Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.

Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin.

A clinical isolate (strain 4037) of Kanagawa phenomenon-negative Vibrio parahaemolyticus was studied. Although the strain was judged to be Kanagawa phenomenon-negative by various conventional tests, it produced a new hemolysin (named Vp-TRH, for thermostable direct hemolysin [Vp-TDH]-related hemolysin) that was related to the Vp-TDH produced by ordinary Kanagawa phenomenon-positive V. parahaemolyticus. Vp-TRH was purified by ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, and Mono Q. The molecular weight of Vp-TRH was estimated as 48,000 by Sephadex G-100 gel filtration, and the molecular weight of the subunit was estimated to be 23,000 by sodium dodecyl sulfate-slab gel electrophoresis. Thus, like Vp-TDH, Vp-TRH seems to be composed of two subunits. The isoelectric point of Vp-TRH was determined to be 4.6. Vp-TRH showed lytic activities different from those of Vp-TDH on erythrocytes from various animals, especially those from calves, chickens, and sheep. The hemolytic activity of Vp-TRH was labile on heat treatment at 60 degrees C for 10 min, unlike that of Vp-TDH. The immunological similarities, but not the identities of Vp-TRH and Vp-TDH, were demonstrated by Ouchterlony, neutralization, and latex agglutination tests. Thus, we conclude that this clinical isolate of Kanagawa phenomenon-negative V. parahaemolyticus produces a new type of hemolysin that is similar, but not identical, to Vp-TDH, which is usually produced by Kanagawa phenomenon-positive V. parahaemolyticus.

Antitumor activity of lipopolysaccharide and radio-detoxified lipopolysaccharide of Vibrio parahaemolyticus.

The antitumor activity of lipopolysaccharide (LPS) and radio-detoxified LPS of Vibrio parahaemolyticus was tested against S180 cells in Swiss mice. The toxicity of the LPS was 200 times less than that of Salmonella typhimurium LPS. The V. parahaemolyticus LPS could be detoxified by exposure to gamma radiation. Both LPS and the irradiated LPS exhibited antitumor activity, though the irradiated LPS was less effective than the native LPS. These observations indicated that exposure to gamma radiation caused significant detoxification of V. parahaemolyticus LPS and the detoxified LPS still possessed considerable antitumor activity.

Effect of D-tryptophan on hemolysin production in Vibrio parahaemolyticus.

Production of the Kanagawa hemolysin by a strain of Vibrio parahaemolyticus isolated from a gastroenteritis patient was found to correlate with the presence in cell lysates of two unidentified compounds, designated X and Y. The two compounds were present in cell lysates of the organism grown in peptone at the optimal pH for hemolysin synthesis but were not present when cell lysates were grown in peptone at a constant pH of 8.0. They were also absent in cells grown in synthetic medium at pH 6.2 without the addition of D-tryptophan, a condition under which hemolysin is not produced. Both X and Y were present intracellularly only from the time that D-tryptophan was added to synthetic medium, a known method of inducing hemolysin synthesis.

Isolation of mucoid Vibrio parahaemolyticus strains.

Mucoid strains of Vibrio parahaemolyticus were isolated from the stools of two asymptomatic carriers and a patient with gastroenteritis. The strains demonstrated biochemical reactions and antibiotic susceptibility typical of nonmucoid strains of V. parahaemolyticus isolated locally. The slime substance was typed by coagglutination and was antigenically similar to the capsular antigen of the same strain. Three different serotypes (O10:K24, O5:K17, and O5:K15) were involved.

Food microbiology update. Emerging foodborne pathogens.

A review of three "emerging" foodborne pathogen groups is presented, including Campylobacter jejuni/coli, Yersinia enterocolitica, and foodborne Vibrio sp.

Isolation and characterization of the outer membrane from Vibrio parahaemolyticus.

The outer membrane of Vibrio parahaemolyticus strain 3283-61 (serotype O2:K3) was isolated from blebs released upon spheroplast formation, in the presence of lysozyme and EDTA, by isopycnic sucrose density gradient centrifugation. SDS-PAGE of the outer membrane fraction prepared from cells grown in nutrient broth containing 3% (w/v) NaCl revealed five major proteins, designated a to e, with apparent approximate molecular weights: a, 44 000; b, 36 000; c, 33 500; d, 26 500; e, 22 000. An increase in NaCl concentration in the growth medium resulted in an increase of proteins b and c, whereas a decrease to 0.5% (w/v) induced two additional major proteins with respective molecular weights of about 35 000 and 32 000. Proteins a and b appeared to be loosely associated with the peptidoglycan layer since they were largely retained after extraction with 2% (w/v) SDS at 50 degrees C for 30 min. Proteins c and/or e may play a role in phage VP1-receptors since phage-resistant mutants derived from strain 3283-61 had significantly diminished amounts of both proteins. The major outer membrane proteins varied in number and molecular weight in strains of V. parahaemolyticus belonging to different K-serotypes.

Isolation and partial properties of a porin-like protein from Vibrio parahaemolyticus cell envelope.

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 M NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.

Sugar composition of O-antigenic lipopolysaccharides isolated from Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a causative bacterium of food poisoning unique for its particular primary association with sea products, is now divided serologically into 11 or 12 O-forms based on agglutination and agglutinin-absorption tests. We determined the sugar composition of the somatic O-antigens, i.e., lipopolysaccharides (LPS), of representative strains of each O-form. Of particular interest is the absence of evidence for the presence of 2-keto-3-deoxy-octonic acid (KDO), a regular sugar component of gram-negative bacterial LPS, in any LPS examined, with the exception of 06. Furthermore, 07 and 012 LPS contained a KDO-like compound that is, however, not identical with KDO. Glucose, glucosamine, and L-glycero-D-mannoheptose were found as common sugar constituents. Three unidentified amino sugars, designated here as P1, P2, and P3, were found. Various combinations of each of these unidentified amino sugars, and of galactose, fucose, arabinose, D-glyucero-D-mannoheptose, galactosamine, KDO, and the KDO-like substance were detected in accordance with the O-form of LPS. On the basis of the sugar composition, LPS of the 12 O-forms of V. parahaemolyticus can be classified into nine chemotypes, because 03, 05, and 011 LPS belong to the same chemotype and 07 and 012 to another chemotype.

Investigation of an intracellular hemolytic agent of Vibrio parahaemolyticus. Lipid fraction of dried cells.

The dried cells of two strains of Vibrio parahaemolyticus were fractionated by extracting first with water and then with organic solvents. The hemolytic activity of the fractions was determined, and some of them were assayed for their effect in mice. The hemolytic agent present in the water-insoluble fraction was extractable in organic solvents such as 70% aqueous ethanol, chloroform-methanol-water (1:2:0.8) and acetone. The extracts showed no toxic effect in mice after intraperitoneal inoculation. No hemolytic activity was observed in the remaining cell residue, which bore the toxicity.

Effect of hemolysin produced by Vibrio parahaemolyticus on membrane conductance and mechanical tension of rabbit myocardium.

The mechanisms of the action of hemolysin extracted from Vibrio parahaemolyticus in the S-A node and right atrium cells of rabbit were studied by means of the single sucrose gap and isometric tension recording methods. Hemolysin caused the membrane to depolarize reversibly without affecting the action potential generating mechanism. Lowering of [Na+]o inhibited membrane depolarization in the presence of hemolysin while the readmission of normal Tyrode solution induced depolarization. Tetrodotoxin (TTX) barely antagonized the depolarizing action of hemolysin but slowed the rate of development of depolarization. Therefore, this depolarization is considered to be primarily due to the increase in conductance to Na which TTX may not block. The dose-response relationship was obtained by measuring a change in membrane resistance. The concentration necessary to yield one-half of the maximum reduction of the membrane was determined to by 7.5 micrograms/ml. Accumulation of Na within the cell may be responsible for an increase of twitch tension observed during the action of a low concentration of hemolysin. On the other hand, a higher concentration of hemolysin seemed to promote exchange of intracellar Na with extracellular Ca, especially when the Na concentration of the perfusing solution was reduced, and led to stronger contracture.

Toxin isolation from a Kanagawa-phenomenon negative strain of Vibrio parahaemolyticus.

Production of a toxin by Vibrio parahaemolyticus Kanagawa-phenomenon negative strains was examined. Ammonium sulfate fractions of broth culture filtrates were dialyzed, concentrated by lyophilization, and tested for toxic effects by mouse intraperitoneal injection. One fraction, which we think is a toxin, was isolated from a broth culture filtrate of V. parahaemolyticus FC 1011 (a Kanagawa-phenomenon negative strain) and consitently produced lethal effects in mice at high concentrations and diarrhea in lower concentrations. The toxin was assayed for mouse LD50 and ability to produce diarrhea via forced feeding in mice. V. parahaemolyticus FC 1011 toxin was found to be protein, to be inactivated by heat or trypsin hydrolysis, and to produce positive skin permeability reactions in rabbits. However, it failed to induce fluid accumulation in ligated ileal loops in rabbits.

Chemical structure of the peptidoglycan of Vibrio parahaemolyticus A55 with special reference to the extent of interpeptide cross-linking.

The chemical structure of the cell wall peptidoglycan of Vibrio parahaemolyticus A55 was studied. Estimation of cross linkages between peptide subunits in the peptidoglycan by dinitrophenylation showed that about 30% of the total 2,6-diaminopimelic acid (A2pm) residues were involved in cross linkages. The presence of interpeptide bridges was also demonstrated by isolating bisdisaccharide peptide subunit dimers from Chalaropsis muramidase digests of the cell wall peptidoglycan by gel filtration followed by ion-exchange column chromatography, although most of the building blocks obtained were uncross-linked disaccharide peptide monomers. The chain length of a glycan moiety of the peptidoglycan obtained by treatment with the L-11 enzyme and gel filtration of the digest was also studied. The chain length varied from 7 to 44, but 30% of the glycan fragments had muramic acid at the reducing end and a chain length of 28 to 44. In conformity with the above structural study it was demonstrated that a particulate enzyme fraction obtained by differential centrifugation of a sonicated preparation of V. parahaemolyticus catalyzed a penicillin-sensitive transpeptidation reaction, using UDP-MurNAc-14C-pentapeptide and UDP-GlcNAc as substrates.

Studies of the cell envelope of Vibrio para-haemolyticus A55: isolation and purification of bag-shaped peptidoglycan (murein sacculus).

The peptidoglycan (PG) component of the envelope of Vibrio parahaemolyticus A55 was studied in relation to the salt dependency of the organism. A bag-shaped PG (murein sacculus) was isolated and purified by digestion of freshly harvested intact whole cells with sodium dodecyl sulfate (SDS) and then protease treatment. Whole cells lyze in salt-deficient or hypotonic conditions and this can be measured by turbidometry. Lysis is prevented by addition of 0.35 M NaCl or acidification to below pH 4.5. The isolated PG or murein sacculi did not seem to require salt for structural integrity. The PG obtained by the same method from subcellular fractions, that is, envelope fractions, prepared either with or without NaCl, was extensively fragmented. The main amino acids and amino sugars of the murein sacculus were glucosamine, muramic acid, alanine, glutamic acid, 2, 6-diamnopimelic acid (A2pm) in a molar ratio of 0.4:0.6:1.7:0.9:1.0. A polyglucose, which was only degraded by Pseudomonas isoamylase,and poly-beta-hydroxybutyrate (PHBA) were found in the murein sacculus. The murein sacculus of the organism is discussed in comparison with PG of other gram negative bacteria and marine or halophilic microorganisms.

Tolerance and other biological properties of Vibrio parahaemolyticus endotoxins.

Biological properties of endotoxins prepared from three strains of Vibrio parahaemolyticus were compared with reference to commercially prepared Salmonella typhi endotoxin. Endotoxin assays performed in rabbits included dermal Shwartzman reactivity, pyrogenicity, heat stability, and ability to induce tolerance as well as cross-tolerance. Mice were used for endotoxin LD50 determinations. Results showed V. parahaemolyticus endotoxins were similar to that of S. typhi strain O901. Induction of tolerance to V. parahaemolyticus strain 11590 endotoxin resulted in complete cross-tolerance to S. typhi endotoxin, and vice versa. Partial cross-tolerance to S. typhi endotoxin was demonstrated with rabbits rendered tolerant to endotoxin from V. parahaemolyticus strains Sak-3 and FC1011. Absorption spectra, nitrogen, phosphorus and carbohydrate analyses revealed additional similarities between endotoxins from V. parahaemolyticus and endotoxin from a member of the Enterbacteriaceae.