1 Laboratoire de Microbiologie, Faculté des Sciences de Sfax, 3018 Sfax, Tunisia
2 Laboratoire d'Océanographie et de Biogéochimie, CNRS UMR6535, Université de la Méditerranée, 163 avenue de Luminy, Case 901, 13288 Marseille cedex 9, France
3 Laboratoire de Microbiologie Marine, CNRS UMR6117, Université de la Méditerranée, 163 avenue de Luminy, Case 907, 13288 Marseille cedex 9, France
Correspondence
Sam Dukan
sdukan{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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Present address: Laboratoire de Chimie Bactérienne IBSM, CNRS UPR 9043, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
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INTRODUCTION |
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The inability to isolate A. hydrophila during the winter months or from cold waters may result not from cell death, but from the entry of cells into a viable but nonculturable (VBNC) state (Hasan et al., 1991). Various Gram-negative bacteria are known to enter such a state, often induced when the bacteria are exposed to adverse environmental conditions (Roszak & Colwell, 1987
; Oliver et al., 1991
; Jiang & Chai, 1996
). For instance, when Vibrio cholerae or Vibrio vulnificus was incubated in sterile seawater at 5 °C, cells entered a VBNC state that was reversed by raising the temperature to 25 °C (Oliver, 2000
). However, more recent results indicated that V. vulnificus cells previously considered as VBNC actually comprised a subpopulation of the culture that failed to reproduce due to starvation-induced injury and hydrogen peroxide sensitivity (Bogosian & Bourneuf, 2001
). Interestingly, A. hydrophila incubated in freshwater also entered a VBNC state at 5 °C (Wai et al., 2000
; Mary et al., 2002
). Controversial results were obtained from attempts to restore culturability via reagents that degrade reactive oxygen species (Wai et al., 2000
; Mary et al., 2002
).
To our knowledge, none of the reported studies addressing the VBNC state concerns A. hydrophila in seawater. The aim of the present work was to investigate the effect of temperature (23 and 5 °C) on the behaviour of A. hydrophila incubated in nutrient-poor seawater. The influence of the cells' growth phase on the entry into the VBNC state, the morphological modifications, and the possible resuscitation from the VBNC state were also considered.
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METHODS |
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Seawater microcosms.
Seawater (salinity 36·5 ; pH 8·1) was collected in the coastal area of Sfax, 7 km from the wastewater outlet, and filtered through a 0·22 µm cellulose nitrate filter (Sartorius). Erlenmeyer flasks (250 ml), containing 90 ml filtered seawater, were inoculated with 108 A. hydrophila c.f.u. ml-1 and incubated in the dark, at 23 or 5 °C, without shaking. To ensure that no biodegradable material was released into the microcosms, the flasks were previously heated at 450 °C for 4 h. Four 1 ml subsamples from all microcosms were collected immediately after inoculation and then periodically during the following 35 days of incubation to assess, individually, changes in culturability, respiratory activity, total count and nucleic acid content, and membrane integrity. In a separate experiment, the effect of chloramphenicol (100 µg ml-1) on the loss of culturability at 23 and 5 °C was tested with cells in the exponential growth phase.
Cell assays and viability tests.
Culturable cells were counted by dilution in 0·85 % (w/v) sterile NaCl solution and mixing 100 µl samples with 20 ml LB agar at 45 °C (in duplicate). Plates were incubated at 37 °C and culturable cells were counted after 48 h. Analysis of counting procedures for culturable cells showed less than 10 % difference between results of the pour-plate method and those of the spread-plating method. When counts fell below the detection limit of 1 c.f.u. per 0·2 ml, 1 ml samples were plated onto ten plates to reduce the detection limit to less than 0·1 c.f.u. ml-1. At this point (3842 days), the cells were considered to be in the nonculturable state and were used for the resuscitation experiments. All experiments were repeated three times; mean c.f.u. ml-1 values were calculated and used for graphical representations. Respiring cells were monitored by the CTC (5-cyano-2,3-ditolyltetrazolium chloride; Polysciences Europe, Eppeilheim, Germany) reduction method (Rodriguez et al., 1992). Counts were performed using an Olympus BH2 epifluorescence microscope (x1000 final magnification). In all cases, cells were counted in at least 10 random fields. Total bacterial counts and discrimination between high (HNA) and low (LNA) nucleic acid content were performed by flow cytometry after single staining with SYBR Green II (Molecular Probes). Cell membrane integrity was assessed by using the nucleic acid double-staining procedure (NADS) of Grégori et al. (2001)
based on the permanent SYBR Green II and the impermanent propidium iodide (PI) fluorescent probes.
All samples stained by the single or double staining procedure were analysed with a Cytoron Absolute (Ortho Diagnostic Systems, Laboratoire d'Océanologie et de Biogéochimie de Marseille) flow cytometer equipped with an air-cooled argon laser emitting 15 mW of blue light at 488 nm and with distilled water as sheath fluid (Elga). Each cell was characterized by five optical parameters: two scatter parameters, namely forward-angle scatter (related to the particle size) and right-angle scatter (related to the cell structure), and three fluorescence parameters measuring fluorescence emission in the red (>620 nm), orange (565592 nm) and green (515530 nm) wavelength ranges. The sample was inserted into the instrument and injected into the measuring chamber by means of a microsyringe controlled by a stepping motor, to provide accurate control of the analysed volume. Data were collected and stored in list mode with the Immunocount software (Ortho Diagnostic Systems) and further analysed with Winlist software (Verity Software, USA). This software provides the cell concentrations (as cells mm-3) of the resolved population.
In vitro resuscitation assays.
Three independent resuscitation experiments were initiated 48 h after the culturable cell numbers reached a level below 0·1 c.f.u. ml-1. The cells stressed at 5 °C were up-shifted to room temperature (23 °C) without shaking. Experiments were carried out in a final volume of 30 ml, sampled from the microcosms and introduced into 250 ml sterile Erlenmeyer flasks with or without chloramphenicol (100 µg ml-1). In a parallel experiment, unstressed bacteria collected in the exponential phase and diluted in nutrient-poor filtered sterilized seawater to about 200 c.f.u. ml-1 were supplemented with 108 dead cells ml-1 and then incubated at 23 °C without shaking. Samples were collected at designated times for plate counts; the mean values of triplicate samples from triplicate experiments were used for graphical representations.
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RESULTS |
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DISCUSSION |
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Culturable cells
The behaviour of A. hydrophila, like that of other bacteria, depends on the cells' history and their ability to respond to environmental changes. In agreement with reports on all other tested bacteria (Kolter et al., 1993), we observed that cells collected in the stationary growth phase were more resistant to various stresses than those collected in the exponential growth phase. However, in sterile seawater at 23 °C, when cells were able to proceed with de novo protein synthesis, cells from the exponential growth phase appeared more resistant over the long term. The low level of nutrients supplemented by seawater and damaged bacteria (Nioh & Furusaka, 1968
; Dukan et al., 1997
) might contribute to a slow de novo protein synthesis that enables bacteria to become more resistant after a few days in sterile seawater. Indeed, when de novo protein synthesis was prevented by chloramphenicol addition, no cell resistance was observed. These results are at odds with the report of Mary et al. (2002)
, who investigated the behaviour of A. hydrophila (ATCC 7966) in fresh sterile water: these authors reported that growth phases had no effect on bacterial survival at 25 or 4 °C. These different results might be explained by the genetic background of strain ATCC 7966.
It is noteworthy that the behaviour of the freshwater bacterium A. hydrophila in nutrient-poor filtered sterilized distilled water is similar to that of the seawater bacterium V. vulnificus in nutrient-poor sterilized seawater (Oliver et al., 1995; Kersters et al., 1996
). At 25 °C under these conditions, A. hydrophila and V. vulnificus both maintained culturability, and at 5 °C, both species became viable but non-culturable after a few weeks.
The nature of the subpopulation which was still active in the environment and which could be recovered by temperature increase has not been identified. Interestingly, we observed that the LNA subpopulation of A. hydrophila was linked to damaged cells (assay of membrane integrity, this study) and to the subsequent loss of culturability (Grégori et al., 2001). In natural samples from aquatic environments, both subpopulations (HNA, LNA) were often observed (Gasol et al., 1999
; Lebaron et al., 2001
) and were interpreted as corresponding to the active and viable cells (HNA) on the one hand, and inactive cells (LNA) on the other. Our results on cultures of a single bacterial species would support these conclusions related to samples from natural aquatic environments.
VBNC bacteria
A. hydrophila became VBNC at both 5 and 23 °C, in response to marine stress conditions. However, our results indicated that VBNC cells lost detectable respiratory activity before they lost membrane integrity, and this loss occurred more rapidly at 23 °C than at 5 °C. Gram-negative bacteria, such as V. cholerae, V. vulnificus and Escherichia coli, have been reported to enter a VBNC state from which they are able to return to a culturable state (Colwell, 2000; Huq et al., 2000
; Oliver, 2000
). However, the concept of the VBNC state as a programmed and adaptive response to nutrient starvation has been controversial (Kell et al., 1998
), and another model suggests that cells become nonculturable due to cellular deterioration and, consequently, are moribund (Dukan & Nyström, 1999
; Bogosian & Bourneuf, 2001
; Desnues et al., 2003
). To date, the best-studied bacterium has been V. vulnificus (Oliver et al., 1995
; Whitesides & Oliver, 1997
) and it has been shown that the switch between VBNC and culturable states was in fact due to the formation of a hydrogen-peroxide-sensitive bacterial subpopulation (Bogosian et al., 2000
). In the presence of catalase, up to 1000-fold higher culturable cells were detected. Interestingly, in our study, this was not the case: we observed no difference with and without catalase. This result was partly due to our experimental conditions, which probably produce a microaerophilic environment around the sampled cells and provide a degree of protection against oxidative stress. However, after the temperature increased from 5 to 23 °C, stressed cells in sterile seawater grew at a significantly higher rate than unstressed cells mixed with the same initial amount of autoclaved cells. We suggest that in addition to culturable A. hydrophila cells that grew after the temperature increase, some damaged cells recovered from injuries that occurred during incubation in sterile seawater. This result raises questions about the properties of the cells responsible for the claimed resuscitation. Our flow cytometry and microscopic analysis revealed that not all the cells from the same initial A. hydrophila population will have the same fate under starvation-survival conditions in natural seawater. Successive loss of growth ability, respiratory activities and membrane integrity were observed (Fig. 1
), leading to the existence of several categories of VBNC cells. We do not know which categories of cells were responsible for the reported resuscitation; it could have been either of the two active subpopulations observed, while the latter are certainly in a state close to death. Our results shed new light on the fate of A. hydrophila in seawater and will be of interest to those concerned with the survival of pathogens in the marine environment.
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Received 10 July 2003;
revised 14 October 2003;
accepted 20 October 2003.