Comparative immunogenicity of outer membrane protein K and whole-cell antigens of Vibrio parahaemolyticus for diagnosis

The immunogenicity of soluble outer membrane protein K (OmpK)- small ubiquitin-like modifier, OmpK inclusion bodies, formalin, and heat-killed Vibrio parahaemolyticus cells were prepared and studied in a mouse model. The results of whole-cell ELISA and Western blot (WB) revealed that the serum against soluble OmpK and OmpK inclusion bodies reacted only with homologous V. parahaemolyticus. Furthermore, recombinant OmpK proteins were not recognized by the serum against whole-cell V. parahaemolyticus antigens. Unexpectedly, the serum against formalin and heat-killed V. parahaemolyticus reacted broadly with homologous (an immunization strain) and heterologous (non-immunization strains) V. parahaemolyticus and Vibrio species. The WB results revealed that the serum against the two V. parahaemolyticus whole-cell antigens primarily reacted with proteins that were approximately 100, 70, 36, 28, and 22 kDa in the cell lysates from different Vibrio strains, rather than the recombinant OmpK. The 70 and 28 kDa proteins exhibited specificity to Vibrio species, while the 22 kDa protein was more specific to V. parahaemolyticus. This study showed the limitation of recombinant OmpK to prepare diagnostic antibodies and revealed several specific Omps of Vibrio sp. and V. parahaemolyticus that were promising in diagnosis and vaccine development.     

An outer membrane protein, OmpK, is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper (Epinephelus coioides)

The outer membrane proteins of the fish pathogen, Vibrio harveyi, have a role in interaction between bacterium and host and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein, OmpK, which serves as the receptor for broad-host-range vibriophage KVP40 in V. harveyi, was isolated and characterized. Then the OmpK gene coding for mature peptide was subcloned into prokaryotic expression vector pBV220 and transformed into Escherichia coli DH5 alpha strain. After temperature induction, a recombinant protein was detected about 28 kDa in molecular weight and accounted for 24.8% of total proteins of whole cell as estimated by SDS-PAGE and scanning analysis of gel image. Polyclonal antibodies were raised in rabbits against the purified protein and the reaction of the antibody was confirmed by western blotting using the purified protein and crude extract of V. harveyi. Orange-spotted groupers (Epinephelus coioides) vaccinated with recombinant OmpK produced specific antibodies, and were highly resistant to infection by virulent V. harveyi. These results indicate that the OmpK is an effective vaccine candidate against V. harveyi in Orange-spotted groupers.     

Vibrios Associated with Litopenaeus vannamei Larvae, Postlarvae, Broodstock, and Hatchery Probionts

Several bacteriological surveys were performed from 1994 to 1996 at different Litopenaeus vannamei hatcheries (in Ecuador) and shrimp farms (in Mexico). Samples were taken from routine productions of healthy and diseased L. vannamei larvae, postlarvae, and their culture environment and from healthy and diseased juveniles and broodstock. In Ecuador, the dominant bacterial flora associated with shrimp larvae showing symptoms of zoea 2 syndrome, mysis mold syndrome, and bolitas syndrome has been determined. Strains were characterized by Biolog metabolic fingerprinting and identified by comparison to a database of 850 Vibrio type and reference strains. A selection of strains was further genotypically fine typed by AFLP. Vibrio alginolyticus is predominantly present in all larval stages and is associated with healthy nauplius and zoea stages. AFLP genetic fingerprinting shows high genetic heterogeneity among V. alginolyticus strains, and the results suggest that putative probiotic and pathogenic strains each have specific genotypes. V. alginolyticus was found to be associated with larvae with the zoea 2 syndrome and the mysis mold syndrome, while different Vibrio species (V. alginolyticus and V. harveyi) are associated with the bolitas syndrome. V. harveyi is associated with diseased postlarvae, juveniles, and broodstock. The identities of the strains identified as V. harveyi by the Biolog system could not be unambiguously confirmed by AFLP genomic fingerprinting. Vibrio strain STD3-988 and one unidentified strain (STD3-959) are suspected pathogens of only juvenile and adult stages. V. parahaemolyticus, Photobacterium damselae, and V. mimicus are associated with juvenile and adult stages.     

Isolation ofVibrio parahaemolyticus andVibrio cholerae (Non-O1 and O139) from moribund shrimp (Penaeus monodon) and experimental challenge study against post larvae and juveniles

Outbreak ofVibrio infection was reported from a shrimp farm near Chennai, south India. Both green and yellowVibrio were isolated from disease outbreak ofPenaeus monodon (Milne Edwards) culture farm and biochemically confirmed asVibrio parahaemolyticus andVibrio cholerae. Randomly selectedV. parahaemolyticus (VP1) andV. cholerae (VC1) were reconfirmed by partial 16S rRNA gene analysis. Both were tested against post larvae and juveniles ofP. monodon by bath challenge test and intramuscular injection respectively. The study revealed that VP1 is more virulent than VC1 isolated from same source againstP. monodon post larvae and juveniles. Phylogenetic tree based on comparison of partial 16S rRNA gene analysis revealed close relationship of VP1 with other shrimp pathogens likeVibrio harveyi andVibrio alginolyticus. There might be some close relationship for disease development among all these strains.     

Inhibition of pathogenic Vibrio by the microalgae Isochrysis galbana

Vibrio alginolyticus, Vibrio campbellii, and Vibrio harveyi were inhibited by Isochrysis galbana in batch cultures. I. galbana reduced the V. alginolyticus, V. campbellii, and V. harveyi counts to undetectable levels in 2, 4, and 7 days (<0.01 Vibrio spp. mL^?1), respectively, remaining so until the end of the experiment on day 15. Other heterotrophic bacteria reached counts of 10⁶ CFU mL^?1 on ZoBell medium at the end of the experiment. Vibrio parahaemolyticus was not inhibited by I. galbana. In all mixed I. galbana and Vibrio spp. cultures, the algal density increased from 3.5 to 4.0?×?10⁷ cells mL^?1, higher than that in I. galbana cultures alone, indicating a lack of an inhibitory effect on microalgae in the mixed cultures. The predominant fatty acids (>82 %) of I. galbana during the stationary growth phase were estearidonic (24.3 %), oleic (15.7 %), myristic (13.8 %), docosahexaenoic (11.0 %), palmitic (10.3 %), and α-linolenic (7.2 %) acids. These results demonstrate that I. galbana synthesizes antibacterial fatty acids that inhibit the growth of pathogenic bacteria such as V. alginolyticus, V. campbellii, and V. harveyi.     

Rapid identification and differentiation of Vibrio parahaemolyticus from Vibrio spp. in seafood samples using developed monoclonal antibodies

Monoclonal antibodies (MAbs) specific to Vibrio parahaemolyticus were successfully generated. According to the specificity of V. parahaemolyticus, MAbs can be classified into 5 groups. The MAbs VP-2D and VP-11H were specific to the O2 and O4 groups of V. parahaemolyticus, respectively. The MAb VP-11B reacted with 11 out of 30 isolates of V. parahaemolyticus used in this study. The MAb VP-516 bound to 27 out of 30 isolates of V. parahaemolyticus and cross reacted with all 10 isolates of V. alginolyticus. The MAb VP-618 demonstrated positive reactivity to 29 out of 30 isolates of V. parahaemolyticus and demonstrated slight cross reactivity to 3 out of 30 isolates of V. harveyi. The sensitivity of the MAbs ranged from 10⁸ to 10⁷ c.f.u. ml^?1 for V. parahaemolyticus obtained from pure cultures and depended on the group of MAbs. However, the detection capability could be improved to be equivalent to that of the PCR technique following pre-incubation of the samples in alkaline peptone water (APW). Using these MAbs along with MAbs specific to V. alginolyticus (VA-165), V. cholerae (VC-63), V. harveyi (VH-9B and VH-20C) and Vibrio spp. (VC-201) from previous studies, V. parahaemolyticus could be identified and differentiated from Vibrio spp. in various seafood samples including shrimp, green mussels, blood clams and oysters by a simple dot blot immunoassay without the requirement for bacterial isolation or biochemical characterization.     

Occurrence of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> in the German Bight over a seasonal cycle

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10³ N l⁻¹ and MPNs up to 240 N g⁻¹ were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.     

Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood

In this study, a single base extension-tag array on glass slides (SBE-TAGS) microarray was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus, Vibrio anguillarum, and Vibrio harveyi. Three multiplex PCR assays were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus, and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi, and V. cholerae, respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.     

Temporal and Spatial Distribution Patterns of Potentially Pathogenic Vibrio spp. at Recreational Beaches of the German North Sea

The number of reported Vibrio-related wound infections associated with recreational bathing in Northern Europe has increased within the last decades. In order to study the health risk from potentially pathogenic Vibrio spp. in the central Wadden Sea, the seasonal and spatial distribution of Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio cholerae were investigated at ten recreational beaches in this area over a 2-year period. V. alginolyticus and V. parahaemolyticus were found to be omnipresent all year round in the study area, while V. vulnificus occurrence was restricted to summer months in the estuaries of the rivers Ems and Weser. Multiple linear regression models revealed that water temperature is the most important determinant of Vibrio spp. occurrence in the area. Differentiated regression models showed a species-specific response to water temperature and revealed a particularly strong effect of even minor temperature increases on the probability of detecting V. vulnificus in summer. In sediments, Vibrio spp. concentrations were up to three orders of magnitude higher than in water. Also, V. alginolyticus and V. parahaemolyticus were found to be less susceptible towards winter temperatures in the benthic environment than in the water, indicating an important role of sediments for Vibrio ecology. While only a very small percentage of tested V. parahaemolyticus proved to be potentially pathogenic, the presence of V. vulnificus during the summer months should be regarded with care.     

Expression and immunogenicity analysis of accessory colonization factor A from Vibrio alginolyticus strain HY9901

The accessory colonization factor A (ACFA) of Vibrio alginolyticus plays an important role in the efficient colonization of the bacterium and is potential candidates for vaccine development. In present study, the acfA gene was cloned, expressed and purified. Western blot analysis revealed protein recognition with the native ACFA in different V. alginolyticus strains. To analyze the immunogenicity of the recombinant ACFA, Lutjanus erythropterus Bloch were immunized by intraperitoneal injection, and the results demonstrated that the recombinant ACFA produced an observable antibody response in all sera of the vaccinated fish. The differential expressions of RAG1 gene in various tissues of L. erythropterus were analyzed by fluorescent quantitative real-time PCR, and the results showed the RAG1 mRNA expression was significantly up-regulated in thymus, head kidney and spleen tissue. Furthermore, the protective property of recombinant ACFA was evaluated through challenge with six heterogeneous virulent V. alginolyticus strains, and the immunohistochemical analysis in different tissues after challenge with V. alginolyticus. The results showed L. erythropterus vaccinated with recombinant ACFA were more tolerant of the infection by virulent V. alginolyticus strains. The data indicate that the recombinant ACFA could provide heterologous protection for the different virulent V. alginolyticus strains.     

Phenotypic characterization and RAPD fingerprinting of Vibrio parahaemolyticus and Vibrio alginolyticus isolated during Tunisian fish farm outbreaks

The genus Vibrio is characterized by a large number of species and some of them are human pathogens causing gastrointestinal and wound infections through the ingestion or manipulation of contaminated fishes and shellfish including Vibrio parahaemolyticus and Vibrio alginolyticus. In this study, we reported the phenotypic and molecular characterization of 9 V. parahaemolyticus and 27 V. alginolyticus strains isolated from outbreaks affecting cultured Gilthead sea bream (Sparus aurata L.) and Sea bass (Dicentrarchus labrax) along the Tunisian coast from 2008 to 2009. All isolates were tested for the presence of DNase, caseinase, protease, lipase, amylase, gelatinase, hemolytic activity and antibacterial resistance to different drugs. Randomly amplified polymorphic DNA was used to examine the genetic relatedness among the V. parahaemolyticus and V. alginolyticus strains.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N₂-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N₂ might explain why they are so abundant in the mucus of different coral species.     

Dynamics of Vibrio with Virulence Genes Detected in Pacific Harbor Seals (Phoca vitulina richardii) Off California: Implications for Marine Mammal Health

Given their coastal site fidelity and opportunistic foraging behavior, harbor seals (Phoca vitulina) may serve as sentinels for coastal ecosystem health. Seals using urbanized coastal habitat can acquire enteric bacteria, including Vibrio that may affect their health. To understand Vibrio dynamics in seals, demographic and environmental factors were tested for predicting potentially virulent Vibrio in free-ranging and stranded Pacific harbor seals (Phoca vitulina richardii) off California. Vibrio prevalence did not vary with season and was greater in free-ranging seals (29 %, n?=?319) compared with stranded seals (17 %, n?=?189). Of the factors tested, location, turbidity, and/or salinity best predicted Vibrio prevalence in free-ranging seals. The relationship of environmental factors with Vibrio prevalence differed by location and may be related to oceanographic or terrestrial contributions to water quality. Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae were observed in seals, with V. cholerae found almost exclusively in stranded pups and yearlings. Additionally, virulence genes (trh and tdh) were detected in V. parahaemolyticus isolates. Vibrio cholerae isolates lacked targeted virulence genes, but were hemolytic. Three out of four stranded pups with V. parahaemolyticus (trh+ and/or tdh+) died in rehabilitation, but the role of Vibrio in causing mortality is unclear, and Vibrio expression of virulence genes should be investigated. Considering that humans share the environment and food resources with seals, potentially virulent Vibrio observed in seals also may be of concern to human health.     

Genes Similar to the Vibrio parahaemolyticus Virulence-Related Genes tdh,tlh, and vscC2 Occur in Other Vibrionaceae Species Isolated from a Pristine Estuary

Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.     

Occurrence, Diversity, and Pathogenicity of Halophilic Vibrio spp. and Non-O1 Vibrio cholerae from Estuarine Waters along the Italian Adriatic Coast

The occurrence, diversity, and pathogenicity of Vibrio spp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.     

Vibrio parahaemolyticus and Its Specific Bacteriophages as an Indicator in Cockles (Anadara granosa) for the Risk of V. parahaemolyticus Infection in Southern Thailand

Correlation between the numbers of Vibrio parahaemolyticus and its specific bacteriophages in cockles was investigated from June 2009 to May 2010 in Hat Yai, Songkhla, Thailand. Cockles obtained monthly from a local market were sampled to determine the numbers of V. parahaemolyticus and bacteriophages that could form plaques on ten strains of pandemic and nonpandemic V. parahaemolyticus. In addition, V. parahaemolyticus isolates from clinical samples from Hat Yai hospital over the same period were investigated. All 139 cockles sampled were positive for V. parahaemolyticus. However, only 76 of them were positive for bacteriophages. During the testing period, the number of bacteriophages was not significantly correlated with the incidence of V. parahaemolyticus-infected patients, but the numbers of V. parahaemolyticus isolates from the cockle samples were closely related to the number of infected patients. The bacteriophages isolated from V. parahaemolyticus also infected Vibrio alginolyticus and Vibrio mimicus, suggesting that the broad host range of phages may be a factor of providing the possibility of their participation in the processes of genetic exchange between V. parahaemolyticus and closely related Vibrio spp. In conclusion, this study indicated that the number of V. parahaemolyticus in cockles may be a useful tool for predicting the relative risk of infection by V. parahaemolyticus in this area of Thailand.     

弧菌外膜蛋白OmpU的免疫交叉反应性和交叉保护性北大核心CSCD

【目的】通过对弧菌外膜蛋白Omp U的克隆、表达以及免疫学特性分析,明确外膜蛋白Omp U是否为弧菌的共同抗原,并具有免疫交叉反应性和交叉保护性。【方法】对弧菌外膜蛋白omp U基因进行克隆和生物信息学分析。分别制备副溶血弧菌、溶藻弧菌、创伤弧菌、拟态弧菌和霍乱弧菌的Omp U重组蛋白抗血清,对Omp U的免疫交叉反应特性以及抗原表位定位情况进行比较分析。以霍乱弧菌的Omp U重组蛋白免疫小鼠后,再以多种弧菌进行攻毒,分析其交叉免疫保护作用。【结果】外膜蛋白Omp U在弧菌种内和种间相似性分别为73.0%–100%和58.6%–89.0%,并至少存在9个保守的B细胞抗原表位。Omp U重组蛋白抗血清在弧菌种内和种间均产生显著的免疫交叉反应,识别弧菌中分子量35–40 k Da的同源蛋白。副溶血弧菌ATCC17802、创伤弧菌ATCC27562和拟态弧菌ATCC33653来源的Omp U重组蛋白抗体能识别供试菌株,提示这些菌株的Omp U抗原表位定位于细胞表面。Omp U重组蛋白对免疫后的小鼠具有交叉免疫保护作用,攻毒实验后小鼠相对存活率(RPS)为43.0%–100%。【结论】上述结果表明,外膜蛋白Omp U是弧菌中一种保守的共同抗原,具有免疫交叉保护性,可以作为弧菌广谱疫苗的候选抗原。     

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     

Heterodisaccharide 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase

Aims: To investigate the attractant effect of 4‐O‐(N‐acetyl‐β‐d ‐glucosaminyl)‐d ‐glucosamine (GlcNAc‐GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc‐GlcN from N,N′‐diacetylchitobiose (GlcNAc)₂. Methods and Results: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane‐migration method. The results demonstrated that GlcNAc‐GlcN functions as an effective chemoattractant in the CE family 4 COD‐producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc‐GlcN appears to stimulate polar flagellum rotation. Conclusions: GlcNAc‐GlcN is a specific chemoattractant for the CE family 4 COD‐producing vibrios, V. parahaemolyticus and V. alginolyticus. Significance and Impact of the Study: It was clarified for the first time that GlcNAc‐GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc‐GlcN from (GlcNAc)₂.