Seasonal Dynamics and Modeling of a <Emphasis Type="Italic">Vibrio</Emphasis> Community in Coastal Waters of the North Sea

Vibrio species are ubiquitously distributed in marine waters all over the world. High genome plasticity due to frequent mutation, recombination, and lateral gene transfer enables Vibrio to adapt rapidly to environmental changes. The genus Vibrio comprises several human pathogens, which commonly cause outbreaks of severe diarrhea in tropical regions. In recent years, pathogenic Vibrio emerged also in coastal European waters. Little is known about factors driving the proliferation of Vibrio spp. in temperate waters such as the North Sea. In this study a quantification of Vibrio in the North Sea and their response to biotic and abiotic parameters were assessed. Between January and December 2009, Vibrio at Helgoland Roads (North Sea, Germany) were quantified using fluorescence in situ hybridization. Vibrio numbers up to 3.4 × 10⁴ cells × mL⁻¹ (2.2% of total microbial counts) were determined in summer, but their abundance was significantly lower in winter (5 × 10² cells × mL⁻¹). Correlations between Vibrio and nutrients (SiO₂, PO₄ ³⁻, DIN), Secchi depth, temperature, salinity, and chlorophyll a were calculated using Spearman rank analysis. Multiple stepwise regression analysis was carried out to analyze the additive influence of multiple factors on Vibrio. Based on these calculations, we found that high water temperature and low salinity best explained the increase of Vibrio cell numbers. Other environmental parameters, especially nutrients and chlorophyll a, also had an influence. All variables were shown to be subject to the overall seasonal dynamics at Helgoland Roads. Multiple regression models could represent an efficient and reliable tool to predict Vibrio abundances in response to the climate change in European waters.     

Kanagawa-Negative, <Emphasis Type="Italic">tdh</Emphasis>- and <Emphasis Type="Italic">trh</Emphasis>-Positive <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> Isolated from Fresh Oysters Marketed in Fortaleza,Brazil

Between October 2008 and June 2009, 15 samples of 10 live oysters each (Crassostrea rhizophorae) measuring 8.31–10.71 cm were purchased from a restaurant on the seashore of Fortaleza, Brazil. The Vibrio count ranged from 75 (estimated) to 43,500 CFU/g. Fourteen species were identified among the 56 isolated Vibrio strains, with V. parahaemolyticus as the most prevalent. Two of the 17 V. parahaemolyticus strains were urease-positive and tdh- and trh-positive on multiplex PCR, but neither produced β-hemolysis halos in Wagatsuma agar. Thus, fresh oysters served in natura in Fortaleza, Brazil, were found to contain Vibrio strains known to cause gastroenteritis in humans.     

In-vitro anti-<Emphasis Type="Italic">Vibrio</Emphasis> spp. activity and chemical composition of some Tunisian aromatic plants

The chemical composition of five aromatic plants (Mentha longifolia, M. pulegium, Eugenia caryophyllata, Thymus vulgaris and Rosmarinus officinalis) frequently used in food preparation in Tunisia was analysed by GC-MS. The antimicrobial effect of the essential oils obtained from these plants was tested against Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio fluvialis strains. Thyme oil exhibited a high level of antimicrobial activities against Vibrio spp. strains. The diameter of the zones of growth inhibition for V. parahaemolyticus species was interestingly high (ranging from 14.66 to 28 mm). The MIC and MBC values were interestingly low for thyme oil (MIC 0.078–0.156 mg/ml) and (MBC >0.31–1.25 mg/ml). These results showed that these plants especially thyme and clove, can be to be used for seafood preparation to protect against contamination by Vibrio spp. strains. An erratum to this article can be found at     

Presence of genes for type III secretion system 2 in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">mimicus</Emphasis> strains

Background  

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.     

<Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and<Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>: Short generation-time marine bacteria

The growth rates of 30 different strains ofVibrio parahaemolyticus andVibrio alginolyticus at 37°C was determined. Each species consists of two major groups, one having a short generation time (12–14 min) and one with a longer generation time (20–25 min). The diversity in generation times of different strains belonging to the same species is discussed. The effect of temperature, salt, and nutrient concentrations on the growth rate of oneV. alginolyticus strain (NCMB 1803) was studied. The most striking is the effect of the temperature; at 39°C the generation time is 10–11 min, while at 21°C it is 60 min. The heat of activation for growth calculating from such data is 22,580 kcal/mole/hr⁻¹. The ecological significance of these results is discussed.     

Isolation of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio splendidus</Emphasis> from captive-bred seahorses with disease symptoms

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.     

Molecular analysis of the emergence of pandemic <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

Vibrio parahaemolyticusis abundant in the aquatic environment particularly in warmer waters and is the leading cause of seafood borne gastroenteritis worldwide. Prior to 1995, numerousV. parahaemolyticusserogroups were associated with disease, however, in that year an O3:K6 serogroup emerged in Southeast Asia causing large outbreaks and rapid hospitalizations. This new highly virulent strain is now globally disseminated.     

Benthic ecology of <Emphasis Type="Italic">Vibrio</Emphasis> spp. and pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> species in a coastal Mediterranean environment (La Spezia Gulf,Italy)

We carried out a 16-month in situ study to investigate the ecology of Vibrio spp. and pathogenic Vibrio species in coastal sediments of the Mediterranean Sea, employing multiple-regression analysis to reveal the major environmental factors controlling their occurrence in the benthic environment. In addition, association between vibrios and sediment-inhabiting meiofauna, which is a major component of benthic ecosystems, was investigated. Culturable and total Vibrio spp. estimates by most-probable-number technique coupled with standard polymerase chain reaction (PCR) and real-time PCR methods, respectively, were at least one order of magnitude higher in sediment than in seawater. In addition, potential human pathogenic species Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus occurred in the sediment with V. parahaemolyticus being the most frequently found. In the pelagic environment, 60% of total variance in culturable Vibrio data was explained by sea surface temperature (40%), salinity (13%) and organic matter concentration (7%). In the benthic environment, sea surface temperature was the only factor that significantly affected culturable Vibrio occurrence although it explained only 25% of total variance, suggesting that additional unexplored factors may play a role as well. No correlation was found between culturable Vibrio spp. concentrations and the abundance of harpacticoid copepods in the sediment whilst a negative correlation was found between Vibrio spp. and nematode abundance which accounted for almost 90% of the total meiofaunal density. Taxonomic analysis revealed that selective bacterial feeders accounted for nearly 50% of the total nematode community and included genera such as Terschellingia, Molgolaimus and Halalaimus, suggesting that top-down control by nematode grazing may be an important factor affecting Vibrio occurrence in these sediments. It is concluded that the benthic marine environment may function as a reservoir of Vibrio spp. and potential pathogenic vibrios whose ecological features appeared substantially different from the ones recognised in the pelagic environment.     

Inhibitory Activity of Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> UTM 126 Against <Emphasis Type="Italic">Vibrio</Emphasis> Species Confers Protection Against Vibriosis in Juvenile Shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>)

The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (10⁵ CFU/g) for 28 days before an immersion challenge with V. harveyi at 10⁵ CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture.     

Expression and purification of two major outer membrane proteins from <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a Gram-negative bacterium, is one of Vibrio pathogens common to human and marine animals. Outer membrane proteins of bacteria play an important role during infection and induction of host immune response. In present research, two outer membrane protein genes (OmpK and OmpW) of V. alginolyticus were cloned and expressed. The open reading frames of OmpK and OmpW contain 846 bp and 645 bp, respectively, the mature proteins consist of 261 and 193 amino acid residues. At the signal peptides positions −3 to −1, the amino acids were V-M-A in OmpK and V-F-A in OmpW, which consistented with the observed sequence V-X-A of the signal peptides of transmembrane OMP. The alignment analysis indicated that both proteins were highly conserved, which could serve as surface antigens for vaccine candidates. SDS-PAGE indicated two genes over-expressed in E. coli BL21 (DE3). By affinity chromatography on Ni²⁺-nitriloaceate resin, the recombinant proteins were purified from inclusion bodies. Western blot analysis revealed that both proteins had immunoreactivity, which provided a base for further study on the evaluation of diagnostication and vaccine candidates.     

Biochemical characteristics and genetic diversity of <Emphasis Type="Italic">Vibrio</Emphasis> spp. and <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> strains isolated from the Lac of Bizerte (Tunisia)

This study characterized the phenotypic and genetic properties of Vibrio spp. and Aeromonas hydrophila strains isolated from seawater and mussels (Mytilus edulis and Crassostrea gigas) cultured in mollusc farm localized in the lac of Bizerte. The 37 strains (31 strains of V. alginolyticus, one strain of V. fluvialis, one strain of V. parahaemolyticus and four strains of A. hydrophila) typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) showed a high polymorphism. Most of the isolates were resistant to at least two antimicrobial agents. All the tested strains were resistant to ampicillin. PCR was used to detect the presence of eight Vibrio cholerae virulence genes in the genome of the Vibrio spp. isolates. The results showed a wide dissemination of these genes in the genome of all Vibrio spp. isolates tested. Differentiation of these strains with the ERIC 2-PCR technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern.     

Detection and Quantification of <Emphasis Type="Italic">Vibrio cholerae,Vibrio parahaemolyticus</Emphasis>, <Emphasis Type="Italic">and Vibrio vulnificus</Emphasis> in Coastal Waters of Guinea-Bissau (West Africa)

V. cholerae, V. parahaemolyticus, and V. vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V. cholerae (ISR 16S-23S rRNA) ranged 0–1.2 × 10⁴ MPN/L, whereas V. parahaemolyticus (toxR) varied from 47.9 to 1.2 × 10⁵ MPN/L. Although the presence of genotypes associated with virulence was found in environmental V. cholerae isolates, ctxA+ V. cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V. parahaemolyticus were detected at concentrations up to 1.2 × 10³ MPN/L. V. vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2 × 10⁵ MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.     

Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus.     

Enzymatic,outer membrane proteins and plasmid alterations of starved <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> cells in seawater

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 μm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.     

Prevalence and Heterogeneity of Hemolysin Gene <Emphasis Type="Italic">vhh</Emphasis> Among Hatchery Isolates of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> in India

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Analysis of RNA Polymerase Beta Subunit (<Emphasis Type="Italic">rpoB</Emphasis>) Gene Sequences for the Discriminative Power of Marine <Emphasis Type="Italic">Vibrio</Emphasis> Species

In the present study, we sequenced the RNA polymerase beta subunit (rpoB) gene of marine Vibrio species and assessed its discriminative power in identifying vibrios. Both the rpoB and 16S rRNA sequences of 29 phenotypically different Vibrio strains isolated from coastal waters were determined. Molecular and phylogenetic comparisons of the sequences of these two genes classified the 29 strains into 11 different species. The resolution of the Vibrio spp. on the rpoB phylogenetic tree was approximately three times greater than that on the 16S rRNA phylogenetic tree. Moreover, by comparing the rpoB sequences of 98 marine γ-Proteobacteria, including 38 marine Vibrio species, Vibrio-specific primers were developed to amplify a 730-bp fragment of the rpoB gene. Using these primers, we successfully detected Vibrio signals in environmental samples and determined their relative abundances via comparisons with known standards. This rpoB-targeting polymerase chain reaction assay can be used efficiently to monitor relative Vibrio abundance in marine waters.     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Hfq regulates the expression of the thermostable direct hemolysin gene in <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

The hfq gene is conserved in a wide variety of bacteria and Hfq is involved in many cellular functions such as stress responses and the regulation of gene expression. It has also been reported that Hfq is involved in bacterial pathogenicity. However, it is not clear whether Hfq regulates virulence in Vibrio parahaemolyticus. To evaluate this, we investigated the effect of Hfq on the expression of virulence-associated genes including thermostable direct hemolysin (TDH), which is considered to be an important virulence factor in V. parahaemolyticus, using an hfq deletion mutant.