Phylogeny and Molecular Identification of Vibrios on the Basis of Multilocus Sequence Analysis
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ABSTRACT We analyzed the usefulness of rpoA , recA , and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae , Photobacteriaceae , Enterovibrionaceae , and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA , recA , and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri , V. logei , and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli -, V. harveyi -, V. splendidus -, and V. tubiashii -related species formed groups within the genus Vibrio . Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA , recA , and pyrH gene sequence similarity, respectively.Keywords:
Polyphyly
Multilocus sequence typing
Vibrio alginolyticus
Hatchery
Plesiomonas shigelloides
Vibrio vulnificus
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Vibrio fischeri belongs to the Vibrionaceae, a large family of marine γ-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus , and Vibrio vulnificus , the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio , including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes . Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.
Vibrio vulnificus
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Vibrio fischeri isolates from Euprymna scolopes are dim in culture but bright in the host. We found the luminescence of V. fischeri to be correlated with external osmolarity both in culture and in this symbiosis. Luminescence enhancement by osmolarity was independent of the lux promoter and unaffected by autoinducers or the level of lux expression, but the addition of an aldehyde substrate for luciferase raised the luminescence of cells grown at high and low osmolarities to the same high level. V. fischeri culture media have lower osmolarities than are typical in seawater or in cephalopods, partially accounting for the bacterium's low light output in culture.
Osmotic concentration
Strain (injury)
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The comparative study of 95 strains of Aeromonas, Plesiomonas and various Vibrio species by 49 characteristics (disregarding those common to all representatives of the Vibrionaceae family) allowed to distinguish a separate group of microorganisms differing from Aeromonas by 17 characteristics, from Plesiomonas by 13 characteristics and from Vibrio by 7 characteristics. The new microogranism was provisionally named Allomonas. The taxonomic study of the new microorganism continues.
Plesiomonas shigelloides
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Polyamine contents of 29 strains of the genus Vibrio, together with 5 strains of other Vibrionaceae, have been determined by a gas chromatographic method to enlarge our knowledge of the occurrence of an "unusual" polyamine, norspermidine, in the genus Vibrio. The results showed that norspermidine was widely distributed in various Vibrio species, except Vibrio fischeri and V. costicola. Besides Vibrio species, Photobacterium leiognathi and P. angustum in Vibrionaceae also contained norspermidine. When the Vibrio strains were grown in the medium supplemented with 1,3-diaminopropane, they synthesized much larger amounts of norspermidine. This ability was specific to the strains which contained norspermidine when grown in the defined medium. These findings suggested that norspermidine, as well as the ability to synthesize much larger amounts of it in the presence of 1,3-diaminopropane, may be useful generic guides to the genus Vibrio.
Polyamine
Photobacterium
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Numerical analysis was used to characterize 111 Vibrionaceae strains. These included 31 reference cultures belonging to the genera Aeromonas, Listonella, and Vibrio and 80 strains isolated from the seasonally cold coastal waters of Newfoundland. The sources of the regional strains were the brown alga Alaria esculenta and the giant scallop Placopecten magellanicus. Most of the regional strains grew at temperatures associated with psychrotrophs, while a small proportion may have been psychrophilic. All of the regional strains grew at 4 °C. A routine incubation temperature of 20 °C was chosen and in tests for utilization of organic components as sole sources of carbon and energy the strains were incubated for 3 weeks rather than the more common 6-day period. The treatment of weak positive results as weak positive, positive, or negative was investigated and it was decided that the general conclusions reached in the study would not be significantly altered by the interpretation of weak positive results. Using numerical analysis it was shown that most of the strains clustered according to source. Most reference cultures were more closely related to each other than they were to the regional strains. Some strains were phenotypically similar to Vibrio splendidus biovar I, which is arginine dihydrolase positive. Although there were differences, some strains were similar to the fish pathogen Vibrio ordalii, which is negative for arginine dihydrolase. Both species are reported to grow at 4 °C. It was shown that most of the regional Vibrionaceae strains studied were different from previously described species belonging to the family Vibrionaceae.Key words: numerical taxonomy, Vibrionaceae, Vibrio, marine bacteria, psychrotroph.
Biovar
Psychrophile
Plesiomonas shigelloides
Vibrio vulnificus
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E.P. IVANOVA, N.V. ZHUKOVA, N.M. GORSHKOVA AND E.L. CHAIKINA. 2001. Aims: To study the phenotypic and chemotaxonomic (i.e. phospholipid and cellular fatty acid composition) characteristics of environmental Aeromonas spp. and Vibrio spp. isolated from a drinking water reservoir near Vladivostok City, and the application of some chemotaxonomic markers for discrimination of the two genera and species. Methods and Results: Presumptive Aeromonas species were dominant in surface water samples (up to 25% of the total number of bacteria recovered). These strains were consistent with respect to the cultural and biochemical properties used to define the species Aeromonas sobria (seven strains) and Aer. popoffii (three strains). Vibrio mimicus (two strains) and Vibrio metschnikovii (one strain) were identified according to phenotypic features and cellular fatty acid composition. Conclusions: Environmental Aer. sobria isolates were atypical in their ability to grow at 42°C, and were haemolytic, proteolytic and cytotoxic. Although it was present in a high proportion in the water samples, atypical Aer. sobria is not an indicator of polluted water. Significance and Impact of the Study: The incidence of Aeromonas in the drinking water reservoirs in the Far East of Russia is reported for the first time.
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Typing and identification of 60 marine psychrophilic and psychrotrophic vibrios isolated from the north-western Pacific Ocean and coastal environment of Japan were performed by restriction fragment length polymorphism analysis on the basis of polymerase chain reaction amplified 16S rDNA. We obtained 15 operational taxonomic units (OTUs) by digestion with four restriction endonucleases (HhaI, DdeI, RsaI, and Sau3AI); four large groups were obtained from the neighbor-joining method. Significant differences were observed in OTU composition between isolates from the deep sea and coastal areas. Vibrio marinus and Photobacterium species were the dominant culturable vibrios in the deep sea areas, while Vibrio splendidus like species were the dominant culturable vibrios in a coastal area of Japan.Key words: restriction analysis, Vibrio, Photobacterium, Vibrio marinus (Moritella marina), Vibrio splendidus.
Photobacterium
Psychrophile
Marine bacteriophage
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Eighty regional strains of Vibrio isolated from the seasonally cold waters of coastal Newfoundland, and a number of Vibrio reference cultures, were studied. The regional strains had been isolated from the brown macroalga Alaria esculenta and the giant scallop Placopecten magellanicus and were known to grow at 4 °C. The strains were grouped according to their arginine-dihydrolase reactions and examined by numerical analysis. According to phenotypic properties the arginine-dihydrolase positive strains closely resembled Vibrio splendidus biovar I. Most clusters of the arginine-dihydrolase negative strains appeared to be unique but the closest phenotypic resemblance among some strains was with Vibrio ordalii. Some strains were examined using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique for fingerprinting and it was shown that the regional strains were significantly different from either V. splendidus biovar I or V. ordalii. Generally, the strains from seaweed clustered separately from those that were from scallops. Strains in some clusters, especially those from the seaweed, were able to utilize most of the compounds that were tested as sole sources of carbon and energy.Key words: numerical taxonomy, marine bacteria, Vibrio, psychrotrophs, RAPD-PCR.
Biovar
Strain (injury)
Marine bacteriophage
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Sixty isolates, comprising nine species of the family Vibrionaceae, were tested with the API 20E 5-h same-day procedure (Analytab Products, Plainview, N.Y.). Included were 27 Aeromonas hydrophila isolates, 10 Aeromonas sobria isolates, 7 Aeromonas caviae isolates, 3 Plesiomonas shigelloides isolates, 3 Vibrio alginolyticus isolates, 3 Vibrio cholerae isolates, 1 Vibrio fluvialis isolate, 5 Vibrio parahaemolyticus isolates, and 1 Vibrio vulnificus isolate. The 5-h profile numbers were specific for the five Vibrio species and the Plesiomonas isolates. The three Aeromonas species shared seven 5-h profile numbers. Of the 63 5-h profile numbers generated by testing each isolate twice, 22 were identical to those found in the overnight analytical profile index. Of these, 20 were correct identifications, and two were incorrect. The remaining 41 5-h profile numbers were not found in the overnight analytical profile index. Because the 5-h analytical profile index does not contain any oxidase-positive organisms, the oxidase value was subtracted from the 63 5-h profile numbers to determine whether misidentifications could occur if the oxidase test was either not performed or not performed correctly. Only five of these factored profile numbers resulted in a possible misidentification. It is feasible, within limitations, to use the 5-h API 20E same-day procedure to identify the more commonly occurring members of the Vibrionaceae. The manufacturer should develop a data base for this purpose.
Plesiomonas shigelloides
Vibrio vulnificus
Vibrio alginolyticus
Aeromonas caviae
Vibrio Infections
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