Serotyping of <Emphasis Type="Italic">Proteus mirabilis</Emphasis> clinical strains based on lipopolysaccharide O-polysaccharide and core oligosaccharide structures

The aim of this work was to serotype Proteus mirabilis urinary tract infection (UTI) strains based on chemically defined O-antigens with the use of two clinical collections from Sweden and Poland consisting of 99 and 24 UTI strains, respectively. A simple two-step serotyping scheme was proposed using enzyme immunoassay with heat-stable surface antigens of Proteus cells and immunoblotting with isolated lipopolysaccharides (LPSs). Using polyclonal anti-P. mirabilis rabbit antisera, 50 Swedish and 8 Polish strains were classified into serogroups O10, O38, O36, O30, O17, O23, O9, O40, O49, O27, O5, O13, O24, O14, and O33. From the Swedish strains, 10 belonged to serogroup O10 and five to each of serogroups O38, O36, and O9. Therefore, none of the O-serogroups was predominant. The majority of the serotyped clinical strains possess acidic O-antigens containing uronic acids and various acidic non-carbohydrate substituents. In immunoblotting, antisera cross-reacted with both O-antigen and core of LPSs. The core region of 19 LPSs bound a single serum, and that of 12 LPSs bound more than two sera. Following bioinformatic analysis of the available sequences, a molecular approach to the prediction of Proteus core oligosaccharide structures was proposed. The identification of the core type of P. mirabilis R110, derived from a serogroup O3 wild strain, using restriction fragments length polymorphism analysis of galacturonic acid transferase is shown as an example. In summary, the most frequent O-serogroups among P. mirabilis UTI stains were identified. The diversity of serological reactions of LPSs is useful for serotyping of P. mirabilis clinical isolates. A possible role of the acidic components of O-antigens in UTI is discussed.     

Vibrio trachuri Sp. Nov., a New Species Isolated from Diseased Japanese Horse Mackerel

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4-diamino-6,7-diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.     

Evidence for the presence of a K antigen in strains ofVibrio anguillarum

ElevenVibrio anguillarum O group 1 strains, isolated from different species of diseased fish, were selected for an immunoelectrophoretic study. Antigenic preparations for immunoelectrophoresis were simple water extracts boiled for 1 h. Using O and OK antisera, immunoelectrophoretic patterns suggested the presence of a K antigen; there is evidence that examined strains possess a common K antigen.     

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.     

Evidence for two newVibrio anguillarum K antigens

Surface polysaccharides from five strains ofVibrio anguillarum were studied by means of immunoelectrophoretic procedures. The study suggested existence of two new K antigens, displaying cross-reactivity, in strains derived from diseased feral fish. The importance of a detailed serologic characterization of isolates for ecologic and epidemiologic studies ofV. anguillarum is considered.     

Extended serotyping scheme forVibrio cholerae

Fifty-seven new O serogroups have been added to the existing serotyping scheme ofVibrio cholerae to extend the scheme from O84 to O140. Prominent new additions were serogroups O139 and O140. The reference strain of O139 was isolated from a patient from an epidemic of cholera-like diarrhea in Madras, Southern India. Serogroup O140 was assigned to a group ofV. cholerae strains which were tentatively named as the Hakata serogroup and which possessed the C (Inaba) factor but not the B (Ogawa) nor the A (major specific antigen of O1 serogroup ofV. cholerae). As all antisera against reference strains ofV. cholerae contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera must be absorbed with the reference rough strain, CA385.     

RpoN gene,RAPD profile,antimicrobial resistance and plasmids of Vibrio anguillarum isolates from vibriosis infected Penaeus monodon

Aim: To evaluate the diversity of Vibrio anguillarum isolates from vibriosis‐infected Penaeus monodon collected from east coast of India. Methods and Results: Thirty‐six V. anguillarum were cultured from specific V. anguillarum medium, further identified using biochemical tests and confirmed by PCR detection of rpoN gene. Randomly amplified polymorphic DNA analysis revealed that in each location, the selected V. anguillarum isolates produced a unique band pattern, indicating that the members of this species are genetically heterogeneous. Antibiotic sensitivity results showed that 85%, 72%, 70%, 58%, 45% and 34% of the isolates were resistant to erythromycin, furazolidone, chloramphenicol, oxolinic acid, ciprofloxacin and nitrofurantoin, respectively. Plasmids were found in 70% of the isolates, and nine different plasmid profiles were observed. Conclusions: Wide ranges of diversity were noted in V. anguillarum isolates collected from P. monodon at different locations of east coast of India. Significance and Impact of the Study: Molecular typing, antibiotic resistance and plasmid profiles of V. anguillarum isolates from shrimp in India enables the prediction of possible risk for diseases in shrimp culture environment and the application of alternative management plans to prevent further spread of antibiotic resistance.     

The Benefit of a Roseobacter Species on the Survival of Scallop Larvae

A marine strain (BS107), identified as a Roseobacter species, was antagonistic to Vibrio species on agar plates. Results suggested that the inhibitory effect was displayed only in the presence of another bacterium. Quantification of the antibacterial activity showed that 48-hour-coculture supernatants from BS107 and another bacterial strain (V. anguillarum 408) reached the highest titers of bacterial inhibition. The antibacterial substance was also liberated when supernatants from V. anguillarum 408 were added to pure cultures of the inhibition-productive bacterium. The presence of a proteinaceous molecule may induce BS107 to display the inhibitory effect. The antibacterial substance was sensitive to trypsin (8000 U/ml) and stable at 100°C. Cell extracts of the isolate BS107 (10⁶ cells/ml) significantly enhanced scallop larval survival, thus being beneficial to the rearing process. Received December 8, 1997; accepted July 15, 1998.     

Surface Display of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> GAPDH in Attenuated <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> to Develop a Noval Multivalent Vector Vaccine

Displaying foreign antigens on the surface of attenuated or avirulent bacteria is an important strategy to develop live multivalent vector vaccines. In our previous work, several efficient surface display systems have been established based on outer membrane anchoring elements, which could successfully display heterologous proteins in attenuated Vibrio anguillarum. In this work, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila LSA34 was fused to seven display systems and introduced into attenuated V. anguillarum strain MVAV6203 (AV) to get seven GAPDH-display strains. The strain AV/pN-gapA showed the best display efficacy of GAPDH and was tested as the multivalent vaccine candidate. Further immune protection evaluation of AV/pN-gapA in turbot (Scophtalmus maximus) demonstrated that the attenuated V. anguillarum with surface-displayed GAPDH of A. hydrophila LSA34 effectively protected turbot from the infections of A. hydrophila and V. anguillarum and showed potential value for further multivalent vaccine development.     

Development of a DNA probe using differential hybridization to detect the fish pathogenVibrio anguillarum

The fish pathogenVibrio anguillarum causes significant economic losses in commercially cultured fish species worldwide. At present, identification ofV. anguillarum requires conventional isolation and culturing techniques. Using differential hybridization, a 310 base pairV. anguillarum-specific DNA fragment was isolated for use as a probe. In specificity studies against 19 different bacterial species, including twoVibrio sp. and fish pathogens, and 223 marine bacterial isolates, the probe hybridized exclusively toV. anguillarum strains. The probe also strongly hybridizes to 7 of 9 serotypes tested, with serotype 09 giving a weak probe reaction and serotype O7 negative. The probe allows rapid and accurate detection of both pathogenic and environmental strains ofV. anguillarum.     

Further antigenic analyses of the fish pathogenic bacteriumVibrio anguillarum

TenVibrio anguillarum strains were selected for an immunoelectrophoretic study. Evidence was provided for existence of two new K antigens which displayed cross-reactivity. The importance of an exact characterization of surface antigens inV. anguillarum is considered.     

Serological Reactivity of Humans to a Vibrio cholerae Common Antigen

Over the course of seven pandemics, Vibrio cholerae serotypes have varied. In 1992 the appearance of a new serotype, O139 Bengal, began the eighth cholera pandemic. Several new O139 antigens have been identified, yet a common V. cholerae antigen has not been described. In this study, a monoclonal antibody specific against an 18.7-kDa outer membrane antigen reacted in dotblot analysis with 292 epidemiologically diverse V. cholerae isolates including O1, non-O1, and O139 serotypes. Serum collected from volunteers experimentally challenged with V. cholerae O139, and rabbit antisera to V. cholerae O1, were reactive with the 18.7-kDa antigen by Western immunoblot. This is the first report that the 18.7-kDa antigen is present in V. cholerae O139. Received: 11 August 1997 / Accepted: 22 September 1997     

Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus > V. anguillarum > M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+ 35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+ 16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus > V. anguillarum > M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.     

Immunoelectrophoretic study of lipopolysaccharide antigens fromVibrio anguillarum strains,serogroup O2

Vibrio anguillarum isolates, derived from feral as well as cultured fish and recorded as serogroup O2 by slide agglutination, were selected for an immunoelectrophoretic study of lipopolysaccharide antigens. Antigenic preparations for the immunoelectrophoretic analyses were simple water extracts, heated to 100°C for 1 h. Two immunoelectrophoretic distinct lipopolysaccharide entities were detected. The analyses did not demonstrate serologic variations in lipopolysaccharide antigens among 16 O group 2 strains. The study also included an O1K1V. anguillarum strain. Antigenic extract from this strain was not precipitated by OK antiserum againstV. anguillarum serogroup O2.     

The detection of fish pathogenVibrio anguillarum in water and fish using a species-specific DNA probe combined with membrane filtration

The marine bacteriumVibrio anguillarum causes disease in fish worldwide and is particularly devastating in aquaculture. Little is known about the ecology ofV. anguillarum in the environment and how this may relate to the pathogenicity of this organism. Combining membrane filtration and a species-specific DNA probe, culturableV. anguillarum cells were detected in water from three habitats and in chinook salmon (Onchorynchus tshawytscha) tissue samples. Results show that different marine habitats have a marked effect on cell numbers and that water temperature may play a role in the culturability and distribution ofV. anguillarum. Vibrio anguillarum was detected from the gills of salmon within 24 h of transfer of fingerlings from freshwater to seawater, with cell numbers reaching a concentration of 1.9 × 10² cells g^–1 tissue 28 days post transfer.Vibrio anguillarum cell numbers were low in the colon throughout the study, andV. anguillarum was not detected in healthy kidney samples. The methodology reported in this paper allows the accurate quantification of culturableV. anguillarum cells and has allowed a preliminary study of the ecology of this species.     

Intraspecific blood group properties in the tilapiine species Oreochromis aureus and O. niloticus and the occurrence of soluble blood group substances

Genetic differences within species of tilapias were investigated. These differences are, for instance, expressed in the molecular composition of the erythrocyte membrane. Specimens of Oreochromis aureus (Steindachner 1864) and O. niloticus (Linné 1766) were immunized with alloantigenic erythrocytes. Results ofagglutination assays with the obtained antisera indicated the occurrence of a minimum offive and seven isoantigens in the investigated groups of O. aureus and O. niloticus, respectively. Comparative tests did not reveal an identity of any of the O.aureus and O. niloticus isoantiens. Apart from these ceflular blood group structures, the existence of soluble blood group substances in the plasma of the two investigated species was indicated by WESTERN blot tests.     

Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity.     

Phenotypic Characteristics and Virulence of Vibrio anguillarum-Related Organisms

The phenotypic, molecular, and virulence properties of 46 Vibrio anguillarum-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of V. anguillarum and the Vibrio splendidus type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from V. anguillarum were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4°C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of V. anguillarum, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of V. anguillarum. The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10² to 10⁶ cells) included V. anguillarum strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10 V. anguillarum serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among V. anguillarum strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of V. anguillarum tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with V. anguillarum in the formulation of vaccines against vibriosis.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

Detection of acylated homoserine lactones produced by Vibrio spp. and related species isolated from water and aquatic organisms

Aims: To assess the diversity in production of acylated homoserine lactones (AHLs) among Vibrio spp and related species. Methods and Results: A total of 106 isolates, with representatives of 28 Vibrio spp and related species, were investigated for the production of AHLs. For this, a rapid method for the screening of AHLs was developed based on the use of bacterial biosensors using a double‐layer microplate assay. At least one bacterial biosensor was activated in 20 species, Agrobacterium tumefaciens being the most frequently activated biosensor. One isolate of Vibrio anguillarum, Vibrio rotiferianus and Vibrio metschnikovii activated the Chromobacterium violaceum biosensor, which is not common among the Vibrionaceae family. For those species with more than one isolate, the biosensor activation profile was the same except for two species, V. anguillarum and V. metschnikovii, which varied among the different isolates. Conclusions: AHL production was observed in the majority of the studied species, with a diverse biosensor activation profile. Significance and Impact of the Study: The high diversity in AHL production is in consistence with the high diversity in ecological niches of the Vibrionaceae family. The absence of AHL detection in eight species warrants further work on their quorum‐sensing systems.