Similarity of Vibrio alginolyticus, V. cholerae and other Vibrio species with respect to the structure of their flagellar apparatus and ribosomal 5S-RNA

Electron microscopic analysis of basal bodies of the flagella Vibrio alginolyticus revealed a structure composed of four discs. The diameters of two discs localized in the cytoplasmic membrane appeared to be twice as little as those of the other two discs. In this respect the basal body of V. alginolyticus resembles that of V. cholerae. The 5S sequence of ribosomal RNA from V. alginolyticus appeared to be similar to those of V. cholerae, V. harveyi and some other vibrios. Comparison of 5S-RNA sequence culminated in a dendrogram of evolutionary relationships of various bacterial species, suggesting that V. alginolyticus is a typical representative of the Vibrionacea family. The data obtained are discussed in terms of the role of Na+ energy metabolism in living cells.     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

Lateral flagella of vibrios: serological classification and genetical similarity.

Lateral (L-) flagella-having vibrios were classified into 13 H-serogroups (flagellar antigen serogroups) by means of H-agglutination test. Vibrio parahaemolyticus was classified into 3 serogroups, HL1 to 3. V. alginolyticus and V. harveyi were classified into 5 and 3 serogroups, respectively, but 2 of those were serogroups common to the both species. V. fluvialis and V. furnissii constituted a same serogroup, HL8. Cross-reactivity between each serogroup was not observed in H-agglutination test, although some cross-reactivity was observed in gel diffusion test. Furthermore, similarity of DNA sequence of L-flagellar structure gene was demonstrated by dot blot hybridization test with a DNA probe of HL2 L-flagellar gene fragment. These results suggest conservation of DNA sequence of the L-flagellar gene of vibrios.     

Vibrio parahaemolyticus and other halophilic vibrios associated with seafood in Hong Kong

The summer prevalence of Vibrio parahaemolyticus and other halophilic vibrios in seafood from Hong Kong markets was investigated. Halophilic vibrios were isolated from all seven types of seafood examined, and comprised 9.1%, 8% and 6.1% of contaminating aerobic heterotrophic bacteria from mussels, clams and oysters respectively. Sucrose-positive vibrios were more common than sucrose-negative varieties. Vibrio alginolyticus was the most frequently isolated species, followed by V. parahaemolyticus, V. harveyi, V. fluvialis, V. vulnificus, V. pelagius, V. campbellii, V. spendidus and V. marinus. Mussels contained the highest concentration of V. parahaemolyticus (4.6 x 10(4)/g); oysters and clams contained 3.4 x 10(4)/g and 6.5 x 10(3)/g respectively. The ubiquity and relatively high concentrations of V. parahaemolyticus and other pathogenic vibrios in shellfish is a potential public health hazard in Hong Kong and other subtropical Asian countries.     

The use of PCR to aid in the rapid identification of Vibrio harveyi isolates

AIMS: Vibrio harveyi is an important pathogen, causing potential devastation to marine aquaculture. This organism, however, is extremely difficult to identify because it is phenotypically diverse. Biochemical identification can involve many tests and take weeks to perform. The aim of this work is to develop a PCR that can reduce the number of biochemical tests, and the time taken, to get a definitive identification of this organism. METHODS AND RESULTS: The PCR was developed using 16S rDNA sequences from a number of V. harveyi strains, and other vibrios. The described test gave positive results for all strains of V. harveyi tested. However, some strains of V. alginolyticus also gave positive results and a small number of biochemical tests were required to differentiate between these two species. This indicated that preisolation of the bacteria was needed and therefore the test was not applicable to the testing of mixed populations directly. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of identification of this species was significantly reduced from a number of weeks to a few days. Hence, diagnosis of affected animals will be faster and earlier treatment can be administered which may increase the survival rate from vibriosis.     

Differences between surface antigenic determinants of polar monotrichous flagella of Vibrio parahaemolyticus and of related species

Polar monotrichous flagella (M-flagella) of Vibrio parahaemolyticus have antigens in common with those of various species of Vibrio including V. cholerae and V. anguillarum, and of Beneckea, revealed by gel diffusion tests with flagelli monomers. However, antiserum against M-flagellin of V. parahaemolyticus did not agglutinate cells of V. cholerae and V. anguillarum, although it did agglutinate cells of V. parahaemolyticus. Agglutination tests after absorption of the antiserum with purified M-flagellar filaments or flagellin monomers and H-agglutination inhibition tests demonstrated that there are two different antigenic determinants in M-flagella as in lateral flagella. One is on the surface of the M-flagella (surface antigenic determinant, SA) and disappears or is buried in dissociated monomers. The other is inside the flagella (internal antigenic determinant, IA) and is exposed when the flagella are dissociated to flagellin monomers. SA of V. parahaemolyticus is different from those of V. cholerae and V. anguillarum, whereas the three species have a common IA.     

Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources.

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V. parahaemolyticus.     

Vibrio parahaemolyticus and other halophilic vibrios associated with seafood in Hong Kong

The summer prevalence of Vibrio parahaemolyticus and other halophilic vibrios in seafood from Hong Kong markets was investigated. Halophilic vibrios were isolated from all seven types of seafood examined, and comprised 9.1%, 8% and 6.1% of contaminating aerobic heterotrophic bacteria from mussels, clams and oysters respectively. Sucrose-positive vibrios were more common than sucrose-negative varieties. Vibrio alginolyticus was the most frequently isolated species, followed by V. parahaemolyticus, V. harveyi, V.fluvialis, V. vulnificus, V. pelagius, V. campbellii, V. spendidus and V. marinus. Mussels contained the highest concentration of V. parahaemolyticus (4.6×10³/g); oysters and clams contained 3.4×10⁴/g and 6.5×10³/g respectively. The ubiquity and relatively high concentrations of V. parahaemolyticus and other pathogenic vibrios in shellfish is a potential public health hazard in Hong Kong and other subtropical Asian countries.     

大黄鱼三种病原弧菌外膜蛋白交叉保护性抗原筛选

弧菌是海水养殖环境中常见的条件性致病菌,弧菌病的暴发给水产养殖业造成了严重损失。鉴于水生动物尤其是鱼类弧菌病的发生常常是多种（血清型或亚种）弧菌的混合感染,筛选具有潜在的交叉保护性蛋白抗原,作为制备多价疫苗或联合疫苗的侯选成分具有重要意义。文中从患病大黄鱼中分离到8株弧菌,经生理生化和分子生物学鉴定分别为6株哈维氏弧菌Vibrio harveyi,1株溶藻弧菌Vibrio alginolyticus和1株副溶血弧菌Vibrio parahaemolyticus。选择典型的不同种的弧菌为代表,提取其外膜蛋白,经SDS-PAGE和Westernblotting分析,确定它们大约在45 kDa、35 kDa、22 kDa处出现了3条共同的免疫印迹条带,表明它们很有可能含有共同的能够彼此交叉保护的抗原。利用双向电泳和免疫印迹相结合的方法,借助于MALDI-TOF-MS质谱分析技术,发现溶藻弧菌V.alginolyticus的一种功能未知的孔蛋白（Porin,GenBank Accession No.ZP₀1260407）和副溶血弧菌V.parahaemolyticus的一种麦芽糖孔蛋白的前体蛋白（Maltoporin precursor,GenBank AccessionNo.NP₈01154）能够和哈维氏弧菌V.harveyi全菌多抗产生免疫反应,表明这两种蛋白可以作为3种弧菌的交叉保护性抗原,以此制备的疫苗可望对3种弧菌的感染产生交叉保护作用。     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong.

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

Development and efficacy of an attenuated Vibrio harveyi vaccine candidate with cross protectivity against Vibrio alginolyticus

Vibrio harveyi is a Gram-negative bacterial pathogen that can infect a wide range of marine animals. In previous studies, we have reported a virulent V. harveyi strain, T4D. In the present study, an attenuated mutant of T4D, T4DM, was obtained by selection of rifampicin resistance. Compared to the wild type, T4DM was different in whole-cell protein profile and much slower in growth rate when cultured in stress conditions caused by iron depletion. Virulence analysis showed that compared to T4D, T4DM exhibited a dramatically increased median lethal dose, impaired tissue dissemination capacity, defective hemolytic activity, and significantly reduced resistance against the killing effect of host serum. To examine the potential of T4DM as a live attenuated vaccine, Japanese flounder (Paralichthys olivaceus) were vaccinated with T4DM via intraperitoneal injection or immersion. The results showed that at one and two months post-vaccination, fish administered with T4DM via both approaches, in particular that of immersion, were effectively protected against not only V. harveyi but also Vibrio alginolyticus, another important fish pathogen. Microbiological analysis showed that following immersion vaccination, T4DM was recovered from the internal organs of the vaccinated fish in a time-dependent manner within the first 6 days post-vaccination. Serum antibodies against V. harveyi and V. alginolyticus were detected in T4DM-vaccinated fish, and, compared to serum from control fish, serum from T4DM-vaccinated fish was significantly enhanced in bactericidal activity. These results indicate that T4DM is an attenuated strain with residual infectivity and that T4DM can induce effective cross-species protection against both V. harveyi and V. alginolyticus when used as a live immersion vaccine.     

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims:  To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins.
Methods and Results:  A gene ( vhhP2 ) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24  V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non- V. harveyi species, including V. parahaemolyticus and V. alginolyticus . A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2 . This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii , which is most closely related to V. harveyi . One of the V. campbellii strains was falsely identified as V. harveyi .
Conclusions:  vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non- V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi . However, this method can not distinguish some V. campbellii strains from V. harveyi .
Significance and Impact of the Study:  the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.     

The mechanism of swarming of Vibrio alginolyticus

Factors leading to swarming of Vibrio alginolyticus cells on solid media were studied. Polar flagellated rods from liquid medium develop into small colonies on solid medium. Byproducts, accumulating in the colony area, induce at certain critical concentrations, the formation of peritrichous flagella and development of long heavily flagellated filaments which swarm away form the high by-product concentrations. Several types of nonswarming mutants were isolated, among them, mutants which lack the capacity to form swarming-inducing pyproducts, but can be induced to swarm by byproducts of other mutants incapable of swarming. Different compounds could replace the natural metabolic byproducts; at very low concentration these compounds induce peritrichous flagella and swarming in some of the nonswarming mutants mentioned above. The natural metabolic byproducts accumulating in yeast-extract-peptone medium are suggested to be volatile acids belonging to the valine and isoleucine pathway. Wild-type V. alginolyticus cells cannot swarm on certain substrates but its mutants, able to swarm on many substrates in minimal media, are easily selected.     

Regulation of polar flagellar number by the flhF and flhG genes in Vibrio alginolyticus

The number and location of bacterial flagella vary with the species. The Vibrio alginolyticus cell has a single polar flagellum, which is driven by sodium ions. We selected mutants on the basis of reduced swarming ability on soft agar plates. Among them, we found two mutants with multiple polar flagella, and named them KK148 and NMB155. In Pseudomonas species, it is known that FlhF and FleN, which are FtsY and MinD homologs, respectively, are involved in regulation of flagellar placement and number, respectively. We cloned homologous genes of V. alginolyticus, flhF and flhG. KK148 cells had a nonsense mutation in flhG; cells expressing transgenic flhG recovered the swarming ability and had a reduced number of polar flagella. NMB155 cells did not have a mutation in either flhF or flhG. In wild-type cells, expression of flhF increased the number of polar flagella; in contrast, expression of flhG reduced both the number of polar flagella and the swarming ability. These results suggest that FlhG negatively regulates the number of polar flagella in V. alginolyticus. KK148 cells expressing both flhF and flhG exhibited fewer polar flagella and better swarming ability than KK148 cells expressing flhG alone, suggesting that FlhG acts with FlhF.