation which is triggering or causing the decline in culturability. Reversing the t e m p e r a t u r e shift can restore a culturable population comparable in numbers to the original population, but this process is largely due to regrowth. A few cells retaining the ability to grow apparently utilize the substrates released by the moribund cells, thus mimicking resuscitation of the whole population.
2. I N T R O D U C T I O N The concept of viability of microorganisms has changed dramatically during the past decades.
Correspondence to: S. Kjelleberg, Department of General and
Marine Microbiology, University of G6teborg, Carl Skottsbergs Gata 22, S-41319 G6teborg, Sweden.
The classical approach using colony counts as an indicator of viability [1] has repeatedly been challenged, and several new techniques to assess viability have been developed [2]. This led to the discovery that colony counts might seriously underestimate the numbers of viable cells both in natural ecosystems and in laboratory microcosms. There is a wealth of information available about bacteria losing their culturability under certain conditions, while maintaining viability in a more general sense [3], as reviewed by Oliver [4]. l/ibrio vulnificus represents one of the most well-studied organisms with respect to the conversion of bacteria into a viable but non-culturable state. In response to incubation at 5°C, Id. vulnificus cells lose their culturability while retaining viability as assayed by such direct viable count techniques as the test for reduction of I N T to the insoluble INT-formazan [5] and the test for elongation after addition of substrate and nalidixic acid [6,7]. Recently, a negative correlation between the duration of prestarvation at room temperature and the time taken for formation of non-culturable ceils was found for Id. vulnificus C7184 [8]. It was suggested that the starvation program inhibits the formation of non-culturable
206
cells in V. t,ulnificus. Further, by reversing the temperature downshift that induced non-culturability, it was observed that 100% of the cells could be recovered as culturable bacteria within a few days [9]. The aim of this study was to provide a more detailed understanding of the processes that lead to non-culturability and resuscitation.
3. M A T E R I A L S A N D M E T H O D S
3.1. Organism The l/ibrio L'ulnificus strain C7184(T) used in this study has been described by Simpson et al. [10] and is identical to the one used by Nilsson et al. [9]. C7184(T) is a spontaneously derived isogenic translucent and non-virulent mutant of the encapsulated (opaque) strain C7184(O), lacking significant amounts of the capsular polysaccharide [10].
3.2. Growth conditions and media Cells were grown at 24°C on a rotary shaker in either VNSS [11] or M M M medium [12] with 0.2% glucose as carbon source. Starvation experiments were performed statically at 24°C or 5°C in either NSS [11] or M M M lacking glucose, phosphate or ammonium ions.
3.3. Determination of colony forming units (cfu) Samples were diluted in the corresponding medium (NSS resp. MMM) which was kept at the microcosm temperature in order to avoid temperature stress during sample handling. Drop plate counts [13] were performed on VNSS-plates which were initially kept at microcosm temperature and then incubated at 30°C. After 20 h, colonies were counted and the plates were left at 24°C for at least another 50 h to check for outgrowth Of previously undetected colonies. Although this precaution was routinely used in all experiments, no colonies were observed to appear after the initial count.
gation of this supernatant was repeated and the second supernatant was collected and stored at 5°C.
3.5. Tests for growth at different temperatures Cells were inoculated from a fresh overnight culture grown at 24°C in VNSS into three aliquots each of fresh, pre-cooled VNSS and M M M medium supplemented with 0.2% glucose. The cultures were incubated on a rotary shaker at 5°C, 9°C and 24°C and growth was monitored both by turbidity and cfu.
3.6. Cold incubation and resuscitation experiments V. ~ulnificus C7184(T) was grown at 24°C and harvested in mid-log phase (OD610=0.15-0.2; 1.0-2.0 × 108 cfu/ml), diluted directly 1:100 or washed and diluted to a final 1.0-2.0 x 10 7 c f u / m l in starvation media. The culture was split; one set of subsamples was left at 24°C, and the other set transferred to 5°C. Microcosms were monitored for cfu during several weeks and left for another 2 or 6 weeks after the cfu counts in the 5°C culture had reached less than one per ml. From the non-culturable suspensions, triplicate samples of 100 ~,1, 1 ml, 5 ml, 10 ml, 20 ml and 50 ml were transferred to precooled containers in a 5°C room in order to avoid rapid temperature changes. An extra set of samples was supplemented with 4 0 / , g ml-~ nalidixic acid, which had been shown to prevent growth (data not shown). Simultaneously, triplicate samples of 10 /,1, 100/~1, 1 ml and 2 ml, and triplicate samples of 10 - 1 t o 10 - 7 dilutions in M M M medium (at 5°C) were added to 5 ml each of fully supplemented MMM(glucose) medium at 5°C. All samples were placed in an insulating container and then transferred to 24°C. During 10 days, samples were withdrawn for cfu determination, and development of turbidity was monitored in the nutrient-supplemented samples.
4. R E S U L T S
3.4. Supernatants Suspensions were centrifuged at 11000 x g at 5°C for 30 rain and supernatants were carefully taken off, leaving a few ml in the tube. Centrifu-
4.1. Growth of V. vulnificus at 24°C, 9°C and 5°C In order to test for growth of V. uulnificus strain C7184(T) at low temperatures, the strain
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was incubated at three different temperatures as described in MATERIALS AND METHODS. NO growth of the strain was observed at 5°C or 9°C during 14 days, whereas the controls grown at 24°C had entered stationary phase after 16 h. VNSS plates, which support excellent growth of V. uulnificus between 24°C and 37°C, were inoculated and incubated at 5°C for 6 months, confirming that the strain used does not grow at temperatures below 10°C, and that mutants displaying a lower minimum growth temperature do not occur in the timescale studied.
4.2. Cuhurability during star~:ation at 24°C and 5°C Cells of C7184(T) were grown at 24°C to an OD610 of 0.2-0.3, harvested in a cooled (5°C) centrifuge, washed twice in NSS and resuspended in 10 × volume of NSS, yielding between 1.25 × 107 and 1.8 x 10 v cells per ml. The culture was then split into identical subsamples of 20 ml each, of which one-third was kept at 24°C during the whole experiment, and one-third was immediately transferred to 5°C. The remaining microcosms were kept at 24°C for 20 h and then transferred to 5°C. Four such experiments were performed independently with between two and four parallels in each treatment. Figure 1 shows the devel-
lOS ~ . ~ . ~
0
5
10 15 20 25 30 Time (days) Fig. 1. Colony forming units during incubation at 24°C (e), 5°C (A) and at 5°C after prestarvation at 24°C for 20 h (©). Data are means of four experiments, with one-sided error bars indicating the standard deviation.
opment of colony forming units during a representative experiment. Each data point is the average of four parallel microcosms, with error bars indicating the scattering of data. Cells directly transferred to 5°C displayed a rapid decline in culturable cells to less than 100 c f u / m l after 11 days. Incubation at 24°C lead to a slow and transient decline to approximately 10 ~ c f u / m l after 15 days, with no further decline below 105 for an additional period of 6 weeks (data not shown). Cells prestarved at 24°C for 20 h showed a delayed response to cold incubation. In the experiment shown in Fig. 1 the culturability of prestarved cells remained at 103 c f u / m l for 22 days. In two independent experiments, prestarved cells maintained more than 103 cfu m l - I for 24 and 14 days, whereas non-prestarved cells had retained less than 103 cells after 10 and 9 days, respectively. Cells prestarved for more than 1 day do not display further maintenance of culturability: prestarvation for 2, 4, 6 and 9 days at 24°C did not yield an additional delay in the loss of culturability (data not shown).
4.3. Killing effect of non-culturable cell supernatants To elucidate the processes that regulate the kinetics of loss of culturability, supernatants were collected from cultures which had been incubated at 5°C for 17 days at which time they retained less than 1 c f u / m l . The supernatant was added in a ratio of 1:1 to duplicate subsamples of two cultures derived from the experiment displayed in Fig. 1, which had been prestarved for 20 h at 24°C and had then been kept at 5°C for 5 days, retaining 2.6 × 105 cfu ml-~. Figure 2 shows the effect of supernatant addition: the supernatanttreated cultures had lost 99.999% (factor 10 _5 ) of their cfu (less than 1 c f u / m l ) within 7 days, whereas the untreated controls retained 5% of their culturable cells (averaging 1.01 × 10 4) during the same time at 5°C. In addition, the latter culture displayed more than 103 c f u / m l for another 4 days. Consequently, the addition of supernatant resulted in an additional decrease in cfu of 99.98% (factor 2 × 10 -4) within 7 days. The same effect was observed when the concentrated pellet fraction or the untreated bacterial
208 10 8
+
10 6
1¢ 100
]
-
0
5
10 15 Time (days)
,
20
25
Fig. 2. Colony forming units during incubation at 5°C; effect of the addition of supernatant from cultures which were non-culturable after 17 days of cold incubation. At day 6 the supernatant was added in a 1:1 ratio to the prestarved cells from the experiment shown in Fig. 1. Both the data from the treated samples (o) and the controls (o) are means of four experiments. In parallel experiments, the pellet from the same culture resuspended in 1/50 of the original volume ([]) and the original cell suspension (z~) were added; in these cases, the data are means of duplicates.
suspension was applied instead of the supernatant. Non-culturable suspensions derived from other experiments gave rise to a comparable induction of loss of cfu within 4 days (data not shown) in both prestarved and non-prestarved samples. Preliminary experiments demonstrated that the supernatant lost most of its killing effect after freezing at - 2 0 ° C . It was also shown that heat treatment of the supernatant abolishes the killing effect (data not shown). The p H in the supernatants was found to be between 6.9 and 7.8 (average 7.40, n = 11), and in the growing, starving and culturable suspensions between 7.1 and 7.53 (average 7.36, n = 9), thus not showing any significant differences. In order to evaluate whether the killing is due to increased susceptibility of the organism at 5°C towards components produced at all temperatures, supernatants obtained from cultures starved at 24°C for 42 days were tested. It was shown that such supernatants did not have any effect on culturability of cells at 5°C during 7 days (data not shown).
4.4. Resuscitation uersus regrowth Initial experiments indicated that resuscitation of non-culturable populations by reverting the temperature shift [9] could not be reproduced reliably. To investigate the processes involved in resuscitation, a series of nine independent resuscitation experiments were run, involving cells grown into mid-log phase in either VNSS or MMM(glucose), and incubated at 5°C after washing or diluting in different media: NSS, M M M lacking carbon-, nitrogen- or phosphorus source, or fully supplemented MMM(glucose)-medium. The rates of loss of culturability were roughly identical in all these experiments, so that after 1 month less than 1 cfu ml - I could be detected (data not shown). Resuscitation experiments were performed with all these cultures as described in MATERIALS AND METHODS. From the nine independent experiments, only one of those which had been non-culturable for 2 weeks showed resuscitation in samples of at least 5 ml. Of the three 5-ml samples only two resuscitated, whereas 20-ml and 50-ml samples resuscitated to 100%. None of the smaller subsamples or dilutions showed any resuscitation or growth in the supplemented aliquots. Addition of nalidixic acid (40 mg 1-1) abolished the resuscitation in the 20-ml and 50-ml samples. The remaining eight experiments displayed neither restored cfu in any of the subsamples nor turbidity in any of the growth assays. In a comparable experiment with the opaque wild-type strain C7184(O), subsamples of 10 ml showed full resuscitation, whereas out of 10 samples of 1 ml each only five showed resuscitation. To investigate the possibility of cryptic growth during resuscitation, 30-50 starved cells of C7184(T) were inoculated into triplicate aliquots of 2 ml from non-resuscitable cultures and transferred to 24°C. Whereas control volumes did not show any cfu development after 7 days at 24°C, the inoculated subsamples grew up to between 1.3 x 10 7 and 4.3 x 10 7 c f u / m l after 2 - 3 days. This amounts to 22-58% of the cfu in the microcosms before cold incubation. It should be stressed that the non-culturable populations employed in this study were derived from washed ceils, so that nutrient carry-over was negligible.
209 5. DISCUSSION
V. vulnificus progressively loses culturability upon cold incubation, forming viable but nonculturable cells as assayed by reduction of INT [5] and direct viable count [6]. This process is independent of the incubation medium [14], while the duration of prestarvation of the cells at growth temperature influences the rate of loss of culturability [8]. The experimental design used in these studies has an important advantage compared with most other survival studies. As V. vulnificus C7184(T) does not grow at 5°C, cryptic growth is excluded by keeping the cells at this temperature. Thus, it is possible to study the same population of cells as long as the temperature does not permit growth. In the experiments described here, prestarvation at room temperature before cold incubation prolongs culturability significantly, but does not offer permanent protection. It is tempting to suggest that the synthesis of stress proteins during starvation might allow for a transient protection of the cells against cold. In fact, it could be shown that the strain employed in this study induced the synthesis of both the DnaK and G r o E L chaperones strongly during the first hours of starvation (unpublished data). By adding the supernatants of non-culturable cells to suspensions which retained culturable cells it was demonstrated that non-culturability can be induced by components released by nonculturable cells. It was concluded that this result is not due to a pH effect. Preliminary experiments showed that supernatants of non-culturable cells lose their activity upon heating and freezing. The labile active component apparently triggers or causes the disappearance of culturable cells in the culture. It is possible that the cells are actually killing themselves, or depriving themselves of the ability to regrow. The nature of the process leading to self-accelerated death in V. vulnificus is so far not elucidated. The lack of killing by supernatants from cultures kept at 24°C for several weeks indicates that the killing is not due to increased susceptibility of the organism at 5°C towards a compound that is produced at all
temperatures. Apparently, low temperature stimulates the activity or induces the synthesis of a killing agent (e.g. a protease, nuclease, lipase, ionophore or prophage) which is not present or active at growth temperature. As we have demonstrated in this paper, it is possible to evoke a response mimicking resuscitation by simply shifting temperature back into the range permitting growth. Our results have shown that during this process extensive regrowth of a few resuscitated or remaining culturable cells is possible. That 'resuscitation' of V. vulnificus [9] may actually represent regrowth rather than true resuscitation is supported by a variety of observations. Firstly, resuscitation occurs only in a certain minimum volume of sample and only within a limited time after entering non-culturability. Thus, the cells leading to restored cfu appear to be extremely few and continuously decreasing in number. Based on our dilution studies, they constituted less than 0.00001% of the original population, as opposed to 100% as reported by Nilsson et al. [9]. The majority of non-culturable cells which are still detectable by acridine orange direct counts (AODC) and direct viable counts do not appear to possess the ability to resuscitate after a temperature shift to 24°C. Secondly, the volumes which are devoid of culturable cells are obviously capable of providing sufficient nutrients for the regrowth of a few resuscitated or culturable cells, restoring up to 58% of the initial cfu. This should be theoretically impossible if the cells are washed prior to cold incubation, because the growth efficiency on lysed cells has been calculated not to exceed 2% [15]. However, it has to be considered that the cells subjected to the temperature downshift are large logarithmic-phase cells, and the cells recovered at the end of the experiment are small cocci, possibly having undergone reductive divisions. Thirdly, two observations can be directly explained by a model involving regrowth of a few cells: the morphology of the resuscitating cells, which is identical to mid-log cells [9], and the difficulties with PCR-amplification of sequences from non-culturable cells [16]. It appears that the cells are progressively los-
210 ing t h e i r ability to r e g r o w d u r i n g cold i n c u b a t i o n , while r e t a i n i n g an intact m o r p h o l o g y with stainable D N A inside their m e m b r a n e , a n d also maintaining t h e i r ability to r e a c t to s u b s t r a t e s in the D V C assay [6]. Both f e a t u r e s could also be att r i b u t e d to m o r i b u n d cells, as has b e e n p o i n t e d out by M a s o n et al. [17]. T o explain the c o n s t a n t n u m b e r of a c r i d i n e o r a n g e direct counts d u r i n g r e s u s c i t a t i o n [9] it can be h y p o t h e s i z e d that the m a j o r i t y of n o n - c u l t u r a b l e cells might u n d e r g o lysis after t e m p e r a t u r e upshift a n d might be rep l a c e d by r e g r o w i n g ceils. It is p e r t i n e n t to ask w h a t significance viable but n o n - c u l t u r a b l e cells might have for the fate of a b a c t e r i a l p o p u l a t i o n in the ecosystem a n d its p o t e n t i a l p a t h o g e n i c i t y if conclusive evidence for r e s u s c i t a t i o n is lacking. T o o u r k n o w l e d g e the p u b l i c a t i o n s that have s u g g e s t e d resuscitation of n o n - c u l t u r a b l e cells [9,18-25] involve the a d d i t i o n or p r e s e n c e of n u t r i e n t s a n d have not i n c l u d e d controls o f the type p r e s e n t e d in this p a p e r . Cryptic growth has only b e e n e x c l u d e d for the b e t a i n e - i n d u c e d r e s u s c i t a t i o n of Escherichia coli a f t e r osmotic shock [26]. H o w e v e r , c o n s i d e r i n g that the inability to r e s u s c i t a t e a b a c t e r i u m in the l a b o r a t o r y might m e r e l y reflect i n a p p r o p r i a t e m e t h o d s , it c a n n o t be e x c l u d e d that true resuscitation occurs in n a t u r e and possibly allows for survival a n d s p r e a d of b a c t e r i a l species in the ecosystems.
ACKNOWLEDGEMENTS F i n a n c i a l s u p p o r t from the N o r d i c Ministry Council, the Swedish N a t i o n a l E n v i r o n m e n t a l P r o t e c t i o n A g e n c y a n d the Swedish A g r i c u l t u r a l R e s e a r c h Council is gratefully a c k n o w l e d g e d . W e a r e i n d e b t e d to Klas Fl~irdh for s t i m u l a t i n g a n d critical discussions.
REFERENCES [I] Postgate, J.R. (1976) In: Proc. Syrup. Soc. Gen. Microbiol. (Gray, T.R.G, and Postgate, J.R., Eds.), pp. 1-18. Cambridge University Press, Cambridge.
[2] Roszak, D.B. and Colwell, R.R. (1987) Microbiol. Rev. 51,365-379. [3l Byrd, J.J., Xu, H.-S. and Colwell, R.R. (1991) Appl. Environ. Microbiol. 57, 875-878. [4] Oliver, J.D. (1993) In: Starvation in Bacteria (Kjelleberg, S,, Ed.), Plenum Press, New York, NY, in press. [5] Zimmermann, R., Iturriaga, R. and Becker-Birck, J. (1978) Appl. Environ. Microbiol. 36, 926-935. [6l Kogure, K., Simidu, U. and Taga, N. (1979) Can. J. Microbiol. 25, 415-420. [7] Kogure, K., Simidu, U., Taga, N. and Colwell, R.R. (1987) Appl. Environ. Microbiol. 53, 2332-2337. [8] Oliver, J.D., Nilsson, E. and Kjelleberg, S. (199l) Appl. Environ. Microbiol. 57, 2640-2644. [9] Nilsson, L., Oliver, J.D. and Kjelleberg, S. (1991) J. Bacteriol. 173, 5054-5059. [10] Simpson, L.M., White, V.K., Zane, S.F. and Oliver, J.D. (1987) Infect. Immun. 55, 269-272. [11] Nystr6m, T., M~rden, P. and Kjelleberg, S. (1986) FEMS Microbiol. Ecol. 38, 285-292. [12] C)stling, J., Goodman, A. and Kjelleberg, S. (1991) FEMS Microbiol. Ecol. 86, 83-94. [13] Hoben, H.J. and Somasegaran, P. (1982) Appl. Environ. Microbiol. 44, 1246-1247. [14] Oliver, J.D. and Wanucha, D. (1989) J. Food Safety 10, 79-86. [15] Postgate, J.R. and Hunter, J.R. (1962) J. Gen. Microbiol. 29, 233-263. [16] Brauns, L.A., Hudson, M.C. and Oliver, J.D. (1991) Appl. Environ. Microbiol. 57, 2651-2655. 117] Mason, C.A., Hamer, G. and Bryers, J.D. (1986) FEMS Microbiol. Rev. 39, 373-401. [18] Roszak, D.B., Grimes, D.J. and Colwell, R.R. (1984) Can. J. Microbiol. 30, 334-338. [19] Jones, D.M., Sutcliffe, E.M. and Curry, A. (1991)J. Gen. Microbiol. 137, 2477-2482. [20] Saha, S.K., Saha, S. and Sanyal, S.C. (1991) Appl. Environ. Microbiol. 57, 3388-3389. [21] Colbourne, LS., Dennis, P.J., Trew, R.M., Berry, C. and Vesey, G. (1988) Water Sci. Technol. 20, 5-10. [22] Allen-Austin, D., Austin, B. and Colwell, R.R. (1984) FEMS Microbiol. Lett. 2l, 143-146. [23] Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Bio/Technology 3, 817-820. [24] Grimes, D.J. and Colwell, R.R. (1986) FEMS Microbiol. Lett. 34, 161-165. [25] Hussong, D., Colwell, R.R,, O'Brien, M., Weiss, E., Pearson, A.D., Weiner, R.M. and Burge, W.D. (1987) Bio/Technology 5, 947-950. [26] Roth, W.G., Leckie, M.P. and Dietzler, D.N. (1988) Appl. Environ. Microbiol. 54, 3142-3146.
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Low temperature induced non-culturability and killing of Vibrio vulnificus D i e t e r W e i c h a r t a, J a m e s D. O l i v e r b a n d S t a f f a n K j e l l e b e r g a o Department of General and Marine Microbiology, University of Gdteborg, G6teborg, Sweden, and b Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina, USA
Received 16 June 1992 Accepted 19 June 1992 Key words: Vibrio vulnificus; Viable but non-culturable; Survival; Resuscitation
1. S U M M A R Y Vibrio vulnificus ceils progressively lose culturability during incubation at 5°C. This process is accelerated by the addition of supernatants from non-culturable cells obtained by incubation at 5°C for 17 days. Thus the organism apparently produces a factor upon cold incubation which is triggering or causing the decline in culturability. Reversing the t e m p e r a t u r e shift can restore a culturable population comparable in numbers to the original population, but this process is largely due to regrowth. A few cells retaining the ability to grow apparently utilize the substrates released by the moribund cells, thus mimicking resuscitation of the whole population.
2. I N T R O D U C T I O N The concept of viability of microorganisms has changed dramatically during the past decades.
Correspondence to: S. Kjelleberg, Department of General and
Marine Microbiology, University of G6teborg, Carl Skottsbergs Gata 22, S-41319 G6teborg, Sweden.
The classical approach using colony counts as an indicator of viability [1] has repeatedly been challenged, and several new techniques to assess viability have been developed [2]. This led to the discovery that colony counts might seriously underestimate the numbers of viable cells both in natural ecosystems and in laboratory microcosms. There is a wealth of information available about bacteria losing their culturability under certain conditions, while maintaining viability in a more general sense [3], as reviewed by Oliver [4]. l/ibrio vulnificus represents one of the most well-studied organisms with respect to the conversion of bacteria into a viable but non-culturable state. In response to incubation at 5°C, Id. vulnificus cells lose their culturability while retaining viability as assayed by such direct viable count techniques as the test for reduction of I N T to the insoluble INT-formazan [5] and the test for elongation after addition of substrate and nalidixic acid [6,7]. Recently, a negative correlation between the duration of prestarvation at room temperature and the time taken for formation of non-culturable ceils was found for Id. vulnificus C7184 [8]. It was suggested that the starvation program inhibits the formation of non-culturable
206
cells in V. t,ulnificus. Further, by reversing the temperature downshift that induced non-culturability, it was observed that 100% of the cells could be recovered as culturable bacteria within a few days [9]. The aim of this study was to provide a more detailed understanding of the processes that lead to non-culturability and resuscitation.
3. M A T E R I A L S A N D M E T H O D S
3.1. Organism The l/ibrio L'ulnificus strain C7184(T) used in this study has been described by Simpson et al. [10] and is identical to the one used by Nilsson et al. [9]. C7184(T) is a spontaneously derived isogenic translucent and non-virulent mutant of the encapsulated (opaque) strain C7184(O), lacking significant amounts of the capsular polysaccharide [10].
3.2. Growth conditions and media Cells were grown at 24°C on a rotary shaker in either VNSS [11] or M M M medium [12] with 0.2% glucose as carbon source. Starvation experiments were performed statically at 24°C or 5°C in either NSS [11] or M M M lacking glucose, phosphate or ammonium ions.
3.3. Determination of colony forming units (cfu) Samples were diluted in the corresponding medium (NSS resp. MMM) which was kept at the microcosm temperature in order to avoid temperature stress during sample handling. Drop plate counts [13] were performed on VNSS-plates which were initially kept at microcosm temperature and then incubated at 30°C. After 20 h, colonies were counted and the plates were left at 24°C for at least another 50 h to check for outgrowth Of previously undetected colonies. Although this precaution was routinely used in all experiments, no colonies were observed to appear after the initial count.
gation of this supernatant was repeated and the second supernatant was collected and stored at 5°C.
3.5. Tests for growth at different temperatures Cells were inoculated from a fresh overnight culture grown at 24°C in VNSS into three aliquots each of fresh, pre-cooled VNSS and M M M medium supplemented with 0.2% glucose. The cultures were incubated on a rotary shaker at 5°C, 9°C and 24°C and growth was monitored both by turbidity and cfu.
3.6. Cold incubation and resuscitation experiments V. ~ulnificus C7184(T) was grown at 24°C and harvested in mid-log phase (OD610=0.15-0.2; 1.0-2.0 × 108 cfu/ml), diluted directly 1:100 or washed and diluted to a final 1.0-2.0 x 10 7 c f u / m l in starvation media. The culture was split; one set of subsamples was left at 24°C, and the other set transferred to 5°C. Microcosms were monitored for cfu during several weeks and left for another 2 or 6 weeks after the cfu counts in the 5°C culture had reached less than one per ml. From the non-culturable suspensions, triplicate samples of 100 ~,1, 1 ml, 5 ml, 10 ml, 20 ml and 50 ml were transferred to precooled containers in a 5°C room in order to avoid rapid temperature changes. An extra set of samples was supplemented with 4 0 / , g ml-~ nalidixic acid, which had been shown to prevent growth (data not shown). Simultaneously, triplicate samples of 10 /,1, 100/~1, 1 ml and 2 ml, and triplicate samples of 10 - 1 t o 10 - 7 dilutions in M M M medium (at 5°C) were added to 5 ml each of fully supplemented MMM(glucose) medium at 5°C. All samples were placed in an insulating container and then transferred to 24°C. During 10 days, samples were withdrawn for cfu determination, and development of turbidity was monitored in the nutrient-supplemented samples.
4. R E S U L T S
3.4. Supernatants Suspensions were centrifuged at 11000 x g at 5°C for 30 rain and supernatants were carefully taken off, leaving a few ml in the tube. Centrifu-
4.1. Growth of V. vulnificus at 24°C, 9°C and 5°C In order to test for growth of V. uulnificus strain C7184(T) at low temperatures, the strain
207
was incubated at three different temperatures as described in MATERIALS AND METHODS. NO growth of the strain was observed at 5°C or 9°C during 14 days, whereas the controls grown at 24°C had entered stationary phase after 16 h. VNSS plates, which support excellent growth of V. uulnificus between 24°C and 37°C, were inoculated and incubated at 5°C for 6 months, confirming that the strain used does not grow at temperatures below 10°C, and that mutants displaying a lower minimum growth temperature do not occur in the timescale studied.
4.2. Cuhurability during star~:ation at 24°C and 5°C Cells of C7184(T) were grown at 24°C to an OD610 of 0.2-0.3, harvested in a cooled (5°C) centrifuge, washed twice in NSS and resuspended in 10 × volume of NSS, yielding between 1.25 × 107 and 1.8 x 10 v cells per ml. The culture was then split into identical subsamples of 20 ml each, of which one-third was kept at 24°C during the whole experiment, and one-third was immediately transferred to 5°C. The remaining microcosms were kept at 24°C for 20 h and then transferred to 5°C. Four such experiments were performed independently with between two and four parallels in each treatment. Figure 1 shows the devel-
lOS ~ . ~ . ~
0
5
10 15 20 25 30 Time (days) Fig. 1. Colony forming units during incubation at 24°C (e), 5°C (A) and at 5°C after prestarvation at 24°C for 20 h (©). Data are means of four experiments, with one-sided error bars indicating the standard deviation.
opment of colony forming units during a representative experiment. Each data point is the average of four parallel microcosms, with error bars indicating the scattering of data. Cells directly transferred to 5°C displayed a rapid decline in culturable cells to less than 100 c f u / m l after 11 days. Incubation at 24°C lead to a slow and transient decline to approximately 10 ~ c f u / m l after 15 days, with no further decline below 105 for an additional period of 6 weeks (data not shown). Cells prestarved at 24°C for 20 h showed a delayed response to cold incubation. In the experiment shown in Fig. 1 the culturability of prestarved cells remained at 103 c f u / m l for 22 days. In two independent experiments, prestarved cells maintained more than 103 cfu m l - I for 24 and 14 days, whereas non-prestarved cells had retained less than 103 cells after 10 and 9 days, respectively. Cells prestarved for more than 1 day do not display further maintenance of culturability: prestarvation for 2, 4, 6 and 9 days at 24°C did not yield an additional delay in the loss of culturability (data not shown).
4.3. Killing effect of non-culturable cell supernatants To elucidate the processes that regulate the kinetics of loss of culturability, supernatants were collected from cultures which had been incubated at 5°C for 17 days at which time they retained less than 1 c f u / m l . The supernatant was added in a ratio of 1:1 to duplicate subsamples of two cultures derived from the experiment displayed in Fig. 1, which had been prestarved for 20 h at 24°C and had then been kept at 5°C for 5 days, retaining 2.6 × 105 cfu ml-~. Figure 2 shows the effect of supernatant addition: the supernatanttreated cultures had lost 99.999% (factor 10 _5 ) of their cfu (less than 1 c f u / m l ) within 7 days, whereas the untreated controls retained 5% of their culturable cells (averaging 1.01 × 10 4) during the same time at 5°C. In addition, the latter culture displayed more than 103 c f u / m l for another 4 days. Consequently, the addition of supernatant resulted in an additional decrease in cfu of 99.98% (factor 2 × 10 -4) within 7 days. The same effect was observed when the concentrated pellet fraction or the untreated bacterial
208 10 8
+
10 6
1¢ 100
]
-
0
5
10 15 Time (days)
,
20
25
Fig. 2. Colony forming units during incubation at 5°C; effect of the addition of supernatant from cultures which were non-culturable after 17 days of cold incubation. At day 6 the supernatant was added in a 1:1 ratio to the prestarved cells from the experiment shown in Fig. 1. Both the data from the treated samples (o) and the controls (o) are means of four experiments. In parallel experiments, the pellet from the same culture resuspended in 1/50 of the original volume ([]) and the original cell suspension (z~) were added; in these cases, the data are means of duplicates.
suspension was applied instead of the supernatant. Non-culturable suspensions derived from other experiments gave rise to a comparable induction of loss of cfu within 4 days (data not shown) in both prestarved and non-prestarved samples. Preliminary experiments demonstrated that the supernatant lost most of its killing effect after freezing at - 2 0 ° C . It was also shown that heat treatment of the supernatant abolishes the killing effect (data not shown). The p H in the supernatants was found to be between 6.9 and 7.8 (average 7.40, n = 11), and in the growing, starving and culturable suspensions between 7.1 and 7.53 (average 7.36, n = 9), thus not showing any significant differences. In order to evaluate whether the killing is due to increased susceptibility of the organism at 5°C towards components produced at all temperatures, supernatants obtained from cultures starved at 24°C for 42 days were tested. It was shown that such supernatants did not have any effect on culturability of cells at 5°C during 7 days (data not shown).
4.4. Resuscitation uersus regrowth Initial experiments indicated that resuscitation of non-culturable populations by reverting the temperature shift [9] could not be reproduced reliably. To investigate the processes involved in resuscitation, a series of nine independent resuscitation experiments were run, involving cells grown into mid-log phase in either VNSS or MMM(glucose), and incubated at 5°C after washing or diluting in different media: NSS, M M M lacking carbon-, nitrogen- or phosphorus source, or fully supplemented MMM(glucose)-medium. The rates of loss of culturability were roughly identical in all these experiments, so that after 1 month less than 1 cfu ml - I could be detected (data not shown). Resuscitation experiments were performed with all these cultures as described in MATERIALS AND METHODS. From the nine independent experiments, only one of those which had been non-culturable for 2 weeks showed resuscitation in samples of at least 5 ml. Of the three 5-ml samples only two resuscitated, whereas 20-ml and 50-ml samples resuscitated to 100%. None of the smaller subsamples or dilutions showed any resuscitation or growth in the supplemented aliquots. Addition of nalidixic acid (40 mg 1-1) abolished the resuscitation in the 20-ml and 50-ml samples. The remaining eight experiments displayed neither restored cfu in any of the subsamples nor turbidity in any of the growth assays. In a comparable experiment with the opaque wild-type strain C7184(O), subsamples of 10 ml showed full resuscitation, whereas out of 10 samples of 1 ml each only five showed resuscitation. To investigate the possibility of cryptic growth during resuscitation, 30-50 starved cells of C7184(T) were inoculated into triplicate aliquots of 2 ml from non-resuscitable cultures and transferred to 24°C. Whereas control volumes did not show any cfu development after 7 days at 24°C, the inoculated subsamples grew up to between 1.3 x 10 7 and 4.3 x 10 7 c f u / m l after 2 - 3 days. This amounts to 22-58% of the cfu in the microcosms before cold incubation. It should be stressed that the non-culturable populations employed in this study were derived from washed ceils, so that nutrient carry-over was negligible.
209 5. DISCUSSION
V. vulnificus progressively loses culturability upon cold incubation, forming viable but nonculturable cells as assayed by reduction of INT [5] and direct viable count [6]. This process is independent of the incubation medium [14], while the duration of prestarvation of the cells at growth temperature influences the rate of loss of culturability [8]. The experimental design used in these studies has an important advantage compared with most other survival studies. As V. vulnificus C7184(T) does not grow at 5°C, cryptic growth is excluded by keeping the cells at this temperature. Thus, it is possible to study the same population of cells as long as the temperature does not permit growth. In the experiments described here, prestarvation at room temperature before cold incubation prolongs culturability significantly, but does not offer permanent protection. It is tempting to suggest that the synthesis of stress proteins during starvation might allow for a transient protection of the cells against cold. In fact, it could be shown that the strain employed in this study induced the synthesis of both the DnaK and G r o E L chaperones strongly during the first hours of starvation (unpublished data). By adding the supernatants of non-culturable cells to suspensions which retained culturable cells it was demonstrated that non-culturability can be induced by components released by nonculturable cells. It was concluded that this result is not due to a pH effect. Preliminary experiments showed that supernatants of non-culturable cells lose their activity upon heating and freezing. The labile active component apparently triggers or causes the disappearance of culturable cells in the culture. It is possible that the cells are actually killing themselves, or depriving themselves of the ability to regrow. The nature of the process leading to self-accelerated death in V. vulnificus is so far not elucidated. The lack of killing by supernatants from cultures kept at 24°C for several weeks indicates that the killing is not due to increased susceptibility of the organism at 5°C towards a compound that is produced at all
temperatures. Apparently, low temperature stimulates the activity or induces the synthesis of a killing agent (e.g. a protease, nuclease, lipase, ionophore or prophage) which is not present or active at growth temperature. As we have demonstrated in this paper, it is possible to evoke a response mimicking resuscitation by simply shifting temperature back into the range permitting growth. Our results have shown that during this process extensive regrowth of a few resuscitated or remaining culturable cells is possible. That 'resuscitation' of V. vulnificus [9] may actually represent regrowth rather than true resuscitation is supported by a variety of observations. Firstly, resuscitation occurs only in a certain minimum volume of sample and only within a limited time after entering non-culturability. Thus, the cells leading to restored cfu appear to be extremely few and continuously decreasing in number. Based on our dilution studies, they constituted less than 0.00001% of the original population, as opposed to 100% as reported by Nilsson et al. [9]. The majority of non-culturable cells which are still detectable by acridine orange direct counts (AODC) and direct viable counts do not appear to possess the ability to resuscitate after a temperature shift to 24°C. Secondly, the volumes which are devoid of culturable cells are obviously capable of providing sufficient nutrients for the regrowth of a few resuscitated or culturable cells, restoring up to 58% of the initial cfu. This should be theoretically impossible if the cells are washed prior to cold incubation, because the growth efficiency on lysed cells has been calculated not to exceed 2% [15]. However, it has to be considered that the cells subjected to the temperature downshift are large logarithmic-phase cells, and the cells recovered at the end of the experiment are small cocci, possibly having undergone reductive divisions. Thirdly, two observations can be directly explained by a model involving regrowth of a few cells: the morphology of the resuscitating cells, which is identical to mid-log cells [9], and the difficulties with PCR-amplification of sequences from non-culturable cells [16]. It appears that the cells are progressively los-
210 ing t h e i r ability to r e g r o w d u r i n g cold i n c u b a t i o n , while r e t a i n i n g an intact m o r p h o l o g y with stainable D N A inside their m e m b r a n e , a n d also maintaining t h e i r ability to r e a c t to s u b s t r a t e s in the D V C assay [6]. Both f e a t u r e s could also be att r i b u t e d to m o r i b u n d cells, as has b e e n p o i n t e d out by M a s o n et al. [17]. T o explain the c o n s t a n t n u m b e r of a c r i d i n e o r a n g e direct counts d u r i n g r e s u s c i t a t i o n [9] it can be h y p o t h e s i z e d that the m a j o r i t y of n o n - c u l t u r a b l e cells might u n d e r g o lysis after t e m p e r a t u r e upshift a n d might be rep l a c e d by r e g r o w i n g ceils. It is p e r t i n e n t to ask w h a t significance viable but n o n - c u l t u r a b l e cells might have for the fate of a b a c t e r i a l p o p u l a t i o n in the ecosystem a n d its p o t e n t i a l p a t h o g e n i c i t y if conclusive evidence for r e s u s c i t a t i o n is lacking. T o o u r k n o w l e d g e the p u b l i c a t i o n s that have s u g g e s t e d resuscitation of n o n - c u l t u r a b l e cells [9,18-25] involve the a d d i t i o n or p r e s e n c e of n u t r i e n t s a n d have not i n c l u d e d controls o f the type p r e s e n t e d in this p a p e r . Cryptic growth has only b e e n e x c l u d e d for the b e t a i n e - i n d u c e d r e s u s c i t a t i o n of Escherichia coli a f t e r osmotic shock [26]. H o w e v e r , c o n s i d e r i n g that the inability to r e s u s c i t a t e a b a c t e r i u m in the l a b o r a t o r y might m e r e l y reflect i n a p p r o p r i a t e m e t h o d s , it c a n n o t be e x c l u d e d that true resuscitation occurs in n a t u r e and possibly allows for survival a n d s p r e a d of b a c t e r i a l species in the ecosystems.
ACKNOWLEDGEMENTS F i n a n c i a l s u p p o r t from the N o r d i c Ministry Council, the Swedish N a t i o n a l E n v i r o n m e n t a l P r o t e c t i o n A g e n c y a n d the Swedish A g r i c u l t u r a l R e s e a r c h Council is gratefully a c k n o w l e d g e d . W e a r e i n d e b t e d to Klas Fl~irdh for s t i m u l a t i n g a n d critical discussions.
REFERENCES [I] Postgate, J.R. (1976) In: Proc. Syrup. Soc. Gen. Microbiol. (Gray, T.R.G, and Postgate, J.R., Eds.), pp. 1-18. Cambridge University Press, Cambridge.
[2] Roszak, D.B. and Colwell, R.R. (1987) Microbiol. Rev. 51,365-379. [3l Byrd, J.J., Xu, H.-S. and Colwell, R.R. (1991) Appl. Environ. Microbiol. 57, 875-878. [4] Oliver, J.D. (1993) In: Starvation in Bacteria (Kjelleberg, S,, Ed.), Plenum Press, New York, NY, in press. [5] Zimmermann, R., Iturriaga, R. and Becker-Birck, J. (1978) Appl. Environ. Microbiol. 36, 926-935. [6l Kogure, K., Simidu, U. and Taga, N. (1979) Can. J. Microbiol. 25, 415-420. [7] Kogure, K., Simidu, U., Taga, N. and Colwell, R.R. (1987) Appl. Environ. Microbiol. 53, 2332-2337. [8] Oliver, J.D., Nilsson, E. and Kjelleberg, S. (199l) Appl. Environ. Microbiol. 57, 2640-2644. [9] Nilsson, L., Oliver, J.D. and Kjelleberg, S. (1991) J. Bacteriol. 173, 5054-5059. [10] Simpson, L.M., White, V.K., Zane, S.F. and Oliver, J.D. (1987) Infect. Immun. 55, 269-272. [11] Nystr6m, T., M~rden, P. and Kjelleberg, S. (1986) FEMS Microbiol. Ecol. 38, 285-292. [12] C)stling, J., Goodman, A. and Kjelleberg, S. (1991) FEMS Microbiol. Ecol. 86, 83-94. [13] Hoben, H.J. and Somasegaran, P. (1982) Appl. Environ. Microbiol. 44, 1246-1247. [14] Oliver, J.D. and Wanucha, D. (1989) J. Food Safety 10, 79-86. [15] Postgate, J.R. and Hunter, J.R. (1962) J. Gen. Microbiol. 29, 233-263. [16] Brauns, L.A., Hudson, M.C. and Oliver, J.D. (1991) Appl. Environ. Microbiol. 57, 2651-2655. 117] Mason, C.A., Hamer, G. and Bryers, J.D. (1986) FEMS Microbiol. Rev. 39, 373-401. [18] Roszak, D.B., Grimes, D.J. and Colwell, R.R. (1984) Can. J. Microbiol. 30, 334-338. [19] Jones, D.M., Sutcliffe, E.M. and Curry, A. (1991)J. Gen. Microbiol. 137, 2477-2482. [20] Saha, S.K., Saha, S. and Sanyal, S.C. (1991) Appl. Environ. Microbiol. 57, 3388-3389. [21] Colbourne, LS., Dennis, P.J., Trew, R.M., Berry, C. and Vesey, G. (1988) Water Sci. Technol. 20, 5-10. [22] Allen-Austin, D., Austin, B. and Colwell, R.R. (1984) FEMS Microbiol. Lett. 2l, 143-146. [23] Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Bio/Technology 3, 817-820. [24] Grimes, D.J. and Colwell, R.R. (1986) FEMS Microbiol. Lett. 34, 161-165. [25] Hussong, D., Colwell, R.R,, O'Brien, M., Weiss, E., Pearson, A.D., Weiner, R.M. and Burge, W.D. (1987) Bio/Technology 5, 947-950. [26] Roth, W.G., Leckie, M.P. and Dietzler, D.N. (1988) Appl. Environ. Microbiol. 54, 3142-3146.
