AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eiler, A.
Right arrow Articles by Bertilsson, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eiler, A.
Right arrow Articles by Bertilsson, S.
Agricola
Right arrow Articles by Eiler, A.
Right arrow Articles by Bertilsson, S.
Applied and Environmental Microbiology, September 2006, p. 6004-6011, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00917-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas){dagger}

Alexander Eiler, Mona Johansson, and Stefan Bertilsson*

Limnology/Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Norbyv. 20, SE-75236 Uppsala, Sweden

Received 18 April 2006/ Accepted 4 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Even if many Vibrio spp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulating Vibrio populations in a salinity gradient along the Swedish coastline, we combined Vibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The total Vibrio abundance ranged from 4 x 103 to 9.6 x 104 cells liter–1, with the highest abundances in the more saline waters of the Skagerrak Sea. Several Vibrio populations were present throughout the salinity gradient, with abundances of single populations ranging from 5 x 102 to 7 x 104 cells liter–1. Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives of V. anguillarum and V. aestuarianus genotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genus Vibrio may also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. A V. cholerae/V. mimicus population was detected in more than 50% of the samples, with abundances exceeding 103 cells liter–1, even in the cold (annual average water temperature of around 5°C) and low-salinity (2 to 4{per thousand}) samples from the Bothnian Bay (latitude, 65°N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potential Vibrio pathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The genus Vibrio is widespread in coastal waters and is comprised of more than 63 species (44). The most well-known and studied species is V. cholerae, which causes cholera epidemics worldwide. In addition to V. cholerae, many other Vibrio spp. are recognized as human pathogens. V. parahaemolyticus (34), V. vulnificus (16), V. mimicus (5), V. alginolyticus, and V. hollisae (38), for example, have been implicated in water- and seafood-related outbreaks of gastrointestinal infections in humans. There are also increasing reports of lethal wound infections linked to several different Vibrio spp. (12, 32). Vibrio-related disease is not restricted to humans; e.g., V. anguillarum and V. splendidus can be potent fish pathogens that are also capable of infecting other marine animals (2), and V. corallilyticus and V. shiloi can infect and kill corals (4, 28).

The presence of V. cholerae and other Vibrio spp. together with related diseases in tropical and temperate environments is well documented (3, 6, 14, 15, 17, 44, 45). However, reports about the occurrence and distribution of Vibrio spp. in northern temperate and boreal waters are rare (11) and restricted to single species (e.g., V. vulnificus from the Danish Coast) (17). There have even been recent cases of fatal V. cholerae non-O1, non-O139 infections in coastal areas surrounding the Baltic Sea (32). The exact sources of the infections remained unclear, but they were all linked to either contact with seawater or consumption of fish. In general, Vibrio spp. tolerate a wide range of salinities and tend to be more common in warm waters, notably when temperatures exceed 17°C (23, 26, 27, 35, 45). Also, host and vector organisms are important for the survival of Vibrio spp. (13, 14, 29). For example, V. cholerae and V. parahaemolyticus can develop biofilms on the cortex of copepods (20, 25), and V. vulnificus can be isolated from gills, intestinal contents, and mucus of fish (17). Several recent studies have corroborated that marine host and vector organisms can promote the survival of Vibrio spp. and hence influence their persistence and distribution in the environment (1, 17, 19, 20, 21, 25, 39, 40, 48).

Most studies on the occurrence of Vibrio spp. in natural environments have been based on culture-dependent techniques. It is well known that many Vibrio spp. enter viable but nonculturable growth states when exposed to low temperatures or otherwise adverse growth conditions (38, 41, 42). Cells in this state evade detection by culture-dependent techniques since they are incapable of growing on conventional media even if they maintain both viability and pathogenicity (38, 41, 42). Hence, surveys using culture-independent molecular methods are needed to fully understand the ecology and distribution of Vibrio spp. in aquatic environments. To address this, a molecular technique combining quantitative competitive PCR (QC-PCR) with denaturant gradient gel electrophoresis (DGGE) for the quantification of individual populations within the genus Vibrio was developed in our laboratory (10). This QC-PCR-DGGE assay can distinguish between closely related Vibrio populations and detect and quantify as little as 200 Vibrio cells captured on a filter.

The aim of the present study was to assess the abundance of populations within the genus Vibrio along a northern temperate and boreal coastline with a culture-independent technique and analyze the relationship between the abundance of different Vibrio populations and environmental-state variables. This was done to both survey the presence and niche separation of Vibrio populations and identify potential environmental factors influencing Vibrio survival and dispersal in coastal waters that are characterized by average annual temperatures below 10°C.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sample collection.
A total of 19 stations situated along the Swedish coastline were sampled one to three times during the summers of 2003 and 2004 (Fig. 1). On each occasion, samples were taken to measure the abundance of phytoplankton and bacteria together with chlorophyll a, salinity, oxygen, total nitrogen, phosphorus, and dissolved organic carbon. Integrated water samples (10 liters) of the upper mixed layer (20 m) were collected in the following ways. For the Skagerrak Sea, Bothnian Sea, and Bothnian Bay samples, a plastic tube (nonpyrogenic) equipped with a weight at the lower end was lowered by hand at a constant rate to 10- to 20-m depth and then sealed at the upper end before being retrieved. Since this equipment was not available on all sampling ships for the Northern Baltic proper, equal volumes of samples retrieved from five discrete depths (0, 5, 10, 15, and 20 m) were combined. For Öresund samples, a plastic tube was used to retrieve an integrated sample from the top 10 m, and this water sample was then mixed (3:1:1:1) with discrete water samples from 12-, 14-, and 20-m depths. For Central Baltic Sea samples, equal volumes of samples retrieved from seven discrete depths (0, 5, 10, 15, 20, 25, and 30 m) were combined. Samples were processed within 2 h, as described below.


Figure 1
View larger version (66K):
[in this window]
[in a new window]
  		FIG. 1. Map of Scandinavia showing the sampling sites. SK indicates Skagerrak stations A to C, Ore indicates Öresund station, CBp indicates Central Baltic proper station, NBp indicates Northern Baltic proper stations A to F, BS indicates Bothnian Sea stations A to C, and BB indicates Bothnian Bay stations A and B.

 
Physical and chemical characteristics.
Water temperature and salinity were measured on site with a YSI conductivity meter (Yellow Spring Instruments, Ohio) in the Skagerrak Sea and a with CTD profiler (Meerestechnik Elektronik GmbH, Germany) at the remaining stations, except for the Central Baltic proper, where only temperature was measured at discrete depths. For nutrient analyses, triplicate water samples (20 ml) were frozen (–20°C) in acid-washed and ultrapure Q-grade water-rinsed plastic bottles. Total organic carbon was measured in a Shimadzu TOC-5000 total organic carbon analyzer after the removal of inorganic carbon by acidifying (10 mM HCl) and purging the samples for 7 min with CO2-free air (8). Total nitrogen was measured using an Antek 9000 high-temperature-combustion total nitrogen analyzer (Antek Instruments Inc.). Total phosphorus was measured by an initial hydrolysis of the sample with potassium peroxysulfate (33), and subsequent detection was done using molybdenum blue spectrophotometry (37).

Chlorophyll a, phytoplankton, and microzooplankton.
For chlorophyll a analyses, phytoplankton from between 1 and 2 liters of water were captured on Whatman GF/F filters (0.7-µm nominal pore size) and immediately frozen at –80°C. Filters were placed in 10 ml ethanol and incubated in the dark at room temperature overnight with intermittent mixing. Extracts were cleared by centrifugation (4,000 rpm, 10 min), and chlorophyll fluorescence was measured on a Fluoromax-2 spectrofluorometer (Spex) equipped with an external microplate reader (18). Fluorescence was calibrated against an ethanol extract of geranium leaves where chlorophyll a had been determined from the absorbance at 665 nm with background correction at 750 nm (22). For identification and counting of phytoplankton and microzooplankton, 150 to 250 ml of integrated water samples was fixed with 1% acid Lugols solution and kept cold (4°C) until analyses. Subsamples (50 to 100 ml) were counted using an inverted microscope (magnification, x200 to x400) in a settling chamber (46). The entire chamber or at least 100 individuals of each species were counted. All dinoflagellates, diatoms, large flagellates (>15 µm), cyanobacteria, and ciliates were identified at the highest possible taxonomic resolution (for details, see reference 24).

Bacterial abundance.
Samples were preserved with borax-buffered and 0.2-µm-filtered formaldehyde (final concentration, 2% [vol/vol]) for analysis of total bacterial abundance by flow cytometry after Syto 13 staining of nucleic acids (7). Counts of selected samples determined microscopically after staining with DAPI (4',6'-diamidino-2-phenylindole) (8) were in excellent agreement with flow cytometry counts (slope, 0.98; R2 = 0.92; n = 9).

DNA sampling and extraction.
Bacterial cells in various volumes of seawater (0.45 to 5 liters) were captured on 0.2-µm (total bacteria) and 0.8-µm (attached bacteria) membrane filters (Supor, Gelman) using a manual vacuum pump and sterile filter manifolds. The funnel and filter holder were soaked in 10% HCl, rinsed in sterile Q-grade ultrapure water, and autoclaved in one piece prior to sampling. Filters were immediately frozen and stored in liquid nitrogen, transported to the laboratory, and transferred to –80°C for long-term storage. DNA was extracted from filters by mechanical disruption using the Ultra Clean soil DNA kit (MoBio Laboratories, Solana Beach, CA) according to the manufacturer's instructions for high DNA yield. These procedures resulted in 50-µl aliquots of high-quality DNA with an average size of ~20 kb, as determined by electrophoretic separation on a 1% agarose gel followed by ethidium bromide staining and detection by UV transillumination (10). Quantification was carried out by comparison of band intensities to a low-DNA-mass ladder (Invitrogen, Carlsbad, CA).

Vibrio identification and quantification.
Vibrio populations were identified and quantified as described elsewhere (10). Briefly, between 10 and 20 ng of environmental DNA was used as a template for PCR amplifications with primers GC567F and 680R (45). Each DNA extract was amended with 1 µl internal competitor DNA corresponding to 20 cells of a V. mimicus strain (GenBank accession number DQ068933). The amplification was performed using SureStart Taq polymerase (Stratagene, La Jolla, CA) as described elsewhere (10, 45). Sequence variants in subsamples containing 10- to 50-ng PCR products were then separated on a 45 to 70% urea-formamide DGGE gel parallel to three standard samples containing mixtures of known Vibrio isolates to enable migration-based identification of indigenous populations (10). The applied separation conditions were stringent enough to separate fragments with single nucleotide differences (10). The gels were stained with SYBR gold (Molecular Probes), and detection of bands by transillumination was carried out using a Spectronics variable-intensity UV source with a diffuser plate and a cooled, 12-bit monochrome charge-coupled-device camera with a SYBR gold filter (Coolsnap Pro CF; Media Cybernetics Inc., Silver Spring, MD). SYBR gold fluorescence of individual DNA bands was quantified and compared using the imaging and gel analysis software Gelpro Analyzer (Media Cybernetics Inc.). The abundance of each Vibrio population observed in a sample was determined from the intensity of its corresponding DGGE band using an experimentally derived titration curve of log-transformed values: log(Pi) = log(APi/AStd) x 0.604 + 3.293, where Pi is the number organisms of population i, APi is the integrated fluorescence signal of the DGGE band corresponding to population i, and AStd is the integrated fluorescence signal of the DGGE band of the internal competitor DNA (10).

Environmental clone library construction and sequence analyses.
A GC567F-680R clone library was constructed from three Skagerrak Sea samples and three Baltic Sea samples. PCR products amplified with the Vibrio-specific primer pair were gel purified and cloned into the pCR-4-TOPO vector and transformed into competent Escherichia coli One Shot TOP10 cells using the TOPO-TA cloning kit for sequencing (Invitrogen, Carlsbad, CA). A total of 20 clones from each library were selected for further analysis. These clones were cultivated in LB with kanamycin at 37°C overnight. Cells were collected by centrifugation, and crude lysates were obtained by dissolving the pellets in sterile Q-grade water and heat lysing the cells at 98°C for 10 min (9).

Inserts from individual clones were amplified with the vector primers M13f and M13r (9). PCR products were quantified by agarose gel electrophoresis and diluted to 10 ng µl–1 before direct sequencing with primer M13f (9). All sequenced inserts were compared to GenBank entries (December 2005) using BLASTN to identify Vibrio strains with identical target sequences. The combined Vibrio community usually cannot be resolved at the species level (for more discussion, see references 10 and 45). Hence, a Vibrio population is defined as all cells that share an identical sequence in the 16S rRNA gene fragment amplified by the Vibrio-specific primers.

Statistical analyses.
Statistica software (StatSoft, Tulsa, OK) was used for calculating correlation coefficients (nonparametric Spearman rank) and stepwise multiple linear regression analysis (MLR) [after testing for normal distribution and homogeneity of variances, variables were log(x + 1) transformed]. Correlations and regressions were considered significant if the P value was <0.05.

Partial least-squares regression analysis (PLS) included in the SIMCA-P+ software (version 10.0.2.0; Umetrics, Umeå, Sweden) was applied to identify environmental variables that could predict the distribution of the total Vibrio community and single Vibrio populations [variables were log(x + 1) transformed].

Nucleotide sequence accession numbers.
The sequences obtained for inserts from individual clones have been deposited in GenBank under the accession numbers DQ351691 to DQ351698.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Environmental variables.
The samples collected along the Swedish coastline represented a salinity gradient from 2 to 30{per thousand} (see Table S1 in the supplemental material). Phosphorus concentrations varied both within and between stations and ranged from 4 to 40 µg P liter–1, with the lowest levels in samples from the Bothnian Sea and Bothnian Bay. Nitrogen concentrations ranged from 0.08 to 0.26 mg N liter–1, with the highest levels in samples from the Northern Baltic proper. Water temperatures ranged from 12 to 18°C during the sampling period (June to September), and the concentration of chlorophyll a ranged from 1.8 to 6.6 µg liter–1, with the highest values in the Northern Baltic proper (see Table S1 in the supplemental material). The Skagerrak phytoplankton community was strongly dominated by dinoflagellates throughout the sampling period (up to 8 x 104 cells liter–1). Also, in Öresund and the Baltic Sea proper, dinoflagellates were the most abundant group of phytoplankton, but there were also high levels of other flagellates and an autotrophic ciliate (Myrionecta rubra) in Öresund and cyanobacteria in the Baltic Sea proper (24). Samples from the Bothnian Sea and Bothnian Bay were even more heterogeneous, with, at most, 40% dinoflagellates. Both small and large ciliates were present in all samples, but the highest abundances were found in June samples from the Skagerrak Sea and August samples from the Northern Baltic proper (>104 cells liter–1) (24). Several dinoflagellate groups were strongly correlated to total dinoflagellate abundance (e.g., Dinophysis, Gymnodinium, and Protoperidinium; P < 0.01), whereas this was not the case for either Ceratium or Prorocentrum. These two groups were most abundant in Skagerrak and also experienced marked seasonal dynamics (24).

Total Vibrio abundance.
The culture-independent QC-PCR-DGGE method detected 16S rRNA genes from bacteria of the genus Vibrio in all samples collected along the Swedish coastline. Total Vibrio abundances (captured on 0.2-µm filters) determined by QC-PCR-DGGE ranged from 4 x 103 to 9.6 x 104 cells liter–1 (Table 1), with the highest levels in samples from the Skagerrak Sea. Vibrio species contributed between 0.002 and 0.015% to the total bacterioplankton cell counts (Table 1; see Table S1 in the supplemental material). Total Vibrio abundances correlated significantly with salinity (R = 0.65; P < 0.01) and the total abundance of dinoflagellates (R = 0.68; P < 0.01) as well as with individual groups of dinoflagellates; e.g., Gymnodinium (R = 0.67; P < 0.01), Protoperidinium (R = 0.86; P < 0.01), Dinophysis (R = 0.55; P < 0.01), and Ceratium (R = 0.69; P < 0.01). A PLS model also revealed similar results, where salinity, phosphorus concentration, and dinoflagellates were important predictors of total Vibrio abundance (Fig. 2).


View this table:
[in this window]
[in a new window]
  		TABLE 1. QC-PCR-DGGE detection and quantification of Vibrio populations in Swedish coastal watersa

 

Figure 2
View larger version (43K):
[in this window]
[in a new window]
  		FIG. 2. Coefficient plot (A) and score plot (B) of a PLS model for total Vibrio abundance (R2 = 0.77 [goodness of fit]; Q2 = 0.57). Variables significant for the PLS model are indicated in boldface type (A). SK indicates Skagerrak stations A to C, Ore indicates Öresund station, CBp indicates Central Baltic proper station, NBp indicates Northern Baltic proper stations A to F, BS indicates Bothnian Sea stations A to C, and BB indicates Bothnian Bay stations A and B. The number indicates the month of sampling.

 
The abundance of attached Vibrio populations (captured on 0.8-µm filters) varied from 8 x 102 to 2.6 x 104 cells liter–1 and contributed between 10 and 100% to individual Vibrio populations (Table 1). The highest absolute abundances of attached Vibrio populations were detected in Skagerrak samples, but the ratio of particle-attached Vibrio abundance to total Vibrio abundance was highest in the Northern Baltic proper, Bothnian Bay, and Bothnian Sea (Table 1). The abundance of attached Vibrio correlated significantly with phosphorus concentration (R = 0.68; P < 0.01), salinity (R = 0.41; P < 0.05), and dinoflagellate abundance (R = 0.61; P < 0.01). MLR models also suggest that ciliate abundance, the presence of chlorophyll a, and nitrogen and phosphorus concentrations were important controlling factors (see Table S2 in the supplemental material). The ratio of attached Vibrio abundance to total Vibrio abundance was positively correlated with chlorophyll a (R = 0.62; P < 0.01).

Quantitative distribution of Vibrio populations.
In this survey, a total of 10 Vibrio populations were detected in the 28 samples analyzed. Between two and seven populations were detected for the individual samples. Cloned 16S rRNA gene sequences were obtained for eight of these populations, and each population represented a unique allele of the PCR-amplified fragment. Most populations matched database entries for more than one Vibrio species (Table 2). Samples from Northern Baltic proper typically contained fewer populations (average of 3.9) than the other four regions (average of 4.3 to 5). Individual Vibrio populations were present in abundances ranging from 1 x 102 to 7 x 104 cells liter–1. Different populations dominated the Vibrio communities in the Skagerrak Sea and the more brackish waters of the Baltic Sea. The high-salinity populations 1, 5, and 7 dominated in the Skagerrak Sea, whereas population of V. anguillarum/V. ordalii (Table 1) and population 6 dominated the Vibrio community of the brackish environments. A low abundance (300 cells liter–1) of a population characteristic for V. vulnificus was detected in a single 0.8-µm sample from the August sample at Skagerrak station C. Another potentially pathogenic population exclusive for V. cholerae and a few strains of V. mimicus was detected in more than 50% of the samples at abundances reaching 104 cells liter–1. The highest abundances were observed in the Skagerrak Sea samples, whereas this population never exceeded 5.5 x 103 cells liter–1 in the Baltic Sea. This V. cholerae population was significantly correlated with chlorophyll a (R = –0.64; P < 0.01), nitrogen concentrations (R = –0.56; P < 0.01), and Prorocentrum abundance (R = 0.69; P < 0.01). However, MLR revealed only weak predictability based on the measured parameters. The abundance of most other Vibrio populations could be predicted from the abundance of various microeukaryotes and total phosphorus and nitrogen concentrations using PLS and MLR (see Table S2 in the supplemental material).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Vibrio populations detected in the present study, GenBank accession numbers for the sequence variants of each population, and GenBank 16S rRNA entries from Vibrio isolates that perfectly match sequences of each detected populationa

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study represents a first attempt at using a culture-independent quantitative method (QC-PCR-DGGE) to determine the abundance of bacteria affiliated with the genus Vibrio as well as individual Vibrio populations in northern temperate and boreal marine and brackish waters. Vibrio populations were present in all samples throughout the natural salinity gradient sampled (2 to 30{per thousand}) and over the sampling season, suggesting that these bacteria are also a natural component of planktonic microbial communities in the cold waters of the Swedish coastline. This observation is supported by previous culture-independent studies where vibrios have been detected in more-southern temperate waters throughout a yearly cycle (11, 45). However, estimated Vibrio abundances in the present study are much lower than values reported for southern temperate waters that were based on either fluorescent in situ hybridization (11, 14) or QC-PCR combined with constant denaturant capillary electrophoresis (45). So far, these three surveys are the only studies that have employed culture-independent techniques to estimate the total Vibrio abundance in coastal waters (11, 14, 45). These studies revealed temporal variations in Vibrio abundance, with lower Vibrio abundances at low temperatures. In fact, total Vibrio abundances in winter samples from Barnegat Bay, New Jersey (45), were similar to abundances observed in the summer samples from the present study. When total Vibrio abundances fell below 105 cells liter–1, water temperatures were always less than 18°C. It is plausible that such low temperatures may limit the abundance of vibrios in coastal waters (23, 26, 27, 35, 45), but it is nevertheless clear that many populations maintain viable populations even under the conditions characteristic of the Baltic Sea, where the annual average temperature rarely exceeds 8°C.

In the present study, bacteria of the genus Vibrio were less abundant in the brackish waters of the Baltic Sea (2 to 6{per thousand} salinity). This may be due to differences in water characteristics other than salinity; MLR and PLS (Fig. 2A; see Table S2 in the supplemental material) models showed that salinity, phosphorus concentration, and the abundance of microeukaryotes such as dinoflagellates were strongly correlated to Vibrio abundance in the coastal waters studied. These results are in agreement with previous culture-dependent surveys in temperate and tropical waters that have shown strong correlations between salinity as well as nutrient concentration and the abundance of Vibrio spp. such as V. vulnificus (17, 27, 35) and V. cholerae (12). Furthermore, the strong correlation between dinoflagellate abundances and total Vibrio abundance suggests that dinoflagellates may promote Vibrio survival and dispersal in Swedish coastal waters or positively affect the growth of Vibrio populations by releasing bioavailable dissolved organic substrates. The observation of significant correlations between total Vibrio abundance and several bloom-forming dinoflagellates, e.g., Gymnodinium, Protoperidinium, Dinophysis, and Ceratium, suggest that not only V. cholerae (21, 36, 47) but also other Vibrio spp. are promoted by these phytoplankton groups.

Different Vibrio populations seem to be regulated by different environmental variables. For example, the population of V. anguillarum/V. ordalii (Table 2) was abundant in the Baltic Sea but mostly absent from the Skagerrak Sea samples (Table 1) and significantly negatively correlated with salinity (R = –0.44; P < 0.05). In contrast, high-salinity populations 1, 5, and 7 were dominant in the Skagerrak Sea samples and significantly positively correlated with salinity (R ranging from 0.58 to 0.70; P < 0.01), indicating that these populations are better adapted to higher salinities than the population of V. anguillarum/V.ordalii. With the exception of V. cholerae and V. mimicus, all Vibrio spp. are believed to require Na+ for growth. Minimum Na+ concentrations for optimal growth vary substantially between species (12). In seawater, Na+ contributes approximately 30% to the total ion concentration, and its relative contribution can be as low as 7% in some freshwaters. Thus, variations in Na+ may be even more pronounced than the observed gradient in salinity. This, combined with the observed differences in Vibrio community composition, suggests that the brackish waters in the Baltic Sea harbor Vibrio communities that are distinctly different from more saline (higher Na+ concentration) marine waters. MLR (see Table S2 in the supplemental material) and correlation analyses suggested that not only salinity but also phosphorus, nitrogen, and vector organisms influenced the various Vibrio populations in contrasting ways. The results suggest that the different Vibrio populations inhabit distinct niches in their aquatic habitat and that some Vibrio populations may, for example, be superior competitors for resources like nitrogen, phosphorus, or phytoplankton-derived dissolved organic matter. For example, the population of V. anguillarum/V. ordallii was significantly correlated with chlorophyll a (R = 0.61; P < 0.01) and cyanobacterial abundance (R = 0.55; P < 0.01), whereas the V. cholerae population was significantly correlated with Prorocentrum abundance (R = 0.69; P < 0.01). Thus, intragenus competition may be an important factor regulating the growth and persistence of pathogenic Vibrio spp. in natural aquatic environments.

A V. cholerae/V. mimicus population was detected along the entire Swedish coastline. Since no serotyping was performed, it is not known whether the detected population included strains of recognized pathogenic serogroups such as serogroups O1 and O139. V. cholerae strains, including pathogenic serotypes (also other than O1 and O139) (32), have been isolated from estuarine and coastal waters along the Pacific, Atlantic, and Gulf coasts of the United States and have also been isolated in Australia, Africa, Asia, and throughout Europe (30). However, to our knowledge, this is the first time that V. cholerae and/or the closely related organism V. mimicus has been detected in boreal brackish waters with an annual average temperature of around 5°C and at a latitude as far north as 65°. It is likely that this Vibrio population has escaped detection in these waters because previous studies have been biased by the use of culture-dependent methods; many Vibrio spp. survive in a viable but nonculturable state when exposed to low temperatures or otherwise adverse growth conditions (38, 41, 42). It cannot be excluded that nonviable bacteria will be detected by our PCR-based method. However, the more than occasional detection of the V. cholerae population in our survey suggests that these organisms are a natural component of the bacterioplankton community in the brackish environment of the Baltic Sea. Previous work corroborates that V. cholerae can maintain viability at temperatures below 15°C and at salinities ranging from 2 to 14{per thousand} in brackish environments (31), and other studies suggested increased survival and proliferation upon associations with eukaryotic microorganisms (1, 48).

V. cholerae and other Vibrio spp. are known to form biofilms (47) and associate with particles as well as organisms (1, 13, 17, 19, 20, 21, 25, 39, 40, 43, 48). However, in our study, typically between 73 and 89% (median of 5 to 21 samples where a population was detected) of the cells from each Vibrio population remained free living rather than particle or plankton attached (e.g., captured on a 0.8-µm filter) (Table 1). The only exception was the V. anguillarum/V. ordalii population, where 43% of the cells were captured on a 0.8-µm filter (median from 21 samples where this population was detected). In marine systems, particle association may not always be advantageous. For example, while particle attachment provides refuge from protozoa and high nutrient concentrations, it may also subject vibrios to sinking and sediment burial or ingestion by larger predators. On the other hand, one might hypothesize that particle ingestion could be beneficial for many vibrios (assuming survival in the eukaryote intestine), since they are then exposed to a nutrient-enriched environment.

In conclusion, environmental factors such as temperature, salinity, phosphorus concentration, and dinoflagellate abundance were important predictors of total Vibrio abundance, and there were marked differences in the distribution patterns of individual populations. There is clearly a need for serological studies, since Vibrio populations that may include pathogenic serotypes were detected along the entire coastline studied.


   ACKNOWLEDGMENTS
 
We thank A. M. Nordqvist for her help with statistical analyses. We are also grateful to staff at the Tjärnö Marine Biological Laboratory, Marine Biology Department, Campus Helsingborg, Lund University; Department of Marine Sciences, Kalmar University; Department of Systems Ecology, Stockholm University; and Umeå Marine Science Centre, Umeå University, for providing help and equipment during sampling.

This work was funded by the Swedish Research Council for Environment, Agricultural Sciences, and Special Planning (FORMAS grant 2004-0634 to S.B. and grant 22.3/2003-0209 to M.J.), the Carl Tryggers Foundation (grant CTS02:26 to S.B.), the Ax:son Johnson Foundation (grant to A.E.), and the Swedish Research Council (grants to S.B.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Limnology/Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Norbyv. 20, SE-75236 Uppsala, Sweden. Phone: 46-18-4712712. Fax: 46-18-531134. E-mail: stebe{at}ebc.uu.se. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Abd, H., A. Weintraub, and G. Sandström. 2005. Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae. Environ. Microbiol. 7:1003-1008.[CrossRef][Medline]
  2. Angulo, L., J. E. López, J. A. Vicente, and A. M. Saborido. 1994. Haemorrhagic areas in the mouth of farmed turbot, Scophthalmus maximus (L.). J. Fish Dis. 17:163-169.[Medline]
  3. Barbieri, E., L. Falzano, C. Fiorentini, A. Pianetti, W. Baffone, A. Fabri, P. Matarrese, A. Casiere, M. Katouli, I. Kühn, R. Möllby, F. Bruscolini, and G. Donelli. 1999. Occurrence, diversity, and pathogenicity of halophilic Vibrio spp. and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast. Appl. Environ. Microbiol. 65:2748-2753.[Abstract/Free Full Text]
  4. Ben-Haim, Y., F. L. Thompson, C. C. Thompson, M. C. Cnockaert, B. Hoste, J. Swings, and E. Rosenberg. 2003. Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis. Int. J. Syst. Evol. Microbiol. 53:309-315.[Abstract/Free Full Text]
  5. Campos, E., H. Bolanos, M. T. Acuna, G. Diaz, M. C. Matamoros, H. Raventos, L. M. Sanchez, O. Sanchez, and C. Barquero. 1996. Vibrio mimicus diarrhea following ingestion of raw turtle eggs. Appl. Environ. Microbiol. 62:1141-1144.[Abstract]
  6. Colwell, R. R. 1996. Global climate and infectious disease: the cholera paradigm. Science 274:2025-2031.[Free Full Text]
  7. Del Giorgio, P. A., A. F. Bird, Y. T. Prairie, and D. Planas. 1996. The flow cytrometric determination of bacterial abundance in lake plankton with the green acid stain SYTO 13. Limnol. Oceanogr. 41:783-789.
  8. Eiler, A., S. Langenheder, S. Bertilsson, and L. J. Tranvik. 2003. Heterotrophic bacterial growth efficiency and community structure at different natural organic carbon concentrations. Appl. Environ. Microbiol. 69:3701-3709.[Abstract/Free Full Text]
  9. Eiler, A., and S. Bertilsson. 2004. Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes. Environ. Microbiol. 6:1228-1243.[CrossRef][Medline]
  10. Eiler, A., and S. Bertilsson. 8 August 2006, posting date. Detection and quantification of Vibrio populations using denaturing gradient gel electrophoresis. J. Microbiol. Methods [Online.] doi:10.1016/j.mimet.2006.04.002.
  11. Eilers, H., J. Pernthaler, F. O. Glöckner, and R. Amann. 2000. Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl. Environ. Microbiol. 66:3044-3051.[Abstract/Free Full Text]
  12. Farmer, J. J., III, J. M. Janda, F. W. Brenner, D. N. Cameron, and K. M. Birkhead. 2005. Genus Vibrio, p. 494-546. In D. J. Brenner, N. R. Krieg, and J. T. Staley (ed.), Bergey's manual of systematic bacteriology, 2nd ed., vol. 2, part B. The Proteobacteria. Springer, New York, N.Y.
  13. Heidelberg, J. F., K. B. Heidelberg, and R. R. Colwell. 2002. Bacteria of the {gamma}-subclass Proteobacteria associated with zooplankton in the Chesapeake Bay. Appl. Environ. Microbiol. 68:5498-5507.[Abstract/Free Full Text]
  14. Heidelberg, J. F., K. B. Heidelberg, and R. R. Colwell. 2002. Seasonality of Chesapeake Bay bacterioplankton species. Appl. Environ. Microbiol. 68:5488-5497.[Abstract/Free Full Text]
  15. Hervio-Heath, D., R. R. Colwell, A. Derrien, A. Robert-Pillot, J. M. Fournier, and M. Pommepuy. 2002. Occurrence of pathogenic vibrios in coastal areas of France. J. Appl. Microbiol. 92:1123-1135.[CrossRef][Medline]
  16. Hlady, W. G., R. C. Mullen, and R. S. Hopkin. 1993. Vibrio vulnificus from raw oysters. Leading cause of reported deaths from foodborne illness in Florida. J. Fla. Med. Assoc. 80:536-538.
  17. Høi, L., J. L. Larsen, I. Dalsgaard, and A. Dalsgaard. 1998. Occurrence of Vibrio vulnificus biotypes in Danish marine environments. Appl. Environ. Microbiol. 64:7-13.[Abstract/Free Full Text]
  18. Holm-Hansen, O., and B. Riemann. 1978. Chlorophyll-a determination: improvement in methodology. Oikos 30:438-447.[CrossRef]
  19. Hood, M. A., and P. A. Winter. 1997. Attachment of Vibrio cholerae under various environmental conditions and to selected substrates. FEMS Microbiol. Ecol. 22:215-223.
  20. Huq, A., and R. R. Colwell. 1995. Vibrios in the marine and estuarine environments. J. Mar. Biotechnol. 3:60-63.
  21. Islam, M. S., M. A. Miah, M. K. Hasan, R. B. Sack, and M. J. Albert. 1994. Detection of non-culturable Vibrio cholerae O1 associated with a cyanobacterium from as aquatic environment in Bangladesh. Trans. R. Soc. Trop. Med. Hyg. 88:298-299.[CrossRef][Medline]
  22. Jespersen, A.-M., and K. Christoffersen. 1987. Measurements of chlorophyll a from phytoplankton using ethanol as extraction solvent. Arch. Hydrobiol. 109:445-454.
  23. Jiang, S. C., and W. Fu. 2001. Seasonal abundance and distribution of Vibrio cholerae in coastal waters quantified by a 16S-23S intergenic spacer probe. Microb. Ecol. 42:540-548.[CrossRef][Medline]
  24. Johansson, M., A. Eiler, L. J. Tranvik, and S. Bertilsson. 2006. Distribution of the dinoflagellate parasite Parvilucifera infectans (Perkinsozoa) along the Swedish coast. Aquat. Microb. Ecol. 43:289-302.
  25. Kaneko, T., and R. R. Colwell. 1975. Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Appl. Environ. Microbiol. 29:269-274.[Abstract/Free Full Text]
  26. Kaneko, T., and R. R. Colwell. 1978. Annual cycle of Vibrio parahaemolyticus in Chesapeake Bay. J. Bacteriol. 113:24-32.
  27. Kaspar, C. W., and M. L. Tamplin. 1993. Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish. Appl. Environ. Microbiol. 59:2425-2429.[Abstract/Free Full Text]
  28. Kushmaro, A., E. Banin, Y. Loya, E. Stackebrandt, and E. Rosenberg. 2001. Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica. Int. J. Syst. Evol. Microbiol. 51:1383-1388.[Abstract]
  29. Lee, K. H., and E. G. Ruby. 1994. Effect of the squid host on the abundance and distribution of symbiotic Vibrio fischeri in nature. Appl. Environ. Microbiol. 60:3897-3903.[Abstract/Free Full Text]
  30. Lipp, E. K., A. Huq, and R. R. Colwell. 2002. Effects of global climate on infectious disease: the cholera model. Clin. Microbiol. Rev. 15:757-770.[Abstract/Free Full Text]
  31. Louis, V. R., E. Russek-Cohen, N. Choopun, I. N. G. Rivera, B. Gangle, S. C. Jiang, A. Rubin, J. A. Patz, A. Huq, and R. R. Colwell. 2003. Predictability of Vibrio cholerae in Chesapeake Bay. Appl. Environ. Microbiol. 69:2773-2785.[Abstract/Free Full Text]
  32. Lukinmaa, S., K. Mattila, V. Lehtinen, M. Hakkinen, M. Koskela, and A. Siitonen. 2006. Territorial waters of the Baltic Sea as a source of infections caused by Vibrio cholerae non-O1, non-O139: report of 3 hospitalized cases. Diagn. Microbiol. Infect. Dis. 54:1-6.[CrossRef][Medline]
  33. Menzel, D. H., and N. Corwin. 1965. The measurement of total phosphorus in seawater based on the liberation of organically bound fractions by persulphate oxidation. Limnol. Oceanogr. 10:280-282.
  34. Miyamoto, Y., T. Kato, Y. Obara, S. Akiyama, K. Takizawa, and S. Yamai. 1969. In vitro hemolytic characteristic of Vibrio parahaemolyticus: its close correlation with human pathogenicity. J. Bacteriol. 100:261-266.
  35. Motes, N. L., A. DePaola, D. W. Cook, J. E. Veazey, J. C. Hunsucker, W. E. Gartright, R. J. Blodgett, and S. J. Chirtel. 1998. Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica). Appl. Environ. Microbiol. 64:1459-1465.[Abstract/Free Full Text]
  36. Mouriño-Pérez, R. R., A. Z. Worden, and F. Azam. 2003. Growth of Vibrio cholerae O1 in red tide waters off California. Appl. Environ. Microbiol. 69:6923-6931.[Abstract/Free Full Text]
  37. Murphy. J., and J. P. Riley. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27:31-36.[Medline]
  38. Oliver, J. D., and J. B. Kaper. 1997. Vibrio species, p. 228-264. In M. P. Doyle, L. R. Beuchat, and T. J. Montville (ed.), Food microbiology. ASM Press, Washington, D.C.
  39. Parvathi, A., H. S. Kumar, I. Karunasagar, and I. Karunasagar. 2005. Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis. Environ. Microbiol. 7:995-1002.[CrossRef][Medline]
  40. Pruzzo, C., G. Gallo, and L. Canesi. 2005. Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. Environ. Microbiol. 7:761-772.[CrossRef][Medline]
  41. Pruzzo, C., R. Tars, L. M. Del Mar, C. Signoretto, M. Zampini, L. Pane, R. R. Colwell, and P. Canepari. 2003. Persistence of adhesive properties in Vibrio cholerae after long-term exposure to seawater. Environ. Microbiol. 5:850-858.[CrossRef][Medline]
  42. Roszak, D. B., and R. R. Colwell. 1987. Survival strategies of bacteria in the natural environment. Microbiol. Rev. 51:365-379.[Free Full Text]
  43. Tamplin, M. L., A. L. Gauzen, A. Huq, D. A. Sack, and R. R. Colwell. 1990. Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters. Appl. Environ. Microbiol. 56:1977-1980.[Abstract/Free Full Text]
  44. Thompson, F. L., T. Iida, and J. Swings. 2004. Biodiveristy of vibrios. Microbiol. Mol. Biol. Rev. 68:403-431.[Abstract/Free Full Text]
  45. Thompson, J. R., M. A. Randa, L. A. Marcelino, A. Tomita-Mitchell, E. Lim, and M. F. Polz. 2004. Diversity and dynamics of a North Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70:4103-4110.[Abstract/Free Full Text]
  46. Utermöhl, H. 1958. Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Mitt. Int. Verein Theor. Angew. Limnol. 29:117-126.
  47. Watnick, P. I., C. M. Lauriano, K. E. Klose, L. Croal, and R. Kolter. 2001. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol. Microbiol. 39:223-235.[CrossRef][Medline]
  48. Worden, A. Z., M. Seidel, S. Smriga, A. Wick, F. Malfatti, D. Bartlett, and F. Azam. 2006. Trophic regulation of Vibrio cholerae in coastal marine waters. Environ. Microbiol. 8:21-29.[CrossRef][Medline]

Applied and Environmental Microbiology, September 2006, p. 6004-6011, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00917-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eiler, A.
Right arrow Articles by Bertilsson, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eiler, A.
Right arrow Articles by Bertilsson, S.
Agricola
Right arrow Articles by Eiler, A.
Right arrow Articles by Bertilsson, S.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals