Temperature and growth-phase effects on Aeromonas hydrophila survival in natural seawater microcosms: role of protein synthesis and nucleic acid content on viable but temporarily nonculturable response

Sami Maalej1, Michel Denis2 and Sam Dukan3,{dagger}

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The behaviour of Aeromonas hydrophila in nutrient-poor filter-sterilized seawater was investigated at 23 and 5 °C with respect to its growth phase. At both temperatures, the culturable A. hydrophila population declined below the detection level (0·1 c.f.u. ml-1) after 3–5 weeks, depending on the initial physiological state of the cells. During the first week, starved A. hydrophila cells appeared more resistant to the seawater stress at 5 °C than cells initially in the exponential growth phase. This difference was not observed at 23 °C, where de novo protein synthesis seemed to be required for long-term adaptation of cells from the exponential growth phase. Over the duration of the experiments, intact and total cell concentrations were not significantly affected, indicating that bacteria had entered a so-called viable but nonculturable state (VBNC). However, the incubated bacteria rapidly became heterogeneous with respect to their nucleic acid content, and their cell size decreased faster at 23 than at 5 °C. Resuscitation of VBNC cells was attempted by a temperature shift from 5 to 23 °C without exogenous nutrient addition. Comparison of the growth rates of the stressed population and of the untreated bacteria growing in the same autoclaved initial cell suspension showed significantly faster growth for the stressed cells, suggesting that in addition to growth of the few culturable stressed cells, a proportion of injured cells became culturable.


Abbreviations: CTC, 5-cyano-2,3-ditolyltetrazolium chloride; HNA, high nucleic acid; LNA, low nucleic acid; NA, nucleic acid; NADS, nucleic acid double staining; PI, propidium iodide; VBNC, viable but non-culturable

{dagger}Present address: Laboratoire de Chimie Bactérienne IBSM, CNRS UPR 9043, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aeromonas hydrophila is an opportunist human pathogen which is widely distributed in aquatic environments (Janda & Abbott, 1998; Schiavano et al., 1998; Rahman et al., 2001). A relationship between changes in water temperature and the incidence of Aeromonas spp. has been reported. In fresh water or seawater within temperate latitudes, aeromonads were found in high numbers in late summer/early autumn when the temperature was around 20–25 °C and were rarely detected during cold seasons (Kersters et al., 1995; Gavriel et al., 1998). In arid regions, the highest densities of aeromonads in fresh water were observed during winter (Maalej et al., 2003; Hassani et al., 1992), probably because of the relatively mild winter and especially hot summer seasons.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strain and culture conditions.
Aeromonas hydrophila strain AH1, used in this study, was originally isolated in the University Hospital of Sfax (Tunisia). It was identified as A. hydrophila with the standard battery of biochemical tests (Baumann & Schubert, 1984), confirmed by using the API 20E system (Analytab product). An overnight culture growing at 37 °C on a 200 r.p.m. shaker in 5 ml Luria–Bertani (LB) broth (Maniatis et al., 1982) supplemented with ampicillin (35 µg ml-1) was used to inoculate 45 ml of the same medium. Cultures in exponential (OD600 2), late exponential (OD600 5) and stationary (OD600 7) phases were harvested by centrifugation (5000 g, 10 min, 4 °C); pellets were washed twice in 0·85 % (w/v) sterile NaCl solution and then resuspended in the same volume of filter-sterilized seawater.

Seawater microcosms.
Seawater (salinity 36·5 {per thousand}; 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 (38–42 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 (565–592 nm) and green (515–530 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Behaviour of A. hydrophila incubated in sterile seawater at 23 and 5 °C
As shown in Fig. 1, incubation in sterile seawater at 23 and 5 °C of A. hydrophila cells collected at the end of the exponential growth phase resulted in a rapid decline of culturable cells. However, during the first week, bacteria incubated at 5 °C were more resistant in terms of culturable cells. Over the experiment, there was no significant decline in the total bacterial counts as determined by flow cytometry. In addition, cells with intact membranes, identified with the NADS protocol of Grégori et al. (2001) (see Methods), decreased by less than 10-fold. Respiring cells, detected by CTC, exhibited a decrease of 100-fold over the whole experiment (Fig. 1). As illustrated by Fig. 2(a), cell size decreased more when bacteria were incubated at 23 °C (53 %) than at 5 °C (41 %). Flow cytometric analysis of the incubated suspensions also revealed marked heterogeneity in nucleic acid content: as illustrated in Fig. 2(b), the initial suspension, which had a rather homogeneous nucleic acid content at day 0, rapidly developed variable nucleic acid content identified as high (HNA) and low (LNA), as shown for day 3, with higher mean values at 5 °C than 23 °C. The cytogram of the suspension at 23 °C on day 3 also shows the pronounced trend of the bacterial population towards smaller size cells. It is noteworthy that the LNA population was associated with a loss of membrane integrity (double staining assay, data not shown) and a concomitant loss of culturability (Grégori et al., 2001).



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Fig. 1. Behaviour of A. hydrophila AH1 in nutrient-poor filtered sterilized seawater during the first week of incubation at 23 °C (a) and 5 °C (b): total bacterial counts, determined by flow cytometry after staining with SYBR Green II ({blacksquare}); cells with an intact membrane determined by the NADS protocol ({blacktriangleup}); respiring cells detected by the CTC method ({triangleup}); and culturable cells (c.f.u.) on LB agar plates ({square}). Each point represents the mean of two determinations and three independent experiments (SD < 15 % of the mean).

 


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Fig. 2. Effect of incubation of A. hydrophila AH1 (in the exponential growth phase) in nutrient-poor filtered sterilized seawater at 23 °C and 5 °C. (a) Effect on cell size ({blacklozenge}, 23 °C; {blacksquare}, 5 °C). (b) Effect on cell nucleic acid content. a.u., arbitrary unit.

 
Growth-phase and temperature effects on the behaviour of A. hydrophila in sterile seawater
Gram-negative bacteria respond to starvation by developing increased resistance to a variety of environmental stresses such as osmotic stress, oxidation or temperature variation (Lange & Hengge-Aronis, 1991; Kjelleberg et al., 1993; Nyström, 1994; Dukan & Touati, 1996). We wondered if starved A. hydrophila were more resistant in sterile seawater at 5 than at 23 °C. As shown in Fig. 3(a), incubation of A. hydrophila in sterile seawater at 23 °C induced the fastest decline in culturable cells when bacteria were collected from the stationary phase. Bacteria collected in the exponential growth phase followed the same decline during the first 3 days, then appeared more resistant (Fig. 3a). However, when bacteria were incubated at 5 °C, cells collected in the stationary phase were the more resistant over 3 weeks (Fig. 3b). The initial growth phase or the incubation temperature (data not shown) did not affect the time-courses of viable cell concentration and total cell count.



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Fig. 3. Effects of growth phase on the behaviour of A. hydrophila AH1 in nutrient-poor filtered sterilized seawater at 23 °C (a) and 5 °C (b). Cells were collected in the exponential ({blacktriangleup}), late exponential ({blacksquare}) and stationary ({bullet}) growth phases. Each point represents the mean of two determinations and three independent experiments (SD < 15 % of the mean).

 
Effect of de novo protein synthesis on the loss of culturability at 5 and 23 °C
To test if differences in A. hydrophila survival at 5 and 23 °C were due to de novo protein synthesis, cells in the exponential growth phase were incubated in the presence of chloramphenicol (100 µg ml-1) in order to prevent potential de novo synthesis. At 23 °C, cells incubated in the presence of chloramphenicol lost resistance to sterile seawater, indicating a requirement for de novo protein synthesis for inducing the adaptation to stress (Fig. 4a). The loss of resistance was also observed when chloamphenicol was added after 2 days of incubation (Fig. 4a), indicating that de novo synthesis was required throughout sterile seawater incubation. Interestingly, at 5 °C, incubation in the presence of chloramphenicol had no effect (Fig. 4b).



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Fig. 4. Effects of chloramphenicol on survival of A. hydrophila AH1 in nutrient-poor filtered sterilized seawater at 23 °C (a) and 5 °C (b). The graphs show culturable cell concentration without chloramphenicol ({bullet}), with chloramphenicol added immediately after inoculation ({blacktriangleup}) and with chloramphenicol added after 2 days ({triangleup}). {blacksquare}, Total bacterial counts in the presence and in the absence of chloramphenicol (results not significantly different for these conditions). Each point represents the mean of three independent experiments (SD < 5 % of the mean).

 
Resuscitation experiments and effects of catalase
Samples (30 ml) were taken from seawater microcosms maintained at 5 °C and containing less than 0·1 c.f.u. ml-1, and shifted to room temperature (23 °C). Culturable cells first appeared after 1 day, then increased to a maximum of 104 c.f.u. ml-1 within 3 days of the temperature shift. When chloramphenicol (100 µg ml-1) was added to the microcosm, the recovery of culturability was completely abolished (Fig. 5a). A comparison of the growth rate of stressed bacteria (µ=0·43 h-1) with those of untreated cells diluted in sterile seawater and supplemented with 108 dead cells ml-1 revealed that the latter population had a lower growth rate (µ=0·25 h-1) (Fig. 5b). However, by using classical methods, we were not able to recover culturable cells from bacteria incubated in seawater. Addition of catalase (100 U ml-1) to LB did not lead to any significant increases in culturable cells for microcosms stored at 5 or 23 °C at any sampling time during the experiments.



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Fig. 5. Growth in nutrient-poor filtered sterilized seawater of A. hydrophila AH1 after a temperature shift from 5 to 23 °C. (a) A 30 ml sample from microcosm at 5 °C with less than 0·1 c.f.u. ml-1 was shifted to 23 °C, and then culturable cells were determined in the presence ({lozenge}) or absence ({blacklozenge}) of 100 µg chloramphenicol ml-1. (b) Unstressed bacteria 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. Culturable cells on LB agar were determined for the assay ({blacklozenge}) and for the control, not supplemented with dead cells ({lozenge}). Each point represents the mean of two determinations and three independent experiments (SD < 15 % of the mean).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have shown that A. hydrophila cells, collected in the exponential or stationary growth phase and incubated at 5 or 23 °C in sterile seawater, entered a VBNC state. Temperature and physiological state affected bacterial behaviour. The apparent recovery of culturability without nutrient addition was largely due to the development of a few culturable cells at the expense of the damaged ones.

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.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 10 July 2003; revised 14 October 2003; accepted 20 October 2003.