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Applied and Environmental Microbiology, September 1999, p. 3896-3900, Vol. 65, No. 9
Danish Institute for Fisheries Research,
Received 6 April 1999/Accepted 21 June 1999
The marine bacterium Shewanella algae, which was
identified as the cause of human cases of bacteremia and ear infections
in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the
water temperature was above 13°C. The bacterium is a typical
mesophilic organism, and model experiments were conducted to elucidate
the fate of the organism under cold and nutrient-limited conditions.
The culturable count of S. algae decreased rapidly from
107 CFU/ml to 101 CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C)
seawater. In contrast, the culturable count of cells exposed to warmer
seawater (10 to 25°C) remained constant. Allowing the bacterium a
transition period in seawater at 20°C before exposure to the 2°C
seawater resulted in 100% survival over a period of 1 to 2 months. The
cold protection offered by this transition (starvation) probably
explains the ability of the organism to persist in Danish seawater
despite very low (0 to 1°C) winter water temperatures. The culturable
counts of samples kept at 2°C increased to 105 to
107 CFU/ml at room temperature. Most probable number
analysis showed this result to be due to regrowth rather than
resuscitation. It was hypothesized that S. algae would
survive cold exposure better if in the biofilm state; however,
culturable counts from S. algae biofilms decreased as
rapidly as did counts of planktonic cells.
Shewanella algae is a
mesophilic marine bacterium and is a recently defined species closely
related to the more psychrotolerant Shewanella putrefaciens
(13). Strains of S. algae probably play an
important role in the environment, e.g., in the turnover of inorganic
material, since the organism is capable of reducing Fe(III) in
anaerobic respiration (4, 36). S. algae may cause disease in humans (11, 19, 29); it has been implicated in cases of ear and wound infections, bacteremia, and sepsis. Strains identified as S. putrefaciens have been isolated from a
number of clinical cases; however, the isolates may have been
misidentified and may rightly belong to S. algae
(13). By traditional phenotypic characterization methods,
including API 20NE, S. algae is identified as S. putrefaciens. S. algae can be differentiated from S. putrefaciens by its ability to grow at 42°C and in 10% NaCl and
its inability to grow at 4°C. Also, its G+C content is 52 to 55%;
that of S. putrefaciens is 43 to 47% (13).
Since S. algae was originally isolated from the marine
environment, it has been suggested that this environment is the source of the organism in diseased humans (11, 14); patients have often reported contact with seawater before infection. In support of
this hypothesis, no significant difference has been found between clinical and water isolates of S. algae, as assessed by
whole-cell protein profiling, ribotyping, and randomly amplified
polymorphic DNA analysis (14). S. algae is a
typical mesophilic bacterium and does not normally grow at temperatures
below 5°C. Infections by mesophilic marine bacteria are not common in
Denmark, but the unusually warm summer in 1994 resulted in S. algae infections (11, 19) as well as infections caused
by other mesophilic marine bacteria, such as Vibrio
vulnificus (7).
Because S. algae occurs in marine waters and is a mesophilic
organism, it must survive many months of low water temperatures during
winter. The organism must also be able to cope with extended periods of
low nutrient and energy availability. Many bacteria undergo a so-called
starvation survival response under oligotrophic conditions
(27). Several starvation proteins are expressed (15, 33), and these proteins may offer cross-protection against other stress factors, such as low temperatures. Thus, the mesophilic marine
bacterium V. vulnificus remains culturable for a
significantly longer period if starved before exposure to low
temperatures (35).
While the starvation survival response is widely recognized as a
mechanism of survival, more controversy exists with regard to the
viable but nonculturable (VBNC) concept (25). In this state,
which is stress induced, the bacteria are unable to grow on laboratory
media but remain alive, as visualized by DNA staining procedures. When
the stressful conditions are reversed, the bacteria regain the ability
to grow on laboratory media. Much debate exists as to whether this
behavior is due to true resuscitation of the nonculturable bacteria
from a dormant state (31, 40) or whether the VBNC cells are
indeed moribund and "resuscitation" is a result of regrowth of a
few surviving cells (12, 23, 39).
The ecology of S. algae in the marine environment has not
been studied before, and the purpose of the present study was to determine the occurrence of S. algae in Danish seawater. As
expected, S. algae was not detected during the colder
months. Model experiments were therefore conducted to study the fate of
the organism under cold and nutrient-limited conditions. It has been
suggested that adhesion to surfaces offers another survival strategy
for starved bacteria in the aquatic environment (8). Also,
bacteria that adhere and form biofilms are in general more resistant to
adverse conditions (6). We therefore also investigated the
survival in seawater of S. algae in the biofilm state.
Collection and investigation of samples.
Ten 200-ml water
samples were collected every second month from September 1996 to
October 1997 at Køge Bugt, approximately 20 km south of Copenhagen.
The seawater temperature was measured with a digital thermometer (Testo
110; Testo GmbH & Co., Lenzkirch, Germany). Since no specific method
exists for the enumeration of S. algae, water samples were
plated in a medium used for detection of hydrogen sulfide production.
Strains were randomly isolated from black (H2S-producing)
colonies and identified (see below). Seawater samples were serially
diluted in physiological saline, and viable counts were estimated by
pour plating in iron agar (Oxoid CM964 [16]).
Thiosulfate, L-cysteine, and ferric citrate were added to
the agar, and hydrogen sulfide-producing bacteria appeared as black
colonies in the agar due to the precipitation of FeS. Two sets of
plates were prepared from each sample; one set was incubated at 4°C
for 14 days to enumerate S. putrefaciens, and one set was
incubated at 37°C for 2 days to enumerate S. algae. The
medium contains no selective agent, so other hydrogen sulfide producers, e.g., Vibrio spp., will also form black colonies.
Isolation and identification of S. algae.
From each of
the 10 samples, four black colonies were isolated from iron agar plates
at 4 and 37°C, yielding a total of 80 H2S-producing
bacteria per sampling point. The isolates were pure cultured on iron
agar plates and tested for glucose metabolism in Hugh-Leifson medium
(19). Fermentative strains were likely to be members of the
Vibrionaceae or the Enterobacteriaceae. Strains which were not fermentative were further identified by testing for the
Gram stain reaction in 3% KOH (17), the oxidase reaction (24), shape, and motility. Strains reducing trimethylamine
oxide and producing H2S were classified as
Shewanella spp. (16). Differentiation between
S. algae and S. putrefaciens was done by testing
for growth in 6 and 10% NaCl at 25°C and at 4 and 42°C (with 0.5%
NaCl) as described by Fonnesbech Vogel et al. (13). Most of
the strains isolated from plates incubated at 37°C were tested for
G+C content (13, 26). A number of strains (September 1997)
could not be directly differentiated by their temperature or NaCl
growth profile and were further tested with the API 20NE
(bioMérieux, Marcy l'Etoile, France).
Survival of S. algae planktonic cells in
seawater.
A strain of S. algae isolated from a patient
with bacteremia (11) was grown in tryptone soya broth (TSB;
Oxoid CM129) at 37 or 20°C for 2 days. Cells from 10 ml were
harvested (5,000 × g for 10 min) and resuspended in 1 ml of phosphate-buffered saline (PBS). Seawater was collected at Køge
Bugt and left in the refrigerator for 3 to 4 days before being
autoclaved in portions of 200 ml. After being cooled to an appropriate
temperature (2, 10, or 25°C), the seawater was inoculated with an
S. algae suspension, yielding an initial cell density of
approximately 107 CFU/ml. Samples were withdrawn weekly for
the determination of culturable counts by spread plating on iron agar.
In samples with low viable counts, total counts were estimated by
4',6'-diamidino-2-phenylindole (DAPI) staining. Briefly, samples were
filtered through black 0.2-µm-pore-size polycarbonate filters
(MicronKlear K02BP02500; MSI) supported by Whatman GF/C glass
microfiber filters (Whatman, Maidstone, United Kingdom) and stained
with 10 µg of DAPI solution per ml. Filters were mounted with
paraffin oil, and counts were determined by use of an Olympus BH2
fluorescence microscope with a 320- to 400-nm excitation filter and a
>420-nm barrier filter. In one experiment, seawater inoculated with
S. algae was kept at 20°C for 2 weeks before transfer and
exposure to 2°C.
Resuscitation of S. algae planktonic cells.
S.
algae cells having been exposed to a low temperature were allowed
to resuscitate by transfer to 25°C. The contents of flasks in which
the counts had decreased 5 log units or more were serially diluted in
2°C cold seawater. The flasks and the dilution series were kept at
25°C for 2 days. Culturable counts in the flasks and in each dilution
were then determined by serial dilution and surface plating on iron
agar. By the initial dilution in cold seawater, an attempt was made to
avoid the nutrient and temperature shock that cells would experience if
plated directly on iron agar. If a large fraction of the population
were able to resuscitate, then culturable bacteria should be found in
several of the higher dilutions.
Survival of S. algae biofilm cells in seawater.
Stainless steel plates (1 by 2 cm2) were thoroughly cleaned
(with detergent and acetone) and placed in a holder ensuring a vertical
position. The holder was placed in a beaker and sterilized. The plates
were covered with 150 ml of TSB, and the medium was inoculated with
S. algae precultured for 24 h in TSB at 20°C. A
magnetic stirrer ensured circulation at 150 rpm. The plates were
incubated for 4 days at room temperature. After being washed for 2 min
in PBS, the plates were individually transferred to tubes with 4 ml of
sterile seawater tempered at either 2 or 20°C. Each week, plates were
removed for enumeration of cells by use of an indirect conductance
measurement with a Malthus instrument (22). Briefly, the
stainless steel plates with bacterial biofilms were placed in tubes
with 3 ml of TSB. Electrodes were places in an inner tube filled with
0.5 ml of 0.1 N NaOH, and the tubes were incubated in a Malthus water
bath at 25°C. CO2 evolving from the respiring cells was
absorbed by the NaOH solution, and this neutralization caused a
decrease in conductance. The time taken from the start of the
measurement until a significant change occurred (detection time) was
inversely correlated with the initial number of bacteria. A standard
curve relating the detection time to colony counts was prepared from a
10-fold serial dilution of 24-h S. algae cells in solution
from a biofilm experiment. Biofilm plates were sampled in duplicate.
Occurrence of S. algae in Danish seawater.
The
water temperature at Køge Bugt varied from
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Occurrence of Shewanella algae in Danish
Coastal Water and Effects of Water Temperature and Culture
Conditions on Its Survival
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.4°C in January 1997 to 19.5°C in August 1997 (Fig. 1). Total counts on iron agar plates
incubated at 4°C were about 103 CFU/ml (from 70 to 5,200 CFU/ml), of which approximately 10% were H2S producing
(Fig. 1). The total counts at 37°C were
not significantly different from the counts at 4°C, and the number of
H2S-producing organisms was approximately 1 log unit lower. The lowest counts were observed when the water temperature was between
0 and 4°C.
View larger version (26K):
[in a new window]
FIG. 1.
Changes in water temperature and bacterial levels in
waters of Køge Bugt from September 1996 to October 1997. TVC, total
viable count on iron agar; H2S, hydrogen sulfide producers
on iron agar.
TABLE 1.
Characterization of nonfermentative hydrogen
sulfide-producing bacteria isolated from Danish seawater in 1996 to 1997a
Survival and resuscitation of planktonic S. algae in seawater. The culturable count of S. algae remained almost constant when cells were exposed to seawater at 10 to 25°C (Fig. 2). The cells changed from a rod shape of approximately 1 by 3 µm to a smaller coccal shape, as assessed by phase-contrast microscopy (data not shown). At 2°C, the culturable count decreased from 107 CFU/ml to 101 to 102 CFU/ml in 4 to 5 weeks (Fig. 2 and 3). Even when the count decreased below 102 CFU/ml, cells at a level of approximately 105 CFU/ml could still be detected by DAPI staining (data not shown). When S. algae cells were exposed to seawater at 20°C before transfer to 2°C, the culturable count remained at 107 CFU/ml for more than 6 weeks (Fig. 3).
|
),
10°C (
), or 25°C (
). Arrows indicate counts below the
detection limit of 10 CFU/ml.
|
and 
, S. algae cultured at 20°C; 
, S. algae cultured at 37°C; Survival of biofilm S. algae in seawater. S. algae readily formed biofilms on stainless steel when grown in TSB. Based on a standard curve relating detection times to colony counts, the number of cells on the plates after 4 days was estimated to be 1.5 × 107 CFU, corresponding to 4 × 106 CFU/cm2. The detection time increased rapidly when plates were exposed to 2°C (Fig. 4), indicating a decrease in the culturable count that was even more rapid than when planktonic cells were exposed to a low temperature. Exposure of biofilms to 25°C caused only a minor reduction in the culturable count, as measured by the detection time (Fig. 4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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S. algae could be readily isolated from Danish coastal waters, and its presence correlated with water temperature in that the organism could not be detected during cold winter and spring months (Fig. 1 and Table 1). Similar findings have been reported for other mesophilic marine bacteria, such as V. vulnificus (18, 28, 32) and Vibrio parahaemolyticus (2, 9), for which the levels in seawater correlate with water temperature. Our study and the study by Høi et al. (18) both assessed the occurrence of mesophilic marine bacteria in Danish waters. Other studies have also evaluated the presence of vibrios in waters with a large yearly temperature fluctuation, such as the Elbe River in Hamburg (2) and Dutch coastal waters (38). Although the winter water temperatures in the Danish, German, and Dutch studies are significantly lower (approximately 0°C) than the lowest temperatures reported in similar incidence studies (9, 28, 32), the levels of approximately 10 to 100 CFU/ml reached during the warm months are equivalent to the levels reached in environments in which the minimum temperatures are significantly higher. These results could indicate that nutrient availability rather than minimum water temperature is important in determining maximum numbers.
The isolation of S. putrefaciens during the cold winter months from plates incubated at 37°C confirmed earlier findings (13) that within this heterogenous psychrotolerant group, strains tolerating the highest temperature (37°C) and a NaCl concentration of 6% have higher G+C contents (46 to 48% versus 43 to 44%) than strains not growing at high temperatures, e.g., strains isolated from spoiling iced fish (13).
In a study of S. algae BrY, a size reduction from 2.2 to 1 µm over 5 to 9 weeks was seen when the bacterium was exposed to sterile PBS at room temperature (5). We similarly found that the exposure of S. algae to sterile seawater led to a size reduction. Such a response to starvation has been found for many other bacteria (21, 27). The exposure of S. algae to sterile seawater resulted in two separate culturable count curves, depending on temperature (Fig. 2). These findings are similar to the results obtained for V. vulnificus, for which culturable counts remained almost constant during starvation at room temperature but decreased rapidly (in 5 to 15 days) when cells were exposed to cold water (12, 39). In other studies, V. vulnificus biotype 2 (1) and V. parahaemolyticus (21) behaved in a similar manner, although the decrease in culturable counts at a low temperature was somewhat slower. Rapid (within hours) temperature downshifts are rarely encountered by bacteria in nature, where they pass through slower temperature changes. Mimicking this situation by allowing S. algae a transition period in 20°C seawater before cold exposure markedly increased its ability to remain culturable (Fig. 3). Also, V. vulnificus remained culturable at a low temperature for longer periods when starved before cold exposure (35, 39). Despite the starvation periods that S. algae must encounter in nature, the culturable count decreased in natural seawater (Fig. 1). Other factors, such as grazing or phage attack, may limit or reduce the population in nature.
Bacteria in the marine environment are associated mostly with surfaces rather than being in the planktonic state. When grown in TSB, S. algae formed a multilayered biofilm on stainless steel surfaces in just a few days. This result was expected, as the organism in other niches is part of biofilm communities, e.g., in activated sludge (5). A crude oil bacterium, "Pseudomonas sp." (strain 200) (30), later identified as S. putrefaciens (10), did not form any significant biofilm on stainless steel coupons in 2 weeks, but a thick bacterial layer was seen after 9 weeks. Thick fibrous exopolysaccharide material entrapped the cells (30). We have seen that S. algae will form a surface layer of slimy growth 1 to 2 mm thick after only 3 to 4 days in nutrient broth at room temperature, indicating that exopolysaccharides required for biofilm formation are indeed readily produced. Since bacteria forming a biofilm are believed to be more resistant to a number of adverse conditions (6), we hypothesized that surface-adherent S. algae would remain culturable at a low temperature for longer periods than planktonic cells. However, our results (Fig. 4) did not support this hypothesis. The Malthus method proved suitable for estimating culturable counts of bacteria adhering to surfaces. However, prolonged lag phases will, due to conversion via a standard curve, result in lower culturable count equivalents than are actually present. This factor should be kept in mind when one is attempting to translate detection times to culturable cells.
The culturable count of mesophilic bacterial cells generally decreases when the cells are exposed to adverse conditions, such as cold temperature, and increases when conditions become favorable, e.g., the temperature is raised (12, 39, 40). Some authors believe that this response is due to true resuscitation of the VBNC part of the population (21, 37, 40), whereas others have found that the phenomenon is due to regrowth of a few surviving cells (3, 12). As pointed out by Kell et al. (23), the majority of studies reporting resuscitation have not eliminated the possibility of regrowth by using an appropriate experimental design. Using a simple most-probable-number-based approach (39), we found that the return of S. algae to culturability was more likely caused by regrowth of a few surviving cells than by resuscitation.
In conclusion, S. algae behaves similarly to other mesophilic marine bacteria when exposed to oligotrophic conditions. The increase in the culturable count when cold, "nonculturable" cells are returned to a warm temperature is likely to be explained by regrowth rather than resuscitation. The occurrence in Danish waters of S. algae is therefore probably due to the prolonged survival in cold water of starved cells at very low levels and subsequent regrowth when the water temperature increases. We could not confirm our hypothesis that biofilm cells are more resistant to cold oligotrophic conditions than planktonic cells.
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ACKNOWLEDGMENTS |
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This work was partly financed by the Danish Food Technology Programme (FØTEK II).
The assistance of Lars Ravn and Christiane Buch is appreciated.
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FOOTNOTES |
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* Corresponding author. Mailing address: Danish Institute for Fisheries Research, Department of Seafood Research, Technical University of Denmark, Bldg. 221, DK-2800 Lyngby, Denmark. Phone: 45 4525 2586. Fax: 45 4588 4774. E-mail: gram{at}dfu.min.dk.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 1999, p. 3896-3900, Vol. 65, No. 9
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