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Vibrio limimangrovi sp. nov., Vibrio phycocola sp. nov. and Vibrio sediminis sp. nov., isolated from coastal habitats.

Four marine bacterial strains of the genus Vibrio, designated WJH972T, RC27, FJH11T, and HA2012T, were isolated from coastal habitats in China. Phenotypic and physiological characterization showed that all four strains are Gram-stain-negative, facultatively anaerobic, short rod-shaped cells, and motile. The 16S rRNA gene sequence similarities between the isolates and their closest relatives were 96.5% - 98.2%, above the genus-level threshold but below the species delineation cutoff. Genomic analysis revealed that strains WJH972T and RC27 shared an average nucleotide identity (ANI) of 98.7% and a digital DNA-DNA hybridization (dDDH) value of 89.9%, confirming their conspecificity. In contrast, the ANI and dDDH values between the four strains and known Vibrio species ranged from 78.6% to 85.8% and from 13.7% to 59.3%, respectively, both falling below the thresholds for species delineation. The biosynthetic gene clusters support bacterial potentials for stress tolerance and antibiotic synthesis, consistent with strain FJH11T's resistance to all 20 antibiotics and other strains' sensitivity to chloramphenicol and penicillin. Based on these polyphasic taxonomic evidence above, three novel species are thus proposed: Vibrio limimangrovi sp. nov. (type strain FJH11T), Vibrio phycocola sp. nov. (type strain WJH972T), and Vibrio sediminis sp. nov. (type strain HA2012T). This study provides novel insights into the systematics, metabolic diversity, antibiotic resistance, and ecological distribution of the genus Vibrio.

Vibrio

Vibrio cholerae flagellar antigens: a serodiagnostic test, functional implications of H-reactivity and taxonomic importance of cross-reactions within the Vibrio genus.

Serodiagnostic tests for all serotypes of Vibrio cholerae using H-antisera were investigated. Activity motile cell lines of 155 stock and international reference cultures of human, animal, fish, and halophilic Vibrios, Aeromonas, Comomonas, Pseudomonas, Salmonella, and Escherichia were investigated. Without exception, all cholera vibrios (including the NAG serotypes) reacted with H sera. Positive reactions were obtained specifically (a) within 2 hrs at 52 degrees C in the tube test using thick formalized suspensions and H antisera at optimal proportion titre and (b) within 30 sec by slide agglutination of fresh cultures. The other vibrios investigated reacted similarly with their homologous H antisera. 2. The rapid diagnostic techniques of fluorescent antibody labeling or immobilization were unsuccessful, V. cholerae flagella being refractive to H sera in these tests. V. cholerae was, however, sensitive in a type-specific manner to O antisera. These and related observations suggest that O antigen has a functional role in Vibrio motility. 3. Interspecies H cross-reactions between V. cholerae and fish and animal vibrios which correlated with bacteriologic similarity, were demonstrated. O antigens of these vibrios were strain specific. Cross-absorption analysis indicated that the H antigens of vibrios were characteristic and homogenous within the species, and therefore a potentially important taxonomic criterion of Vibrio species.

Adsorption

Incidence of Vibrio parahaemolyticus bacteriophages and other Vibrio bacteriophages in marine samples.

Vibrio bacteriophages were isolated by enrichment from 177 of 643 samples of marine molluscan shellfish, crustaceans, seawater, and sediments. The predominant bacteriophage types isolated were specific for some strains of Vibrio parahaemolyticus. A high frequency of phage isolations was also observed with strains of agar-digesting vibrios (21 of 56) and psychrophilic vibrios (14 of 72) that were originally isolated from non-shellfish growing areas. No bacteriophages were isolated against V. alginolyticus and only rarely for V. anguillarum even though these were the two most abundant species found in near-shore environments. No V. cholerae phages were isolated. It was also determined from quantitative studies on the Pacific oyster (Crassostrea gigas) obtained from two environments in Washington and Oregon that the titers of V. parahaemolyticus bacteriophages increased with increasing seasonal water temperatures and that this was proportional to the increase in numbers of mesophilic vibrios and not with the incidence of V. parahaemolyticus. Titers of V. parahaemolyticus bacteriophages occasionally exceeded 10(6) per g of oyster during the summer months. Specific V. parahaemolyticus bacteriophages were also isolated from market seafoods and other marine samples that originated in cold environments where no mesophilic vibrios are expected to be found. The possibility that V. parahaemolyticus bacteriophages originate from Vibrio spp. other than V. parahaemolyticus and the role of these bacteriophages in the ecology of marine vibrios are discussed.

Animals

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.

Agar

Vibrio phycocola sp. nov. and Vibrio phycohabitans sp. nov., Isolated from the Phycosphere of Marine Algae.

Two Gram-stain-negative, facultatively aerobic, oxidase- and catalase-positive, motile (by means of a polar flagellum) rod-shaped bacterial strains, designated BS-M-Sm-2T and MA40-2T, were isolated from marine algae. Growth was optimal at pH 7.0-8.0 and 2.0-3.0% (w/v) NaCl, with temperature optima of 25°C for BS-M-Sm-2T and 25-30°C for MA40-2T. Ubiquinone-8 was the sole respiratory quinone. The major fatty acids common to both strains were C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), while BS-M-Sm-2T additionally contained C12:0 and C14:0. The predominant polar lipids were phosphatidylethanolamine and phosphatidylglycerol, with diphosphatidylglycerol also detected in strain MA40-2T. The DNA G+C contents of strains BS-M-Sm-2T and MA40-2T were 44.2 and 39.8 mol%, respectively. The 16S rRNA gene sequence similarity, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) values between the two strains were 93.8%, 71.4%, and 23.2%, respectively. Phylogenetic and phylogenomic analyses placed both strains within the genus Vibrio, forming distinct lineages. Comparisons with closely related Vibrio type strains yielded ANI and dDDH values below 91.6% and 44.3%, respectively, further supporting their classification as novel species. Genome analyses revealed genes potentially involved in algal symbiosis, including those for polysaccharide degradation and vitamin biosynthesis. Based on comprehensive genomic, phylogenetic, phenotypic, and chemotaxonomic evidence, strains BS-M-Sm-2T and MA40-2T represent two novel species, for which the names Vibrio phycocola sp. nov. (BS-M-Sm-2T =KACC 24066T =DSM 119941T) and Vibrio phycohabitans sp. nov. (MA40-2T =KACC 24064T = DSM 119942T) are proposed.

RNA, Ribosomal, 16S

[Phage types of Vibrio cholerae and non agglutinable Vibrio isolated in Algeria from 1975 to 1977].

1 285 strains of Vibrio cholerae and 556 strains of non agglutinable Vibrio were isolated either from stool specimens or water in Algeria from 1975 to 1977. These strains were phage-typed with Basu and Mukerjee's 5 phages for the Vibrio biotype El Tor and with Mukerjee's phage IV for the Vibrio biotype cholerae. The results obtained are discussed in terms of choleraphages used for the diagnosis and epidemiological study of cholera.

Agglutination Tests

Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios.

A thermolabile direct hemolysin from an El Tor cholera vibrio strain has been isolated and partially characterized as a simple protein of ca. 20,000 molecular weight. In addition to its hemolytic activity, the hemolysin is cytotoxic, cardiotoxic, and rapidly lethal. In these respects it resembles the thermostable direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial hemolysins, although there are other significant differences. Because identical diseases are produced by both hemolytic and nonhemolytic cholera vibrios, the El Tor hemolysin may be presumed to be pathogenetically irrelevant. These observations raise the question of "When is a toxic substance also a toxin?"

Adrenal Glands

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions.

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25 degrees C. Replacement of K+ with Na+ effectively prevented the lysis by SCN-.K+ salts of NO3-, Br- and I-, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN- than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN- is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na+ observed here was considered to be manifested by specific interactions of Na+ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.

Bacteriolysis

[Study of Vibrio cholerae fractions. II. A comparison of the immunochemical and biological properties of preparations of alkaline extracts (vibrionin) and the endotoxin of Vibrio cholerae].

Comparative study of immuno-chemical and biological properties of vibrionin and cholera vibrio endotoxin showed vibrionin to contain up to 73.5% of protein, whereas endotoxin preparations contained only 25% of protein. Use of disc electrophoresis in polyacrylamide gel permitted to determine the relative electrophoretic mobility of the protein component bands of both preparations. Residual quantities of O-antigenic cholera vibrio determinants were revealed serologically in the vibrionin preparations. Study of biological properties of vibrionin (cholera allergen) demonstrated a possibility of using vibrionin in the capacity of a new bacterial allergen.

Animals

[Isolation under the influence of tetracycline of L-forms of Menchikov's vibrio and the NAG vibrio and their biological properties].

Stable L-forms were for the first time obtained under the effect of tetracycline on the Mec thnikov vibrio and NAG vibrio. The process of conversion of the L-forms under the effect of tetracycline was the same as under the action of penicilline. Three types of the L-forms were obtained: stable tetracycline-resistant L-forms, stable tetracyclin-superresistant and tetracycline-dependent L-forms. Under the reversion of the unstable L-forms strains with a typical complex of biological properties and atypical revertants were obtained.

Bacteriological Techniques

Adhesive properties of Vibrio cholerae: nature of the interaction with intact mucosal surfaces.

Two companion papers in this series have characterized the interaction between Vibrio cholerae and the surfaces of eukaryotic cells. The present paper reports studies of the association between vibrios or Salmonella enteritidis and intact slices of intestinal tissue. A significant number of differences were noted in the characteristics of bacterial adhesion in these systems. The results are interpreted to indicate the presence of at least two receptors for vibrio adhesion on the mucosal surface of the rabbit small intestine. The receptor mediating the adhesion of salmonella appeared to be distinct from these. A primary role for bacterial motility in the process of adhesion of vibrios to mucosal surfaces could not be demonstrated in the assay systems studied. Rather, loss of motility in mutant vibrios appeared to be correlated with the simultaneous loss of adhesive factors (adhesins) from the bacterial surface. The inhibition of vibrio adhesion to slices of intestinal tissues by antibody to the heat-stable antigens of V. cholerae occurred in the absence of bacterial agglutination. Agglutination in this assay system appeared to be an artifact in that it could be observed only in experiments where extremely high concentrations of vibrios were used. We speculate that such high vibrio concentrations are not likely to be present in humans at the time of infection and that agglutination in the lumen of the intestine might therefore play only a minor role in prophylactic immunity against natural cholera and other enteric infections of humans.

Agglutination

Superoxide dismutase and catalase levels in halophilic vibrios.

Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance viruulence by allowing the organisms to survive in oxygenated environments.

Aerobiosis

Non-cholera vibrio infections in the United States. Clinical, epidemiologic, and laboratory features.

Non-cholera vibrios are organisms that are biochemically indistinguishable from Vibrio cholerae but do not agglutinate in vibrio 0 group 1 antiserum. Since 1972 there has been a dramatic increase in the number of these organisms referred to the Center for Disease Control for identification. Clinical, epidemiologic, and laboratory data were analyzed for 26 of 28 patients with isolates identified between January 1972 and March 1975. Thirteen (50%) of the isolates were obtained from feces of patients who had an acute diarrheal illness; no other pathogens were isolated from their feces, and all patients survived. Four (15%) patients had non-cholera vibrios isolated from other gastrointestinal or biliary tract sites; none of these patients had acute illness definitely attributable to non-cholera vibrios. Nine (35%) patients had non-cholera vibrios isolated from other tissues and body fluids; four deaths occurred in this group. Patients with acute diarrhea frequently had a history of recent shellfish ingestion or foreign travel, whereas some patients with systemic non-cholera vibrio infection had a history of recent occupational or recreational exposure to salt water.

Adolescent