Vibriosis

Mol Microbiol, 1999 Feb, 31(4), 1197 - 204
Quorum sensing in Vibrio fischeri: elements of the luxl promoter; Egland KA et al.; Although cell density-dependent regulation of the luminescence genes in Vibrio fischeri is a model for quorum sensing in Gram-negative bacteria, relatively little is known about the promoter of the luminescence operon . The luminescence operon is activated by the LuxR protein, which requires a diffusible acylhomoserine lactone signal . The lux box, a 20 bp inverted repeat, is located in the luxl promoter region and is required for LuxR-dependent induction of the luminescence genes . Using primer extension, we mapped the LuxR-dependent transcriptional start site of the lux operon to 19 bp upstream of the luxl start codon . This indicates that the lux box is centred at -42.5 bp from the start of transcription . To gain evidence about the location of the -10 sequence, we placed a consensus -35 hexamer at different locations relative to the luxl transcriptional start site and measured constitutive levels of luminescence in recombinant Escherichia coli . The strongest constitutive promoter contained a TATAGT hexamer 17 bp from the -35 consensus sequence and 6 bp from the transcriptional start site . We propose that this is the -10 hexamer . Also in recombinant E . coli, both half-sites of the lux box were required for LuxR-dependent gene activation and for activation by an autoinducer-independent, monomeric LuxR deletion protein . LuxR-dependent activation of luminescence was eliminated when the lux box was centred at -47.5, -52.5 and -62.5 with respect to the luxl transcriptional start site . Our evidence, taken together with other information, points to a model in which a LuxR dimer overlaps the -35 region of the luxl promoter and functions as an ambidextrous activator with each LuxR subunit interacting with a different region of RNA polymerase.

Protein Expr Purif, 1999 Apr, 15(3), 381 - 8
Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli; Karamohamed S et al.; ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli . The synthesis of ATP sulfurylase was directed by an expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri . A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E . coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration . The specific activity of the purified enzyme was estimated to 140 U/mg . The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical subunits (the molecular mass of a single subunit is 59.3 kDa) . The ATP sulfurylase activity was monitored in real time by a very sensitive bioluminometric method .

Mil Med, 1999 Mar, 164(3), 198 - 201
Skin and soft-tissue infections after injury in the ocean: culture methods and antibiotic therapy for marine bacteria; Reed KC et al.; Isolated organisms from two common Indo-Pacific marine animals (Echinometra mathaei urchins and Acanthaster planci sea stars) likely to cause puncture wounds to recreational beachcombers, diverse, or operational military forces during amphibious assaults demonstrate why practitioners should consider their first choice for potential antibiotic therapy differently from their usual favorite antibiotics . The effects of thiosulfate-citrate-bile-sucrose (TCBS) agar, varying salt concentrations in the standard media, and comparison of room temperature incubation versus use of the 30 degrees C (86 degrees F) incubator are reviewed . The yield of pathogenic marine bacteria is increased if TCBS agar is used and more than one temperature is used for incubation . A potentially significant human pathogen, Vibrio vulnificus, appears to be ubiquitous.

Infect Immun, 1999 Apr, 67(4), 2030 - 4
Expanded safety and immunogenicity of a bivalent, oral, attenuated cholera vaccine, CVD 103-HgR plus CVD 111, in United States military personnel stationed in Panama; Taylor DN et al.; To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa) . The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer . A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(7) CFU, (b) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(6) CFU, (c) CVD 103-HgR alone at 10(8) CFU, (d) CVD 111 alone at 10(7) CFU, or (e) inactivated Escherichia coli placebo . Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools . None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea . CVD 111 was detected in the stools of 46% of the 103 volunteers who received it . About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers . The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550 . Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300) . The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable . While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.

Infect Immun, 1999 Apr, 67(4), 1694 - 701
In vivo expression and immunoadjuvancy of a mutant of heat-labile enterotoxin of Escherichia coli in vaccine and vector strains of Vibrio cholerae; Ryan ET et al.; Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) of Escherichia coli, the latter when expressed in V . cholerae . Both toxins are also potent immunoadjuvants . Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E . coli have been developed . One such mutant LT molecule has the substitution of a glycine residue for arginine-192 {LT(R192G)} . Live attenuated strains of V . cholerae that have been used both as V . cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed . In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo by V . cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains of V . cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors . We found that LT(R192G) was expressed from pCS95 in vitro by both E . coli and V . cholerae strains but that LT(R192G) was detectable in the supernatant fraction of V . cholerae cultures only . In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the three V . cholerae vaccine strains alone and compared to groups inoculated with the V . cholerae vaccine strains supplemented with purified CT as an oral immunoadjuvant or V . cholerae vaccine strains expressing LT(R192G) from pCS95 . We found that mice continued to pass stool containing V . cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated . We found that inoculation with V . cholerae vaccine strains containing pCS95 resulted in anti-LT(R192G) immune responses, confirming in vivo expression . We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant . We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that received V . cholerae vaccine strains expressing LT(R192G) from pCS95 compared to the responses in animals that received V . cholerae vaccine strains alone . These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains of V . cholerae and that such expression can result in an immunoadjuvant effect.

Antibiot Khimioter, 1998, 43(11), 6 - 10
{Evaluation of antibiotic sensitivity of pathogenic vibrios of various species}; Danilkina EB et al.; The recent increase of the number of antimicrobials and isolation of antibiotic resistant strains from humans and environmental objects is indicative of the necessity of further investigation of antibiotic susceptibility of the representatives of the genus Vibrio pathogenic for man to provide rational therapy of the diseases due to them . Susceptibility of 160 strains of pathogenic vibrios of 9 species to 11 antibiotics and chemotherapeutic drugs was assayed by the method of serial dilutions in agar media . The isolates were shown to be highly susceptible to chloramphenicol, doxycycline, cefotaxime, nalidixic acid and ciprofloxacin which made it possible to consider them as the drugs of choice in the treatment of the diseases caused by the microorganisms . A tendency to form polyantibiotic resistant strains within every species of tested pathogenic vibrios was observed . It conditioned the prospects of further profound study of the phenomenon with the analysis of the genetic determination of antibiotic resistance markers in pathogenic vibrios.

An Med Interna, 1998 Sep, 15(9), 485 - 6
{Vibrio vulnificus septicemia in Spain}; Garcia Cuevas M et al.; Vibrio vulnificus is a virulent marine organism, able to contaminate sea-food . It usually produces bacteremia associated with secondary skin lesions in patients with underlying conditions, such as hepatic cirrhosis . We report a case of septic shock and characteristic skin lesions, due to Vibrio vulnificus in a patient with cirrhosis, who had eaten raw oysters . The patient survived in spite of the severity of the clinical picture . We conclude that Vibrio vulnificus infection must be considered in the differential diagnosis of sepsis and skin lesions.

FEMS Microbiol Lett, 1999 Mar 1, 172(1), 73 - 7
The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source; Miyoshi S et al.; Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins . This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX) . In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-alpha,beta,gamma,delta-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source . When inoculated into a medium containing Fe-TPPS, V . vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply . Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS . The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS . The data suggest that, V . vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme.

Bull Soc Pathol Exot, 1998, 91(5 Pt 1-2), 412 - 5
{The current status of research on a cholera vaccine}; Fournier JM; Cholera remains today a major health problem in most developing countries . The long-term control of cholera depends on the improvement of hygiene but this is a distant goal for many countries . The availability of an effective cholera vaccine is thus important for the prevention of cholera in such countries . More than a century after the first attempt to vaccinate against cholera by Ferran in Spain, there is still no truly effective cholera vaccine . A bacterial fraction vaccine, referred to as CH1 +2 was prepared by Professor A . Dodin . A field trial of this vaccine was carried out in Zaire in 1983 . Significant protection was observed but this vaccine was not evaluated in additional trials . Two other oral cholera vaccines, developed in Sweden and in the USA, were widely experimented on human beings: a combination of cholera toxin B-subunit and inactivated bacterial cells, and a live attenuated vaccine containing the genetically manipulated Vibrio cholerae O1 strain CVD 103-HgR . Despite their efficiency as evaluated in field trials (inactivated vaccine) or on volunteers (live vaccine), these vaccines have drawbacks that may limit their usefulness as practical vaccines . Protection induced by the inactivated vaccine was transient in young children, lasting only approximately for six months . One of the safety concerns associated with live vaccines is a possible reversion to virulence . Efforts should be continued to find a better cholera vaccine . A new vaccine development program based upon the hypothesis that immunoglobulin G directed to the O-specific polysaccharide of Vibrio cholerae O1 could confer protective immunity to cholera . This program may lead to the development of a cholera conjugate vaccine to elicit protection in infants.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 3183 - 7
Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae; Hase CC et al.; The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription . To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V . cholerae carrying a chromosomally integrated toxT::lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl beta-D galactoside (X-gal) . Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH-ubiquinone oxidoreductase (NQR) . In V . alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation . Inhibition of NQR enzyme function in V . cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT::lacZ activity . Increased toxT::lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H . However, the ability of the TcpP/H proteins to activate the toxT::lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium . The flagellar motor of V . cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT::lacZ expression . Thus, the regulation of the main virulence factors of V . cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way.

J Clin Microbiol, 1999 Apr, 37(4), 1173 - 7
Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene; Kim YB et al.; The DNA colony hybridization test with the polynucleotide probe for Vibrio parahaemolyticus toxR gene was performed . All 373 strains of V . parahaemolyticus gave positive results, and the strains belonging to four other Vibrio species including Vibrio alginolyticus gave weakly positive results, suggesting that toxR sequence variation may reflect the phylogenetic relationships of Vibrio species . We then established a toxR-targeted PCR protocol for the specific detection of V . parahaemolyticus.

J Bacteriol, 1999 Mar, 181(6), 1927 - 30
The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force; Kojima S et al.; Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle . Motility is thought to be an important factor for the virulence of V . cholerae . The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V . cholerae . The energy source of its flagellar motor was investigated . We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V . cholerae strains and present the evidence that the polar flagellar motor of V . cholerae is driven by an Na+ motive force.

Dis Aquat Organ, 1999 Jan 7, 35(1), 77 - 80
Isolation and characterization of Vibrio parahaemolyticus causing infection in Iberian toothcarp Aphanius iberus; Alcaide E et al.; High mortality among laboratory cultured Iberian toothcarp Aphanius iberus occurred in February 1997 in Valencia (Spain) . The main signs of the disease were external haemorrhage and tail rot . Bacteria isolated from internal organs of infected fish were biochemically homogeneous and identified as Vibrio parahaemolyticus . The bacteria were haemolytic against erythrocytes from eel Anguilla anguilla, amberjack Seriola dumerili, toothcarp A . iberus and humans, and were Kanagawa-phenomenon-negative . Infectivity tests showed that the virulence for A . iberus was dependent on salinity . Finally, all strains were virulent for amberjack and eel.

Am J Trop Med Hyg, 1999 Feb, 60(2), 271 - 6
Transmission of epidemic Vibrio cholerae O1 in rural western Kenya associated with drinking water from Lake Victoria: an environmental reservoir for cholera?
Shapiro RL, Otieno MR, Adcock PM, Phillips-Howard PA, Hawley WA, Kumar L, Waiyaki P, Nahlen BL, Slutsker L.
Sub-Saharan Africa has the highest reported cholera incidence and mortality rates in the world . In 1997, a cholera epidemic occurred in western Kenya . Between June 1997 and March 1998, 14,275 cholera admissions to hospitals in Nyanza Province in western Kenya were reported . There were 547 deaths (case fatality rate = 4%) . Of 31 Vibrio cholerae O1 isolates tested, all but one were sensitive to tetracycline . We performed a case-control study among 61 cholera patients and age-, sex-, and clinic-matched controls . Multivariate analysis showed that risk factors for cholera were drinking water from Lake Victoria or from a stream, sharing food with a person with watery diarrhea, and attending funeral feasts . Compared with other diarrheal pathogens, cholera was more common among persons living in a village bordering Lake Victoria . Cholera has become an important public health concern in western Kenya, and may become an endemic pathogen in the region.

Vaccine, 1999 Feb 26, 17(7-8), 949 - 56
Diarrheagenicity evaluation of attenuated Vibrio cholerae O1 and O139 strains in the human intestine ex vivo; Burgos JM et al.; The recent spread of El Tor cholera in Latin America highlights the need for a safe and economical vaccine . The main approach for developing live recombinant vaccines has been to disarm known pathogenic strains of cholera toxin leaving intact antigens involved in protection . These recombinant vaccine candidates do not cause severe diarrhea, but they are too reactogenic for wide scale usage . We describe here a test capable of determining the diarrheagenic potential of attenuated V . cholerae strains . The functional test consists in the simultaneous recording of net water movement, electrical potential difference and short-circuit current across the human intestine ex vivo . We found that human tissues incubated with supernatants from the attenuated 638, 413 and 251a V . cholerae strains caused no changes in the ion conductances and water absorption in ileal and colon tissues allowing them to be assayed in volunteers.

Clin Diagn Lab Immunol, 1999 Mar, 6(2), 276 - 8
Phagocytosis of Vibrio cholerae O139 Bengal by human polymorphonuclear leukocytes; Albert MJ et al.; Capsulated bacteria exhibit serum (complement) resistance and resistance to phagocytosis, which result in disseminated infections . Vibrio cholerae O139 strains possess a thin capsule and have been found to be partially serum resistant in a previous study . In the present study, compared to a standard capsulated Klebsiella pneumoniae strain, which showed total resistance to killing by phagocytosis, V . cholerae O139 strains were shown to be only partially resistant, with most strains showing <40% survival . These findings may explain the relative rarity of V . cholerae O139 bacteremia in cholera caused by this organism.

Curr Opin Microbiol, 1998 Apr, 1(2), 183 - 9
Self perception in bacteria: quorum sensing with acylated homoserine lactones; Fuqua C et al.; A variety of Gram-negative bacteria produce membrane permeant, acylated homoserine lactone (HL) pheromones that act as cell density cues . Synthesis and response to these factors requires proteins homologous to the Luxl acylhomoserine lactone synthase and the LuxR transcription factor from Vibrio fischeri . Recent genetic and biochemical studies have begun to provide a mechanistic understanding of acyl HL dependent gene regulation . Examination of the role of acyl HLs in diverse bacteria positions LuxR-Luxl type systems within an increasingly broad regulatory context and suggests that, in some bacteria, they comprise a global regulatory circuit.

No To Shinkei, 1999 Jan, 51(1), 41 - 7
{The spinal somatosensory evoked potentials in amyotrophic lateral sclerosis in relation to the spinal cord conduction velocities}; Matsumoto A et al.; The lumbar-to-cervical conduction velocity (spinal cord conduction velocity, SCCV) was electrophysiologically studied in 14 patients with amyotrophic lateral sclerosis (ALS) . The age of these patients ranged from 37 to 63, averaging 51.0 years old . We recorded the spinal somatosensory evoked potentials (SSEPs) from the surface electrodes at the level of the C2 spine and the T12 spine by the simultaneous stimulation of bilateral posterior tibial nerves . SCCV from the lumbar to cervical was measured from the latency difference between both SSEPs elicited at the each position . As the results, SCCVs were in the range of 50.6-66.6 (58.6 +/- 4.7: mean +/- SD) m/sec in normal age matched controls (18 adult volunteers, 46-63 years old, averaging, 52.7) . On the other hand, in ALS patients, SCCVs were in the range of 42.1-67.1 (53.5 +/- 7.8: mean +/- SD) m/sec, values of which were lowered compared to those in normal subjects . These examination documented 4 out of 14 patients with ALS (28.6%) showing abnormalities beyond standard deviation . The vibration sense was checked by using 128 Hz tuning fork at the ankles, and for the quantitative measurement, a newly designed vibriometer being attached the piezoelectric accelerometer to the end of 128 Hz tuning fork was applied in 14 ALS patients . The vibration sense at the ankles was diminished in 6 patients, and 3 patients showed the abnormalities beyond 2 standard deviations . The degree of lowering in SCCVs among ALS patients were correlated with the degree of diminution of impaired vibration sense and the duration of illness, but were not correlated with the H/M ratio and the latency difference between T wave and H wave . Since SSEP impulses are transmitted in dorsal columns and dorsolateral fasciculus predominantly by large diameter and fast-conduction fibers, our results may suggest that, in ALS patients, spinal cord conduction velocities of ascending fibers mediating the dorsal columns and dorsolateral fasciculus are disturbed compared to those in normal subjects, and that the functional disturbance of ascending fibers mediating the dorsal columns and dorsolateral fasciculus plays the important role in the high rates of impaired vibration sense among ALS patients.

J Appl Microbiol, 1999 Feb, 86(2), 337 - 47
Clonality of Vibrio anguillarum strains isolated from fish from the Scandinavian countries, Sweden, Finland and Denmark; Pedersen K et al.; In order to investigate whether outbreaks of vibriosis in the Baltic region were caused by the spread of certain pathogenic clones, 291 Vibrio anguillarum isolates from Finland (n = 156), Sweden (n = 88) and Denmark (n = 47) were studied with respect to serogroup, ribotype, plasmid content, and biochemical phenotypes as expressed with the PhenePlate (PhP) typing system . For comparison, 54 V . anguillarum serogroup O1 from other countries worldwide were included . Most isolates from Finland, Sweden and Denmark belonged to serogroup O1 (255), followed by O2 (30) . Four Finnish isolates cross-reacted strongly with antisera against two new serogroups VaNT2 and VaNT4, whereas two strains were non-typeable . The serogroup O1 isolates displayed ten different ribotype patterns, whereas the other strains were considerably more diverse with respect to ribotypes . Most of the O1 isolates carried the 67 kb virulence plasmid and a group of Finnish isolates, in addition, carried an 86 kb plasmid . Additional plasmids with molecular weights of 63, 76, 135 or 260-290 kb were found in single O1 isolates . With few exceptions, strains of serogroup O2 either had no plasmids or carried one or two small plasmids . PhenePlate typing revealed considerable diversity within the species, serogroup O1 being the most homogeneous . A few PhP types were dominant, whereas other types were observed only in one to four isolates . The prevalence of the different types changed significantly from one year to another but in Finland, one clonal lineage became increasingly important from 1992 (20% of isolates) to 1996 (80%) . Remaining clones were mostly restricted to specific geographic areas . By cluster analysis, it was demonstrated that most of the isolates from Finland, Sweden and Denmark belonged to two clusters, and most of the strains from Southern Europe fell into two other, distinct clusters . Most isolates from the UK, North America, Chile and Tasmania grouped together in a distinct cluster . For the typing of V . anguillarum, O-serotyping should be the primary method . For isolates belonging to serogroups other than O1, plasmid profiling in combination with ribotyping gives a very good discrimination between strains, whereas for serogroup O1, another method is required . It is concluded that PhP typing is a tool that provides a good discrimination between O1 isolates.

J Control Release, 1999 Mar 29, 58(2), 123 - 31
Optimization of preparative conditions for poly-DL-lactide- polyethylene glycol microspheres with entrapped Vibrio cholera antigens; Deng XM et al.; Poly-dl-lactide-polyethylene glycol (PELA) with different contents of polyethylene glycol(PEG) were synthesized and the PEG content was estimated according to the integral height of hydrogen shown in 1H-NMR . PELA microspheres containing V . cholera antigen, outer membrane protein (OMP) were prepared by a water-in-oil-in-water (W/O/W) based on solvent evaporation procedure . Antigen microspheres with smooth surface, suitable size for oral administration (0.5-5 microm), high loading efficiency (about 60%) and low level of residual solvent (lower than 20ppm) were obtained . Microspheres prepared from PELA with PEG content of about 10% achieved the highest loading efficiency among PELA copolymers and poly-dl-lactide (PLA) homopolymer, which suggested that microspheres size, morphology and the precipitation rate of polymer showed considerable relations with OMP loading efficiency . The regulation of the solvent components of the oil phase contributes to a stable emulsion W/O, and it is concluded that the stable emulsion W/O plays a significant role in improving the protein loading efficiency of obtained microspheres . The addition of stabilizer, such as gelatin and polyvinyl alcohol, into the internal water phase before emulsification produced no significant difference in OMP entrapment and microspheres size . A higher OMP loading efficiency was achieved by adding NaCl or adjusting the pH at the iso-electric point of OMP in the external water phase . It was indicated in vitro that PELA microspheres with smaller size showed larger extent of initial release and higher release rate, whereas microspheres with the diameter of 2.17 microm showed no apparent burst effect.

FEBS Lett, 1999 Feb 12, 444(2-3), 170 - 2
Mechanosensitive channel functions to alleviate the cell lysis of marine bacterium, Vibrio alginolyticus, by osmotic downshock; Nakamaru Y et al.; The mechanosensitive channel with large conductance of Escherichia coli is the first to be cloned among stretch-activated channels . Although its activity was characterized by a patch clamp method, a physiological role of the channel has not been proved . The marine bacterium, Vibrio alginolyticus, is sensitive to osmotic stress and cell lysis occurs under osmotic downshock . We introduced an mscL gene into Vibrio alginolyticus, and the mechanosensitive channel with large conductance functions was found to alleviate cell lysis by osmotic downshock . This is the first report to show a physiological role of the mechanosensitive channel with large conductance.

Appl Environ Microbiol, 1999 Mar, 65(3), 1352 - 5
Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan; Amaro C et al.; The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work . These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum . Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.

Appl Environ Microbiol, 1999 Mar, 65(3), 1348 - 51
Role of surface proteins in Vibrio cholerae attachment to chitin; Tarsi R et al.; The role of surface proteins in Vibrio cholerae attachment to chitin particles in vitro was studied . Treatment of V . cholerae O1 ATCC 14034 and ATCC 14035 with pronase E reduced the attachment of bacteria to chitin particles by 57 to 77% . A statistically significant reduction was also observed when the attachment to chitin was evaluated in the presence of homologous Sarkosyl-insoluble membrane proteins (MPs) (67 to 84%), N-acetylglucosamine (GlcNAc) (62%), the sugar that makes up chitin, and wheat germ agglutinin (40 to 56%), a lectin that binds GlcNAc . The soluble oligomers N,N'-diacetylchitobiose or N,N', N"-triacetylchitotriose caused an inhibition of 14 to 23% . Sarkosyl-insoluble MPs able to bind chitin particles were isolated and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; two of these peptides (molecular sizes, 36 and 53 kDa) specifically bind GlcNAc.

Appl Environ Microbiol, 1999 Mar, 65(3), 1145 - 51
Arbitrarily primed PCR to type Vibrio spp . pathogenic for shrimp; Goarant C et al.; A molecular typing study on Vibrio strains implicated in shrimp disease outbreaks in New Caledonia and Japan was conducted by using AP-PCR (arbitrarily primed PCR) . It allowed rapid identification of isolates at the genospecies level and studies of infraspecific population structures of epidemiological interest . Clusters identified within the species Vibrio penaeicida were related to their area of origin, allowing discrimination between Japanese and New Caledonian isolates, as well as between those from two different bays in New Caledonia separated by only 50 km . Other subclusters of New Caledonian V . penaeicida isolates could be identified, but it was not possible to link those differences to accurate epidemiological features . This contribution of AP-PCR to the study of vibriosis in penaeid shrimps demonstrates its high discriminating power and the relevance of the epidemiological information provided . This approach would contribute to better knowledge of the ecology of Vibrio spp . and their implication in shrimp disease in aquaculture.

Appl Environ Microbiol, 1999 Mar, 65(3), 1141 - 4
Randomly amplified polymorphic DNA analysis of clinical and environmental isolates of Vibrio vulnificus and other vibrio species; Warner JM et al.; Vibrio vulnificus is an estuarine bacterium that is capable of causing a rapidly fatal infection in humans . A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed for use in detecting V . vulnificus, as well as other members of the genus Vibrio . The resulting RAPD profiles were analyzed by using RFLPScan software . This RAPD method clearly differentiated between members of the genus Vibrio and between isolates of V . vulnificus . Each V . vulnificus strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous . All of the vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while the V . vulnificus strains had an additional two molecular weight range bands in common . All of the V . vulnificus strains isolated from clinical specimens produced an additional band that was only occasionally found in environmental strains; this suggests that, as is the case with the Kanagawa hemolysin of Vibrio parahaemolyticus, the presence of this band may be correlated with the ability of a strain to produce an infection in humans . In addition, band pattern differences were observed between encapsulated and nonencapsulated isogenic morphotypes of the same strain of V . vulnificus.

Appl Environ Microbiol, 1999 Mar, 65(3), 1133 - 40
Isolation from estuarine sediments of a Desulfovibrio strain which can grow on lactate coupled to the reductive dehalogenation of 2,4, 6-tribromophenol; Boyle AW et al.; Strain TBP-1, an anaerobic bacterium capable of reductively dehalogenating 2,4,6-tribromophenol to phenol, was isolated from estuarine sediments of the Arthur Kill in the New York/New Jersey harbor . It is a gram-negative, motile, vibrio-shaped, obligate anaerobe which grows on lactate, pyruvate, hydrogen, and fumarate when provided sulfate as an electron acceptor . The organism accumulates acetate when grown on lactate and sulfate, contains desulfoviridin, and will not grow in the absence of NaCl . It will not utilize acetate, succinate, propionate, or butyrate for growth via sulfate reduction . When supplied with lactate as an electron donor, strain TBP-1 will utilize sulfate, sulfite, sulfur, and thiosulfate for growth but not nitrate, fumarate, or acrylate . This organism debrominates 2-, 4-, 2,4-, 2,6-, and 2,4,6-bromophenol but not 3- or 2,3-bromophenol or monobrominated benzoates . It will not dehalogenate monochlorinated, fluorinated, or iodinated phenols or chlorinated benzoates . Together with its physiological characteristics, its 16S rRNA gene sequence places it in the genus Desulfovibrio . The average growth yield of strain TBP-1 grown on a defined medium supplemented with lactate and 2,4,6-bromophenol is 3.71 mg of protein/mmol of phenol produced, and the yield was 1.42 mg of protein/mmol of phenol produced when 4-bromophenol was the electron acceptor . Average growth yields (milligrams of protein per millimole of electrons utilized) for Desulfovibrio sp . strain TBP-1 grown with 2,4,6-bromophenol, 4-bromophenol, or sulfate are 0.62, 0.71, and 1.07, respectively . Growth did not occur when either lactate or 2,4,6-bromophenol was omitted from the growth medium . These results indicate that Desulfovibrio sp . strain TBP-1 is capable of growth via halorespiration.

Appl Environ Microbiol, 1999 Mar, 65(3), 1117 - 26
Effects of salinity and temperature on long-term survival of the eel pathogen Vibrio vulnificus biotype 2 (serovar E); Marco-Noales E et al.; Vibrio vulnificus biotype 2 (serovar E) is a primary eel pathogen . In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium . We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30 degrees C) . Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25 degrees C for microcosms at 0.3 and 0.5% and 12 degrees C for microcosms at 1.5 to 3.8%) . In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis . Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years . The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones . Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice . Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced . In conclusion, V . vulnificus biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water . Moreover, the aquatic ecosystem is one of its reservoirs.

Antimicrob Agents Chemother, 1999 Mar, 43(3), 693 - 6
Antibiotic resistance conferred by a conjugative plasmid and a class I integron in Vibrio cholerae O1 El Tor strains isolated in Albania and Italy; Falbo V et al.; Multidrug-resistant Vibrio cholerae O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance . All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine) . Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the aadA1 gene cassette located in the bacterial chromosome within a class 1 integron . The resistance to trimethoprim and O/129 was conferred by the dfrA1 gene, which was present on the plasmid . Although the dfrA1 gene is known to be borne on an integron cassette, class 1, 2, or 3 intI genes were not detected as part of the plasmid DNA from the strains studied.

Mol Microbiol, 1999 Feb, 31(3), 763 - 71
A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon; Skorupski K et al.; The expression of the ToxR virulence regulon is dependent upon the regulatory proteins ToxR/ToxS, TcpP/TcpH and ToxT . We describe here a previously unidentified gene in Vibrio cholerae, aphA (activator of tcpP and tcpH expression), which is required for the transcription of the tcpPH operon . Under conditions normally optimal for virulence gene expression, an in frame aphA deletion decreased the expression of a cholera toxin promoter fusion (ctx-lacZ) and prevented the production of the toxin co-regulated pilus (TCP) . Plasmids producing ToxT or TcpP/H, but not ToxR, restored ctx-lacZ expression and TCP production in the delta aphA strain, suggesting that the mutation interferes with toxT expression by influencing the transcription of tcpPH . Indeed, the expression of a chromosomal tcpP-lacZ fusion was reduced in the delta aphA mutant and increased in both V . cholerae and Escherichia coli by introducing aphA expressed from an inducible promoter . These results support a model in which AphA functions at a previously unknown step in the ToxR virulence cascade to activate the transcription of tcpPH . TcpP/TcpH, together with ToxR/ToxS, then activate the expression of toxT, resulting ultimately in the production of virulence factors such as cholera toxin and TCP.

Microbiol Immunol, 1998, 42(12), 837 - 43
Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells; Kim JS et al.; Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection . The injection of living bacteria or purified V . vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability . In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC . Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH) . At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP . VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S . These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells . At low concentrations, VVC does not induce the release of stored histamine from damaged cells . The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death . However, no increase in blood histamine level was detected . This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.

Microbiol Immunol, 1998, 42(12), 823 - 8
Detection of genes encoding cholera toxin (CT), zonula occludens toxin (ZOT), accessory cholera enterotoxin (ACE) and heat-stable enterotoxin (ST) in Vibrio mimicus clinical strains; Shi L et al.; A total of 51 clinical strains of Vibrio mimicus were searched for the presence of virulence-associated genes, like ctx, zot or ace genes which locate in "cholera virulence cassette," and the st gene by polymerase chain reaction . Moreover, the pathological potential of each clinical strain was also examined by rabbit ileal loop (RIL) . Three strains showed to have the ctx gene, of which only one strain was zot gene-positive . Meanwhile, one other strain was zot+ but ctx- . All of these four strains were found to have the ace gene and to belong to serogroup O115 . Nine strains showed to carry the st gene . However, none of these ST-gene-positive strains was indicated to contain the genes located in the "cholera virulence cassette." It is of interest to note that all of the RIL-positive and/or virulence gene-positive strains were restricted to three serogroups, O20, O41 and O115 . These results suggest a significant association between O antigens and enterotoxic activities in V . mimicus clinical strains, and clearly demonstrate multifactorial virulence potentials of this human pathogen.

Epidemiol Infect, 1998 Dec, 121(3), 535 - 45
Ribotypes of clinical Vibrio cholerae non-O1 non-O139 strains in relation to O-serotypes; Dalsgaard A et al.; The emergence of Vibrio cholerae O139 in 1992 and reports of an increasing number of other non-O1 serogroups being associated with diarrhoea, stimulated us to characterize V . cholerae non-O1 non-O139 strains received at the National Institute of Infectious Diseases, Japan for serotyping . Ribotyping with the restriction enzyme BglI of 103 epidemiological unrelated mainly clinical strains representing 10 O-serotypes yielded 67 different typing patterns . Ribotype similarity within each serotype was compared by using the Dice coefficient (Sd) and different levels of homogeneity were observed (serotypes O5, O41 and O17, Sd between 82 and 90%: serotypes O13 and O141 Sd of 72; and O2, O6, O7, O11, O24 Sd of 62-66%) . By cluster analysis, the strains were divided into several clusters of low similarity suggesting a high level of genetic diversity . A low degree of similarity between serotypes and ribotypes was found as strains within a specific serotypes often did not cluster but clustered with strains from other serotypes . However, epidemiological unrelated O5 strains showed identical or closely related ribotypes suggesting that these strains have undergone few genetic changes and may correspond to a clonal line . Surprisingly, 10 of 16 O141 strains studied contained a cholera toxin (CT) gene, including 7 strains recovered from stool and water samples in the United States . This is to our knowledge the first report of CT-positive clinical O141 strains . The closely related ribotypes shown by eight CT-positive strains is disturbing and suggest that these strains may be of a clonal origin and have the potential to cause cholera-like disease . Despite the low degree of correlation found between ribotypes and serotypes, both methods appears to be valuable techniques in studying the epidemiology of emerging serotypes of V . cholerae.

Lett Appl Microbiol, 1999 Jan, 28(1), 66 - 70
Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus; McCarthy SA et al.; The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive . To reduce the time and effort necessary to verify the identity of V . parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed . An alkaline phosphatase (AP)-labelled probe was evaluated for specificity against 26 strains of V . parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non-vibrio species . Of the 124 isolates tested, the probe hybridized only with the 26 strains of V . parahaemolyticus, indicating species specificity . Two hundred and six suspect V . parahaemolyticus isolates from oysters were tested by this probe and API-20E diagnostic strips; there was 97% agreement between results . A digoxigenin (DIG)-labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP-labelled probe . When tested on 584 suspect V . parahaemolyticus isolates, results obtained with the AP- and DIG-labelled probes were in 98% agreement . These results suggest that the probes are equivalent for detection of the V . parahaemolyticus tlh gene.

J Appl Microbiol, 1999 Jan, 86(1), 125 - 34
Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast; Arias CR et al.; A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media . Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences . This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar . None of the V . vulnificus isolates identified corresponded to serovar E . Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V . splendidus below 20 degrees C and V . harveyi and V . mediterranei above that temperature . Low percentages of several pathogenic vibrios were recorded but V . vulnificus was never recovered at this incubation temperature . The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.

Mol Microbiol, 1999 Jan, 31(2), 665 - 77
A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi; Freeman JA et al.; Two independent quorum-sensing systems control the expression of bioluminescence (lux) in the marine bacterium Vibrio harveyi . Each system is composed of an autoinducer (AI-1 or AI-2) and its cognate sensor (LuxN or LuxQ) . The sensors are two-component hybrid kinases, containing both sensor kinase domains and response regulator domains . Sensory information from the two systems is relayed by a phosphotransfer mechanism to a shared integrator protein called LuxO . LuxO is a member of the response regulator class of the two-component family of signal transduction proteins, and LuxO acts negatively to control luminescence . In this report, missense and in frame deletion mutations were constructed in luxO that encoded proteins mimicking either the phosphorylated or the unphosphorylated form, and these mutations were introduced into the V . harveyi chromosome at the luxO locus . Phenotypical analyses of the resulting mutant V . harveyi strains indicate that the phosphorylated form of LuxO is the repressor, and that the unphosphorylated form of the protein is inactive . Analysis of the lux phenotypes of V . harveyi strains containing single and double luxN and luxQ mutations indicate that LuxN and LuxQ have two activities on LuxO . They act as LuxO protein kinases at low cell density in the absence of autoinducers, and they switch to LuxO protein phosphatases at high cell density in the presence of autoinducers . Furthermore, the timing and potency of inputs from the two systems into regulation of quorum sensing are different.

Infect Immun, 1999 Mar, 67(3), 1393 - 404
Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae; Fullner KJ et al.; The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons . The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12 . This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved . The pilA gene encodes a putative type IV pilus subunit . However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes . The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences . Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice . Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences . Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD . Taken together, these data demonstrate the effectiveness of the V . cholerae genome project for rapid identification and characterization of potential virulence factors.

Infect Immun, 1999 Mar, 67(3), 1287 - 91
Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens; Marinaro M et al.; Zonula occludens toxin (Zot) is produced by toxigenic strains of Vibrio cholerae and has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier . In the present study, we investigated whether Zot could be exploited to deliver soluble antigens through the nasal mucosa for the induction of antigen-specific systemic and mucosal immune responses . Intranasal immunization of mice with ovalbumin (Ova) and recombinant Zot, either fused to the maltose-binding protein (MBP-Zot) or with a hexahistidine tag (His-Zot), induced anti-Ova serum immunoglobulin G (IgG) titers that were approximately 40-fold higher than those induced by immunization with antigen alone . Interestingly, Zot also stimulated high anti-Ova IgA titers in serum, as well as in vaginal and intestinal secretions . A comparison with Escherichia coli heat-labile enterotoxin (LT) revealed that the adjuvant activity of Zot was only sevenfold lower than that of LT . Moreover, Zot and LT induced similar patterns of Ova-specific IgG subclasses . The subtypes IgG1, IgG2a, and IgG2b were all stimulated, with a predominance of IgG1 and IgG2b . In conclusion, our results highlight Zot as a novel potent mucosal adjuvant of microbial origin.

Infect Immun, 1999 Mar, 67(3), 1139 - 48
Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells; Fabbri A et al.; Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide . A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production . TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species . Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability . In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria . In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle . The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.

Infect Immun, 1999 Mar, 67(3), 1116 - 24
Evolutionary relationships of pathogenic clones of Vibrio cholerae by sequence analysis of four housekeeping genes; Byun R et al.; Studies of the Vibrio cholerae population, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S . Gulf Coast clones . However, the relationship of the pathogenic clones to environmental V . cholerae isolates remains unclear . A previous study to determine the phylogeny of V . cholerae by sequencing the asd (aspartate semialdehyde dehydrogenase) gene of V . cholerae showed that the sixth-pandemic, seventh-pandemic, and U.S . Gulf Coast clones had very different asd sequences which fell into separate lineages in the V . cholerae population . As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the mdh (malate dehydrogenase) and hlyA (hemolysin A) genes from representatives of environmental and clinical isolates of V . cholerae and found that the mdh and hlyA sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion in hlyA in the sixth-pandemic clone . Identical sequences were obtained, despite average nucleotide differences in the mdh and hlyA genes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related . To extend these observations, segments of the recA and dnaE genes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones . The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.

Infect Immun, 1999 Mar, 67(3), 1025 - 33
Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae; Chakrabarti S et al.; The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen . The central regulator of several virulence genes of V . cholerae is ToxR, a transmembrane DNA binding protein . The V . cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity . Using Northern blot analysis and toxR transcriptional fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed from toxR . This may be due to increased heat shock induction in the dnaK mutant . In vivo, however, although expression from heat shock promoters in the dnaK mutant was similar to that observed in vitro, expression of both toxR and htpG was comparable to that by the parental strain . In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease in htpG expression was observed . These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro.

Biochim Biophys Acta, 1999 Feb 16, 1444(2), 269 - 75
Cloning and characterisation of a novel ompB operon from Vibrio cholerae 569B; Tow LA et al.; The ompB operon of Vibrio cholerae 569B has been cloned and fully sequenced . The operon encodes two proteins, OmpR and EnvZ, which share sequence identity with the OmpR and EnvZ proteins of a variety of other bacteria . Although the order of the ompR and envZ genes of V . cholerae is similar to that of the ompB operon of E . coli, S . typhimurium and X . nematophilus, the Vibrio operon exhibits a number of novel features . The structural organisation and features of the V . cholerae ompB operon are described.

J Cell Biochem, 1999 Mar 15, 72(4), 445 - 57
Halide peroxidase in tissues that interact with bacteria in the host squid Euprymna scolopes; Small AL et al.; An enzyme with similarities to myeloperoxidase, the antimicrobial halide peroxidase in mammalian neutrophils, occurs abundantly in the light organ tissue of Euprymna scolopes, a squid that maintains a beneficial association with the luminous bacterium Vibrio fischeri . Using three independent assays typically applied to the analysis of halide peroxidase enzymes, we directly compared the activity of the squid enzyme with that of human myeloperoxidase . One of these methods, the diethanolamine assay, confirmed that the squid peroxidase requires halide ions for its activity . The identification of a halide peroxidase in a cooperative bacterial association suggested that this type of enzyme can function not only to control pathogens, but also to modulate the interactions of host animals with their beneficial partners . To determine whether the squid peroxidase functions under both circumstances, we examined its distribution in a variety of host tissues, including those that typically interact with bacteria and those that do not . Tissues interacting with bacteria included those that have specific cooperative associations with bacteria (i.e., the light organ and accessory nidamental gland) and those that have transient nonspecific interactions with bacteria (i.e., the gills, which clear the cephalopod circulatory system of invading microorganisms) . These bacteria-associated tissues were compared with the eye, digestive gland, white body, and ink-producing tissues, which do not typically interact directly with bacteria . Peroxidase enzyme assays, immunocytochemical localization, and DNA-RNA hybridizations showed that the halide-dependent peroxidase is consistently expressed in high concentration in tissues that interact bacteria . Elevated levels of the peroxidase were also found in the ink-producing tissues, which are known to have enzymatic pathways associated with antimicrobial activity . Taken together, these data suggest that the host uses a common biochemical response to the variety of types of associations that it forms with microorganisms.

Hereditas, 1998, 129(2), 131 - 42
Structure and organization of a 25 kbp region of the genome of the photosynthetic green sulfur bacterium Chlorobium vibrioforme containing Mg-chelatase encoding genes; Petersen BL et al.; A region comprising approximately 25 kbp of the genome of the strictly anaerobic and obligate photosynthetic green sulfur bacterium Chlorobium vibrioforme has been mapped, subcloned and partly sequenced . Approximately 15 kbp have been sequenced in it's entirety and three genes with significant homology and feature similarity to the bchI, -D and -H genes and the chlI, -D and -H genes of Rhodobacter and Synechocystis strain PCC6803, respectively, which encode magnesium chelatase subunits, have been identified . Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX, and is the first enzyme unique to the (bacterio)chlorophyll specific branch of the porphyrin biosynthetic pathway . The organization of the three Mg-chelatase encoding genes is unique to Chlorobium and suggests that the magnesium chelatase of C . vibrioforme is encoded by a single operon . The analyzed 25 kbp region contains five additional open reading frames, two of which display significant homology and feature similarity to genes encoding lipoamide dehydrogenase and genes with function in purine synthesis, and another three display significant homology to open reading frames with unknown function in distantly related bacteria . Putative E . coli sigma 70-like promoter sequences, ribosome binding sequences and rho-independent transcriptional stop signals within the sequenced 15 kbp region are related to the identified genes and orfs . Southern analysis, restriction mapping and partial sequencing of the remaining ca . 10 kbp of the analyzed 25 kbp region have shown that this part includes the hemA, -C, -D and -B genes (MOBERG and AVISSAR 1994), which encode enzymes with function in the early part of the biosynthetic pathway of porphyrins.

Diagn Microbiol Infect Dis, 1999 Jan, 33(1), 63 - 4
Vibrio cholerae O1 serotype Ogawa in a neonate; Uppal B et al.; In India, cholera is endemic and affects usually the 3 to 5-year-old age group . There have been occasional reports in the neonatal period with Vibrio cholerae O139 Bengal . We report here a case of Vibrio cholerae O1 diarrhea in a 2-day-old, breastfed male, who had been delivered in the hospital and developed severe dehydration.

Anal Chem, 1999 Feb 1, 71(3), 633 - 41
Differentiation of microorganisms based on pyrolysis-ion trap mass spectrometry using chemical ionization; Barshick SA et al.; The ability to differentiate microorganisms using pyrolysision trap mass spectrometry was demonstrated for five Gram-negative disease-causing organisms: Brucella melitensis, Brucella suis, Vibrio cholera, Yersinia pestis, and Francisella tularensis . Bacterial profiles were generated for gamma-irradiated bacterial samples using pyrolytic methylation and compared for electron ionization and chemical ionization using several liquid reagents with increasing proton affinities . Electron ionization combined with pyrolysis caused extensive fragmentation, resulting in a high abundance of lower mass ions and diminishing the diagnostic value of the technique for compound identification and bacterial profiling . Chemical ionization reduced the amount of fragmentation due to ionization while enhancing the molecular ion region of the fatty acids . As the proton affinity of the reagent increased, the protonated molecular ions of the fatty acids became the predominant ions observed in the mass spectrum . As a result, chemical ionization was shown to be more effective than electron ionization in bacterial profiling . Whereas the bacteria could be distinguished at the Genera level using electron ionization, further differentiation to the subspecies level was possible using chemical ionization . The greatest separation among the five test organisms, in terms of Euclidean distances, was obtained using ethanol as the chemical ionization reagent and using pooled masses representing specific fatty acid biomarkers rather than total ion profiles.

Indian J Public Health, 1997 Apr-Jun, 41(2), 61 - 7
Epidemiological study of an outbreak of cholera in Delhi cantonment; Tilak VW et al.; An epidemiological study was undertaken to investigate an outbreak of cholera in Delhi Cantonment during May 1991 . The study design was a hybrid design using a retrospective Case-Control method superimposed on a population based cross-sectional approach . A total of 9 cases of cholera, confirmed in the laboratory as Vibrio cholerae, 0-1, Eltor, Ogawa were identified using population based survey and compared with 33 controls from the same source population . The overall Incidence rate was 0.71% and showed a significant rising trend with age . There was no morality . Assessment of water supply, sanitary conditions of cook houses and disposal system of night soil could not provide any clue to the source of infection . Subsequently, all the food handlers were subjected to rectal swab examination . Two of them, working in the same messes from where cases had occurred, were found positive for Vibrio cholerae (0-1, Eltor, Ogawa) . Immediate control measures by way of isolation and treatment of carriers promptly abated the outbreak . Role of carriers in outbreak of cholera has been highlighted.

Eur J Pharmacol, 1999 Jan 22, 365(2-3), 267 - 72
Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin; Kook H et al.; Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V . vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase . We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations . Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation . The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor . However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane . Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it . These results suggest that V . vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).

FEMS Microbiol Lett, 1999 Feb 1, 171(1), 49 - 55
Toxin-co-regulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence of a third allele of pilin gene; Novais RC et al.; Polymerase chain reaction has been used to detect the presence of the virulence associated gene, tcpA and part of the promoter distal region of the toxin-co-regulated pilus cluster in non-O1, non-toxigenic, Vibrio cholerae . The amplified regions were characterised by restriction fragment length polymorphism and heteroduplex motility assay . We describe the nucleotide sequence of the tcpA gene fragment from non-toxigenic vibrios from clinical and environmental sources . The present study shows that there are at least three types of the tcpA gene among V . cholerae and the primers specific for the classical tcpA gene, amplify all biotypes . A sequence similarity in other regions of the toxin-co-regulated pilus cluster is suggested . The evidences for the presence of this cluster among non-toxigenic vibrios is, to our knowledge, reported for the first time . The use of restriction fragment length polymorphism for typing the tcpA and studying the alleles distribution is proposed.

Mol Microbiol, 1999 Jan, 31(1), 305 - 17
ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation; Dziejman M et al.; ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae . Previous experiments using lambda repressor-ToxR chimeric proteins and a lambda repressor-controlled reporter system (OR1 PR-lacZY) established that ToxR sequences can effectively dimerize the amino-terminal domain of lambda repressor in Escherichia coli . However, in E . coli, ToxR does not respond to environmental signals that control virulence gene expression in V . cholerae . Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V . cholerae also modulate a monomer to dimerization transition of lambda-ToxR chimeras . When the OR1 PR-lacZY reporter fusion and chimeric proteins were transferred to V . cholerae, we unexpectedly found that lambda-ToxR chimeras did not dimerize significantly . Interestingly, experiments evaluating the ability of lambda-ToxR proteins to form tetramers in E . coli suggested that lambda-ToxR dimers could act co-operatively . Using a redesigned reporter system containing multiple lambda operator sites (OR1 OR2 OR3 PR-lacZY), we found that lambda-ToxR could dimerize quite efficiently in V . cholerae . These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V . cholerae and suggest that ToxR dimers might be capable of co-operative interactions . However, we falled to correlate a monomer-dimer transition of the lambda-ToxR chimeras with changes in virulence gene expression in response to environmental signals in V . cholerae . Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re-evaluated . This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E . coli and V . cholerae . These assays suggest that, in V . cholerae, deletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain . In addition, we noted differences in chimeric protein activity when expressed in E . coli and V . cholerae . A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E . coli . Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E . coli, both chimeras were able to produce wild-type levels of toxin in V . cholerae . These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E . coli . In V . cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.

J Clin Microbiol, 1999 Mar, 37(3), 734 - 41
Cholera in Vietnam: changes in genotypes and emergence of class I integrons containing aminoglycoside resistance gene cassettes in vibrio cholerae O1 strains isolated from 1979 to 1996; Dalsgaard A et al.; The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995 . Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983 . Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies . All cases were associated with Vibrio cholerae O1 . Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991 . In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype . Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette ant(3")-1a encoding resistance to streptomycin and spectinomycin . This is, to our knowledge, the first report of class I integrons in V . cholerae . The development of cholera and the changes in the phenotypic and genotypic properties of V . cholerae O1 shown in the present study highlight the importance of monitoring V . cholerae O1 in Vietnam as in other parts of the world . In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.

J Clin Microbiol, 1999 Mar, 37(3), 581 - 90
Genetic diversity and population structure of Vibrio cholerae; Beltran P et al.; Multilocus enzyme electrophoresis (MLEE) of 397 Vibrio cholerae isolates, including 143 serogroup reference strains and 244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II) . Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic of division I but not in subdivision Ia, which includes 76% of the ETs . Despite this evidence of relatively frequent recombination, clonal lineages may persist for periods of time measured in at least decades . In addition to the pandemic clones of serogroups O1 and O139, which form a tight cluster of four ETs in subdivision Ia, MLEE analysis identified numerous apparent clonal lineages of non-O1 strains with intercontinental distributions . A clone of serogroup O37 that demonstrated epidemic potential in the 1960s is closely related to the pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference strain is not . A strain of serogroup O22, which has been identified as the most likely donor of exogenous rfb region DNA to the O1 progenitor of the O139 clone, is distantly related to the O1/O139 clones . The close evolutionary relationships of the O1, O139, and O37 epidemic clones indicates that new cholera clones are likely to arise by the modification of a lineage that is already epidemic or is closely related to such a clone.

J Bacteriol, 1999 Feb, 181(4), 1110 - 7
Genetic and transcriptional analyses of the Vibrio cholerae mannose-sensitive hemagglutinin type 4 pilus gene locus; Marsh JW et al.; The mannose-sensitive hemagglutinin (MSHA) of the Vibrio cholerae O1 El Tor biotype is a member of the family of type 4 pili . Type 4 pili are found on the surface of a variety of gram-negative bacteria and have demonstrated importance as host colonization factors, bacteriophage receptors, and mediators of DNA transfer . The gene locus required for the assembly and secretion of the MSHA pilus has been localized to a 16.7-kb region of the V . cholerae chromosome . Sixteen genes required for hemagglutination, including five that encode prepilin or prepilin-like proteins, have been identified . Examination of MSHA-specific cDNAs has localized two promoters that drive expression of these genes . This evidence indicates that the MSHA gene locus is transcriptionally organized into two operons, one encoding the secretory components and the other encoding the structural subunits, an arrangement unique among previously characterized type 4 pilus loci . The genes flanking the MSHA locus encode proteins that show homology to YhdA and MreB of Escherichia coli . In E . coli, the yhdA and mreB genes are adjacent to each other on the chromosome . The finding that the MSHA locus lies between these two E . coli homologs and that it is flanked by a 7-bp direct repeat suggests that the MSHA locus may have been acquired as a mobile genetic element.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Nov-Dec, (6), 30 - 2
{Analysis of Vibrio cholerae hemolysins using monolayer continuous cell cultures}; Telesmanich NR et al.; The comparative study of the preparations of V.cholerae hemolysins of different serovars with the use of continuous cell lines CHO-K1, Vero, Hela, L-929 was carried out . The preparations of hemolysins isolated from such strains as V.cholerae 569B, V.eltor 9949, V.cholerae O-mut 461/67-34 differed in their biological activity on experimental animal models and had different cytotoxic activity . The preparations exhibiting no activity when tested in vivo (V.cholerae and V.eltor hemolysins) were cetotoxins, but in lower doses (1000 and 100 times respectively) than the preparation of V.cholerae O-mut hemolysin, active in vivo . Hemolysins induced the formation of anticytotoxic antibodies in low titers.

MMWR Morb Mortal Wkly Rep, 1999 Jan 29, 48(3), 48 - 51
Outbreak of Vibrio parahaemolyticus infection associated with eating raw oysters and clams harvested from Long Island Sound--Connecticut, New Jersey, and New York, 1998; Identification of a vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage; Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USAWe identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro . This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V . cholerae genome . We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity . The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif . Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD . In V . cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity . Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity . Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence . Furthermore, the RTX toxin of V . cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.

J Biolumin Chemilumin, 1998 Nov-Dec, 13(6), 365 - 9
LuxR controls the expression of Vibrio fischeri luxCDABE clone in Escherichia coli in the absence of luxI gene; Ulitzur S; We have recently suggested that the expression of V . fischeri right lux operon is initiated from two sites, the first located upstream of the luxI gene, while the second seems to be located upstream of the luxC gene . The transcription from both sites is negatively controlled by H-NS protein . E . coli MC4100 rpoS hns mutant harbouring the V . fischeri luxCDABE genes showed constitutive mode and 70,000-fold higher luminescence than the wild-type cells . The present study shows that the expression of luxCDABE genes in E . coli MC4100 wild-type cells is also controlled by LuxR protein in the absence of the autoinducer . The co-presence of a ptac-controlled luxR gene in a trans position to a plasmid carrying the luxCDABE genes resulted in 100,000 times higher luminescence . In the absence of the autoinducer, the presence of the luxR gene under its own regulated control resulted in about 100-200-fold increase of luminescence from the luxC upstream site . Taken together, it seems that the LuxR protein initiates the formation of the V . fischeri lux system cloned in E . coli from two sites located upstream and downstream of the luxI gene . Only the activation of the first site requires the presence of the autoinducer, whereas the second site is fully activated by LuxR protein in the absence of the autoinducer.

Appl Environ Microbiol, 1999 Feb, 65(2), 856 - 8
Comparative study of biological properties and electrophoretic characteristics of lipopolysaccharide from eel-virulent and eel-A virulent Vibrio vulnificus strains; Biosca EG et al.; In Vibrio vulnificus, virulence for eels is associated with serovar E strains . In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains . Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.

Biol Bull, 1998 Dec, 195(3), 326 - 36
Late postembryonic development of the symbiotic light organ of Euprymna scolopes (Cephalopoda: Sepiolidae); Montgomery MK et al.; The symbiotic light organ of the sepiolid squid Euprymna scolopes undergoes significant anatomical, morphological, and biochemical changes during development . Previously we described the embryonic organogenesis and early postembryonic development of the light organ . During embryogenesis, tissues are developed that will promote the onset of an association with Vibrio fischeri, the light organ symbiont . Upon inoculation, and in response to the first interactions with the bacterial symbionts, the light organ undergoes a dramatic morphogenesis during the first 4-5 days of postembryonic development . Here we describe the final developmental stage of the light organ system, a period of late postembryonic development in which particular tissues of the light organ mature that eventually mediate the functional symbiosis . The maturation of the light organ occurs within 1 to 2 weeks posthatch and entails two principal processes: (1) changes in the shape of the organ and elaboration of the accessory tissues that modify the bacterially produced light; and (2) branching of the epithelial crypts, where the bacterial symbionts reside, and restriction of epithelial cell proliferation to the deepest branches of the crypts . The gross morphological changes of the organ occur in the absence of V . fischeri, although rudiments of the ciliated field of the hatchling remain in animals not exposed to the microbial symbiont.

Rev Panam Salud Publica, 1998 Dec, 4(6), 371 - 4
{Survival of Vibrio cholerae 01 in freshwater surface and endemic cholera: a geological hypothesis}; Borroto RJ; The danger that cholera is becoming endemic in Latin America makes it imperative to know the geographic location of aquatic environments where ecological conditions favor long-term survival of the toxigenic Vibrio cholerae O1 El Tor biotype, and such aquatic environments should be sampled to determine if they harbor this microorganism . For efficient and effective sampling, it would be useful to know what kinds of waters are ecologically suitable for the survival of this pathogen during periods between epidemics, and where these bodies of water are located . This paper presents the hypothesis that toxigenic V . cholerae O1's ability to survive in surface freshwaters tends to be inversely related to the altitude above sea level of these freshwaters.

Curr Microbiol, 1999 Mar, 38(3), 183 - 9
Extended serotyping scheme for Vibrio anguillarum with the definition and characterization of seven provisional O-serogroups; Pedersen K et al.; The present paper summarizes the serotyping scheme of the fish pathogenic bacterium Vibrio anguillarum and defines seven additional O-serogroups . Strains, collected in our laboratories that were nontypable with antisera against the previously defined 16 O-serotypes, were used for generating new antisera and were characterized further by means of LPS profiles, Western blots, and serological reactions . On the basis of the results, it is suggested that the seven new O-serogroups are to be included in the existing serotyping system as serotypes O17-O23 . However, the existence of further V . anguillarum strains that were not typable with any of the 23 O-antisera suggested the existence of additional O-serotypes within this species . The relevance of the description of additional O-serotypes for the species V . anguillarum is discussed.

Curr Microbiol, 1999 Mar, 38(3), 168 - 75
Adaptive response to cold temperatures in Vibrio vulnificus; Bryan PJ et al.; The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation . When a V . vulnificus culture was shifted from 35 degrees C to 6 degrees C conditions, it underwent transition to a non-culturable state . Cells adapted to 15 degrees C prior to change to 6 degrees C condition, however, remain viable and culturable . In addition, cultures adapted to 15 degrees C were able to survive better upon freezing at -78 degrees C compared with cultures frozen directly from 35 degrees C . Inhibition of protein synthesis by addition of chloramphenicol in a V . vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance . These results suggest that cold-adaptive "protective" proteins may enhance survival and tolerance at cold temperatures . In addition, removal of iron from the growth medium by adding 2,2'-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels . This suggests that iron plays an important role in adaptation at cold temperatures . Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with 35S-methionine during exposure at 15 degrees C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein . These results suggest that, for V . vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA . In this study, for the first time we have shown that exposure to an intermediate cold temperature (15 degrees C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6 degrees C) . This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V . vulnificus infection from consuming raw oysters.

J Bacteriol, 1999 Feb, 181(3), 899 - 906
Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi; Freeman JA et al.; Vibrio harveyi regulates the expression of bioluminescence (lux) in response to cell density, a phenomenon known as quorum sensing . In V . harveyi, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal . The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO . Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers . At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor . In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU . LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1 . A critical His residue (His 58) of LuxU is required for phosphorelay function.

J Bacteriol, 1999 Feb, 181(3), 879 - 83
The mutK gene of Vibrio cholerae: a new gene involved in DNA mismatch repair; Bhakat KK et al.; A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified . The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants . The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V . cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent . The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product . The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank . ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E . coli vsr gene . ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria . Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified . The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V . cholerae strain from which the gene was isolated (10).

Res Microbiol, 1998 Nov-Dec, 149(10), 745 - 55
Molecular characterization of Vibrio cholerae O1 strains isolated in Romania; Damian M et al.; A collection of 89 Vibrio cholerae O1 strains, isolated in Romania between 1977 and 1994, and 6 strains from the Republic of Moldavia, was characterized by ribotyping, toxin gene restriction pattern (toxinogenotype) and distribution of cholera toxin gene (ctx), accessory toxin gene (ace) and zonula occludens toxin gene (zot) . After Bg/I endonuclease restriction of chromosomal DNA, a total of 18 ribotypes and 21 toxinogenotypes were distinguished . Deletions in the core region of the toxin gene cassette were found in 20% of strains; however, with the exception of one strain, all the isolates contained the ctx gene . Used in association, the three methods of molecular typing provided an accurate characterization of V . cholerae O1 isolates.

Toxicon, 1999 Jan, 37(1), 85 - 108
Characterisation of cholera toxin by liquid chromatography--electrospray mass spectrometry; van Baar BL et al.; Cholera toxin, one of the toxins that may be generated by various strains of the bacterium Vibrio cholerae, can be considered as a substance possibly used in biological warfare . The possibilities of characterising the toxin by liquid chromatography electrospray mass spectrometry (LC-ES-MS) were investigated . The toxin can be detected by flow-injection (FIA) ES-MS of a dialysed solution and observation of the charge envelope signals of its A-unit and B-chain protein; sufficient information for identification by the molecular mass of either protein could be obtained for quantities in the order of 10 fmol . Confirmatory analysis was carried out by 2-mercaptoethanol reduction and FIA-ES-MS detection of the product proteins or by tryptic digest LC-ES-MS with ion chromatogram detection of most of the tryptic fragments of the A-unit and B-chain from the singly, doubly or triply charged ion signals . The confirmatory tryptic digest LC-ES-MS analysis could be achieved with quantities as low as 1 pmol . Possible biovariations in the toxin can mostly be determined by sequencing, where the amino acid composition of tryptic fragments of the A1-chain, T5 and T15, and of the B-chain, T1, T4 and T5, cover all known biovariations . Partial sequencing of cholera toxin, originating from a classical strain, O1/569B, was achieved by LC-ES-MS/MS of most tryptic fragments larger than three amino acid residues.

J Med Microbiol, 1999 Jan, 48(1), 51 - 7
Response of wild-type mutants of Vibrio cholerae O1 possessing different combinations of virulence genes in the ligated rabbit ileal loop and in Ussing chambers: evidence for the presence of additional secretogen; Koley H et al.; Five wild-type mutant strains of Vibrio cholerae serogroup O1 that lacked the CTX virulence cassette, or contained a natural deletion of a virulence gene within the CTX virulence cassette, or possessed an additional virulence gene, along with a prototype toxigenic strain representing the El Tor classical biotypes were examined by in-vivo and in-vitro methods to determine their enterotoxic potential . The ability of whole cells and culture supernates of the strains to cause fluid accumulation in the rabbit ileal loop model revealed a pattern consistent with the presence of the various virulence gene(s), with those possessing the intact CTX virulence cassette being the most secretogenic . Culture supernates of strains without the CTX virulence cassette or the strain with an incomplete cassette were also able to evoke mild to moderate fluid accumulation in the rabbit ileal loop . Of the various media used, AKI and brain heart infusion broth appeared to support the production of a hitherto unknown secretogenic factor, because culture supernates of the non-toxigenic V . cholerae O1 strains showed higher fluid accumulation ratios when grown in these media than in the others . To confirm that the fluid accumulation elicited by these strains in the ileal loop was due to enterotoxin activity, the effect of supernate of the strains was examined in rabbit small intestine mounted on Ussing chambers . Increases in short circuit current and tissue conductance, as compared with the medium control, were observed even with the strains that did not possess the CTX virulence cassette, confirming their ability to disrupt the function of intestinal tissue . From these studies, it was concluded that strains of V . cholerae O1 devoid of the CTX virulence cassette were still able to elicit a secretory response in the ileal loop and displayed enterotoxic activity in an in-vitro experimental model.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 237 - 42
Purification and characterization of an Aeromonas caviae metalloprotease that is related to the Vibrio cholerae hemagglutinin/protease; Toma C et al.; A zinc metalloprotease (AP34) from Aeromonas caviae was purified by ammonium sulfate precipitation and subsequent gel filtration through Sephadex G-100 and Sephadex G-50 Superfine . The molecular mass was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 34 kDa . The protease showed maximum activity at pH 7.0 and was stable at 60 degrees C . AP34 was completely inactivated by EDTA and Zincov . The N-terminal amino acid sequence of AP34 showed a high degree of homology with a range of proteases within the family Vibrionaceae, including the hemagglutinin/protease (HA/P) of Vibrio cholerae . Immunologic relatedness of AP34 and HA/P was demonstrated by Western blotting . AP34-like protease was widely distributed among the aeromonad strains.

Infect Immun, 1999 Feb, 67(2), 976 - 80
rfb mutations in Vibrio cholerae do not affect surface production of toxin-coregulated pili but still inhibit intestinal colonization; Chiang SL et al.; The toxin-coregulated pilus (TCP) of Vibrio cholerae is essential for colonization . It was recently reported that rfb mutations in V . cholerae 569B cause the translocation arrest of the structural subunit of TCP, raising the possibility that the colonization defects of lipopolysaccharide mutants are due to effects on TCP biogenesis . However, an rfbB gene disruption in either V . cholerae O395 or 569B has no apparent effect on surface TCP production as assessed by immunoelectron microscopy and CTX phage transduction, and an rfbD::Tn5lac mutant of O395 also shows no defect in TCP expression . We conclude that the colonization defect associated with rfb mutations is unrelated to defects in TCP assembly.

Infect Immun, 1999 Feb, 67(2), 539 - 45
Preliminary assessment of the safety and immunogenicity of a new CTXPhi-negative, hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate; Benitez JA et al.; Vibrio cholerae 638 (El Tor, Ogawa), a new CTXPhi-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers . In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638 . Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools . The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers . V . cholerae 638, at doses ranging from 4 x 10(7) to 2 x 10(9) vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.

Dis Aquat Organ, 1998 Nov 30, 34(3), 161 - 6
Vibrio viscosus in farmed Atlantic salmon Salmo salar in Scotland: field and experimental observations; Bruno DW et al.; Winter mortality occurred in market-sized (2 to 3 kg) Atlantic salmon Salmo salar reared in sea cages in Scottish waters . Many of the fish had skin ulcers . Internally prominent dark-brown petechiae or ecchymotic haemorrhage was observed . Splenomegaly was associated with congestion and widespread necrosis . A Vibrio sp . was isolated from internal organs . Biochemically isolates of the bacterium were similar to a previously described bacterium, Vibrio viscosus, recorded in a phenotypic study from farmed salmon in Norway . This work examines the occurrence of V . viscosus in marine-reared Atlantic salmon for the first time in Scottish waters . An experimental study reproduced the field observations and Koch's postulates were fulfilled . The histopathology associated with natural infection was compared with that in laboratory-infected fish.

Biochim Biophys Acta, 1999 Jan 8, 1415(2), 297 - 305
Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein; Ikigai H et al.; Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa . These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes . We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical . We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH . To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer . This 190-kDa oligomer consisted of only the 50-kDa subunits . Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers . These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

J Hand Surg {Br}, 1998 Dec, 23(6), 808 - 10
Hand infections due to non-cholera Vibrio after injuries from St Peter's fish (Tilapia zillii); Said R et al.; We report 49 patients with a wide variety of hand infections, which developed after injuries from St Peter's fish (Tilapia zillii) . Twenty-eight of 36 patients who had been operated on had non-cholera Vibrio infections, all identified as Vibrio vulnificus . The course in these patients was characterized by rapid spread of the infection with progressive necrosis of the tendon sheath, subcutaneous tissues and the skin . Two of them required amputations but the others had satisfactory functional results . Thirteen other patients were managed nonoperatively with intravenous antibiotics and all of them recovered completely.

Mol Pharmacol, 1999 Jan, 55(1), 179 - 85
Site-directed mutagenesis of predicted active site residues in glutamate carboxypeptidase II; Speno HS et al.; Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate . GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate . The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined . These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28 . Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members . In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II . Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis . In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function . Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu) . The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.

Glycoconj J, 1998 Jul, 15(7), 663 - 9
Synthesis and evaluation of N-acetylneuraminic acid-based affinity matrices for the purification of sialic acid-recognizing proteins; Ciccotosto S et al.; The synthesis of 2-S-(2-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-galacto-2-nonulopyranosidonic acid (1) has been successfully achieved from the precursors methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thio-D-glyce ro-alpha-D-galacto-2-nonulopyranosonate (2) and 2-bromo-N-(tert-butoxycarbonyl)-ethylamine (5) . Compounds 1 and 2 were coupled, via amino and thioglycosidic linkages, respectively, to epoxy-activated Sepharose 6B . The resultant affinity adsorbents have proved efficient in purifying the sialic acid-recognizing enzyme Vibrio cholerae sialidase, in a one-step process with yields in the order of 60%.

J Biol Chem, 1999 Jan 15, 274(3), 1375 - 80
Oligomerization of Vibrio cholerae cytolysin yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in the target membrane; Zitzer A et al.; Vibrio cholerae cytolysin permeabilizes animal cell membranes . Upon binding to the target lipid bilayer, the protein assembles into homo-oligomeric pores of an as yet unknown stoichiometry . Pore formation has been observed with model liposomes consisting of phosphatidylcholine and cholesterol, but the latter were much less susceptible to the cytolysin than were erythrocytes or intestinal epithelial cells . We here show that liposome permeabilization is strongly promoted if cholesterol is combined with sphingolipids, whereby the most pronounced effects are observed with monohexosylceramides and free ceramide . These two lipid species are prevalent in mammalian intestinal brush border membranes . We therefore propose that, on its natural target membranes, the cytolysin has a dual specificity for both cholesterol and ceramides . To assess the stoichiometry of the pore, we generated hybrid oligomers of two naturally occurring variants of the toxin that differ in molecular weight . On SDS-polyacrylamide gel electrophoresis, the mixed oligomers formed a pattern of six distinct bands . Ordered by decreasing electrophoretic mobility, the six oligomer species must comprise 0 to 5 subunits of the larger form; the pore thus is a pentamer . Due to both lipid specificity and pore stoichiometry, V . cholerae cytolysin represents a novel prototype in the class of bacterial pore-forming toxins.

FEMS Immunol Med Microbiol, 1998 Dec, 22(4), 303 - 8
Antisera to selected outer membrane proteins of Vibrio cholerae protect against challenge with homologous and heterologous strains of V . cholerae; Das M et al.; Each year cholera epidemics occur in various places around the world . Though there is no effective vaccine against cholera, people who recover from an infection usually have prolonged immunity to the disease . Sera from convalescent patients contain antibodies to a number of outer membrane proteins (OMPs) of V . cholerae . We isolated several OMPs (43, 42, 30, and 22 kDa) from V . cholerae V86 E1 Tor Inaba, sequenced their amino-termini, and generated hyperimmune sera against them in rabbits . Antisera to the 43-, 42-, and 22-kDa OMPs, but not the preimmune sera, significantly reduced V . cholerae-induced fluid secretion seen in rabbit intestinal loops challenged with the homologous strain . In addition, a combination of antisera to the different OMPs reduced the fluid secretion induced by challenge with heterologous V . cholerae Ogawa and O139 strains . These results have significance in the development of vaccines to V . cholerae, as the hyperexpression of these OMP encoding genes in vaccine strains may improve the efficacy of cholera vaccines.

Xenobiotica, 1998 Nov, 28(11), 1061 - 73
Pharmacokinetics of a discontinuous absorption process of oxolinic acid in turbot, Scophthalmus maximus, after a single oral administration; Poher I et al.; 1 . The pharmacokinetics of oxolinic acid have been studied in 500 g turbot (Scophthalmus maximus) . The fish were kept in seawater at 16 degrees C with a 15 h/9 h photoperiod . Oxolinic acid was administered orally via a stomach tube at a single dose of 10 mg/kg of body weight . Serum concentrations of oxolinic acid were determined by a (HPLC) using liquid phase extraction with an internal standard and a fluorescence detection . 2 . The pharmacokinetic process was not significantly sex-influenced . The short elimination phase of the oxolinic acid in turbot after oral administration was similar to the elimination after intravascular administration . The serum concentration profile of oxolinic acid was better described by a discontinuous absorption model than by compartment models using continuous absorption processes . The absorption of oxolinic acid in turbot was characterized by two distinct phases after a lag time of about 2 h . A time (Tmax) of 12 h was necessary to reach the peak serum concentration (Cmax) of 1.41 microg/ml . The oral bioavailability was 27.9% . 3 . Based on the minimum inhibitory concentration for susceptible strains, and especially Vibrio anguillarum, the oxolinic acid could be effective in turbot after an oral treatment of 10 mg/kg/day.

Appl Environ Microbiol, 1999 Jan, 65(1), 336 - 8
Sources of Vibrio mimicus contamination of turtle eggs; Acuna MT et al.; Vibrio mimicus contamination of sand increased significantly during the arrival of the olive ridley sea turtles (Lepidochelys olivacea) at Ostional anidation beach, Costa Rica . Statistical analysis supports that eggs are contaminated with V . mimicus by contact with the sand nest . V . mimicus was isolated from eggs of all nests tested, and ctxA+ strains were found in 31% of the nests, all of which were near the estuary.

Appl Environ Microbiol, 1999 Jan, 65(1), 73 - 9
A mechanism of resistance to hydrogen peroxide in Vibrio rumoiensis S-1; Ichise N et al.; A possible mechanism of resistance to hydrogen peroxide (H2O2) in Vibrio rumoiensis, isolated from the H2O2-rich drain pool of a fish processing plant, was examined . When V . rumoiensis cells were inoculated into medium containing either 5 mM or no H2O2, they grew in similar manners . A spontaneous mutant strain, S-4, derived from V . rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H2O2 . These results suggest that catalase is inevitably involved in the resistance and survival of V . rumoiensis in the presence of H2O2 . Catalase activity was constitutively present in V . rumoiensis cells grown in the absence of H2O2, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli . The presence of the HP II-type catalase in V . rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism . A notable difference between V . rumoiensis and E . coli is that catalase is accumulated at very high levels ( approximately 2% of the total soluble proteins) in V . rumoiensis, in contrast to the case for E . coli . When V . rumoiensis cells which had been exposed to 5 mM H2O2 were centrifuged, most intracellular proteins, including catalase, were recovered in the medium . On the other hand, when V . rumoiensis cells were grown on plates containing various concentrations of H2O2, individual cells had a colony-forming ability inferior to those of E . coli, Bacillus subtilis, and Vibrio parahaemolyticus . Thus, it is suggested that when V . rumoiensis cells are exposed to high concentrations of H2O2, most cells will immediately be broken by H2O2 . In addition, the cells which have had little or no damage will start to grow in a medium where almost all H2O2 has been decomposed by the catalase released from broken cells.

J Appl Microbiol, 1998 Dec, 85(6), 1073 - 7
Note: characterization of Vibrio cholerae O139 Bengal isolated from water in Malaysia; Son R et al.; Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis . All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid . The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene . Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates . RAPD proved to be more sensitive . These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.

J Lipid Res, 1999 Jan, 40(1), 160 - 3
A simple, nonenzymatic method for desialylating polysialylated ganglio-N-tetraose series gangliosides to produce GM1; Schengrund CL et al.; Dowex-50W-H+ was used to catalyze the highly selective desialylation of polysialylated ganglio-N-tetraose series gangliosides to yield primarily GM1 . High performance thin-layer chromatographic analysis of recovered lipid indicated that 60-70% of the recovered ganglioside was GM1 . Identification of the major product as GM1 was confirmed by proton NMR spectra and lack of sialic acid release by Vibrio cholerae sialidase.

FEMS Microbiol Lett, 1998 Dec 15, 169(2), 331 - 9
Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease; Mitra R et al.; The non-membrane-damaging cytotoxin which causes dramatic cell rounding of cultured HeLa cells was purified to homogeneity from a clinical strain (WO5) of non-toxigenic Vibrio cholerae O1 Inaba belonging to the E1 Tor biotype . The purified protein has a denatured molecular weight of 35 kDa and a native molecular weight of approximately 37 kDa indicating the monomeric nature of the protein . The 15 N-terminal amino acid sequence of non-membrane-damaging cytotoxin showed complete homology to the hemagglutinin protease previously purified and characterized from V . cholerae O1 . Purified non-membrane-damaging cytotoxin from V . cholerae O1 was immunologically and biochemically identical to that previously purified from V . cholerae O26 . Non-membrane-damaging cytotoxin was found to be enterotoxic in rabbit ileal loop assay inducing accumulation of non-hemorrhagic fluid at 100 micrograms and elicited a concentration dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on Ussing chambers . A significant serum immunoglobulin G response against non-membrane-damaging cytotoxin was elicited by patients infected with V . cholerae O139 but not with V . cholerae O1 . These properties make non-membrane-damaging cytotoxin a potential virulence factor of V . cholerae which should be taken into consideration while making live, attenuated recombinant vaccine strains against cholera.

Ann Intern Med, 1998 Dec 1, 129(11), 932 - 9
Management of hemochromatosis . Hemochromatosis Management Working Group; Barton JC et al.; The complications of iron overload in hemochromatosis can be avoided by early diagnosis and appropriate management . Therapeutic phlebotomy is used to remove excess iron and maintain low normal body iron stores, and it should be initiated in men with serum ferritin levels of 300 microg/L or more and in women with serum ferritin levels of 200 microg/L or more, regardless of the presence or absence of symptoms . Typically, therapeutic phlebotomy consists of 1) removal of 1 unit (450 to 500 mL) of blood weekly until the serum ferritin level is 10 to 20 microg/L and 2) maintenance of the serum ferritin level at 50 microg/L or less thereafter by periodic removal of blood . Hyperferritinemia attributable to iron overload is resolved by therapeutic phlebotomy . When applied before iron overload becomes severe, this treatment also prevents complications of iron overload, including hepatic cirrhosis, primary liver cancer, diabetes mellitus, hypogonadotrophic hypogonadism, joint disease, and cardiomyopathy . In patients with established iron overload disease, weakness, fatigue, increased hepatic enzyme concentrations, right upper quadrant pain, and hyperpigmentation are often substantially alleviated by therapeutic phlebotomy . Patients with liver disease, joint disease, diabetes mellitus and other endocrinopathic abnormalities, and cardiac abnormalities often require additional, specific management . Dietary management of hemochromatosis includes avoidance of medicinal iron, mineral supplements, excess vitamin C, and uncooked seafoods . This can reduce the rate of iron reaccumulation; reduce retention of nonferrous metals; and help reduce complications of liver disease, diabetes mellitus, and Vibrio infection . This comprehensive approach to the management of hemochromatosis can decrease the frequency and severity of iron overload, improve quality of life, and increase longevity.

Indian J Pathol Microbiol, 1998 Oct, 41(4), 419 - 22
Study of proteases and other enzymes of Vibrio cholerae 01 E2 Tor and 0139 serotypes isolated in Yavatmal (Maharashtra); Ingole KV et al.; V . cholerae 01 E1 Tor isolated during Cholera epidemic of 1994 and V cholerae 0139 serotype isolated during 1993 epidemic were subjected to the study of proteases and other enzymes . Out of 26 strains of V . cholerae 01 studied, gelatinase and caseinase activity was seen in 100 and 69.23 percent strains respectively . All strains showed catalase and oxidase activity . Of the other enzymes studied 19.23, 65.38 and 57.69 percent strains were positive for DNAse, lipase and phosphatase respectively . None of the strains showed lecithinase activity . Similar findings were observed in 22 strains of V . cholerae 0139 except all strains were positive for phosphatase activity . Role of enzymes in virulence is suggested.

Infect Immun, 1999 Jan, 67(1), 294 - 301
Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide; Boesen HT et al.; The sensitivity of Vibrio anguillarum to the bactericidal effect of rainbow trout serum was investigated with different strains of serogroups O1 and O2a, which are the most frequently found serogroups in clinical outbreaks of vibriosis . All of the V . anguillarum strains were able to activate complement in rainbow trout serum, but smooth strains of V . anguillarum serogroup O1 were resistant to complement-mediated killing in the absence of specific antibodies . In the case of V . anguillarum serogroup O2a strains, 80% of the analyzed strains were resistant to rainbow trout serum even when specific antibodies were present . Analysis of the lipopolysaccharide structures of the tested V . anguillarum strains showed a positive correlation between the O-antigen size of the lipopolysaccharide and resistance to serum killing . The classical complement pathway was responsible for the antibody-dependent serum killing of susceptible V . anguillarum strains . When serum-resistant V . anguillarum serogroup O2a strains were grown in glucose-enriched Lennox L broth, they produced lipopolysaccharide molecules with fewer high-molecular-weight O-antigen units than did strains grown in broth without the addition of glucose . Strains grown in glucose-enriched medium became sensitive to rainbow trout serum killing, indicating that the high-molecular-weight O-antigen side chains prevented the activated complement from damaging the bacterium.

Infect Immun, 1999 Jan, 67(1), 148 - 54
Resurgent Vibrio cholerae O139: rearrangement of cholera toxin genetic elements and amplification of rrn operon; Khetawat G et al.; The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis . The reappearance of V . cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V . cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits . With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V . cholerae O139 (O139R) strains were examined . Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates . Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS . While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B . The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains . The recent isolates of V . cholerae O139 have thus gained one rrn operon . This variation in the number of rrn operons within a serogroup has not been reported for any other organism . The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.

Biotechniques, 1998 Dec, 25(6), 1030 - 5
Chitobiase, a new reporter enzyme; Kalabat DY et al.; N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions . This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine . The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-beta-D-glucosaminidase activities are missing in E . coli strains, (ii) chitobiase can be monitored using blue/white colony indicator plates and (iii) convenient substrates for this enzyme are commercially available . The use of chitobiase as a reporter enzyme is generally applicable to the study of gene expression in those bacteria that do not contain N-acetyl-beta-D-glucosaminidases . We constructed plasmid vectors containing a multiple cloning site for producing in-frame fusions to chitobiase, the attP of lambda phase for movement into the bacterial chromosome for single-copy analysis, the gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 origin of replication and the rrnBt1t2 terminator region upstream of the chb gene to prevent read-through from other promoters . In-frame fusions between the dnaA gene and chb were moved to the chromosome by site-specific recombination with the chromosomal attB site . These single-copy fusions were assayed for chitobiase to examine the effects of a deletion in the dnaA regulatory region.

Carbohydr Res, 1998 Nov, 313(1), 15 - 20
Studies towards neoglycoconjugates from the monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa using the diethyl squarate reagent; Zhang J et al.; The effect of reaction time, concentration and molar excess of hapten upon the efficiency of the conjugation of carbohydrates to proteins using the diethyl squarate reagent has been studied using chicken serum albumin (CSA) as the carrier protein and a linker-equipped D-glucose derivative as the hapten . A high degree of incorporation of the latter into CSA was achieved with high efficiency, and the use of a large excess of the ligand was not necessary . Conjugation of the immunodominant monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, bearing the same spacer, followed a similar pattern, showing that the nature of the carbohydrate does not substantially affect the outcome of the conjugation and that a predicted degree of antigen-loading onto carrier protein is possible to achieve.

Infection, 1998 Nov-Dec, 26(6), 399 - 401
Necrotizing fasciitis caused by Vibrio vulnificus: first published infection acquired in Turkey is the second time a strain is isolated in Germany; Horre R et al.; Vibrio vulnificus, a marine vibrio, has recently been recognized as a potential human pathogen . It causes human infections with mortality rates up to 60% . Until 1991, most human isolations were reported from the USA, Japan and Taiwan . The second strain isolated in Germany is documented and a significant case of V . vulnificus infection acquired in Turkey is published for the first time.

Gene, 1998 Nov 26, 223(1-2), 269 - 82
Genetic organization of the regions associated with surface polysaccharide synthesis in Vibrio cholerae O1, O139 and Vibrio anguillarum O1 and O2: a review; Stroeher UH et al.; Vibrio cholerae and V . anguillarum are recognized as aquatic-borne human and fish pathogens, respectively . Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related . Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species . The most recent findings suggest that IS1358 is associated with the rfb region in V . cholerae O1 and O139 and in V . anguillarum O1 and O2 . In addition, the rfb region in both V . cholerae serogroups and in V . anguillarum O1 is limited at one end by gmhD . These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD . Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions . This review examines the genetics and biosynthesis of the O-antigen and capsule of V . cholerae O1 and O139, as well as the V . anguillarum serogroup O1 and the role of IS1358 . Throughout this review we have used the new nomenclature for rfb genes proposed by.

Microb Ecol, 1999 Jan, 37(1), 3 - 12
Factors Affecting Predation by Cyclidium sp . and Euplotes sp . on PAH-Degrading and Nondegrading Bacteria; Tso SF et al.; Abstract If predators select for or against contaminant-degrading bacteria, it will affect bacterial survival and has important implications for bioremediation . Protozoa are important predators of bacteria . In order to determine whether protozoa preyed differentially on bacteria with different degradation abilities, two ciliates (Euplotes sp . and Cyclidium sp.) and three strains of PAH-degrading bacteria (Vibrio spp., degrading naphthalene, anthracene, or phenanthrene) were isolated from sediment from New York/New Jersey Harbor . By manipulating growth conditions, bacterial strains with different PAH-degradation abilities and different cell properties were produced . Stepwise regression models were used to analyze how clearance rates on suspended bacteria and grazing rates on bacteria attached to particles were affected by bacterial size, hydrophobicity, C:N ratio, protein content, and PAH-degradation ability . Clearance rates ranged from 0 to 49 nl ciliate-1 h-1 for Euplotes sp . and from 0 to 1.7 nl ciliate-1 h-1 for Cyclidium sp . Clearance rates of both ciliates were positively correlated with bacterial size, hydrophobicity, and protein content, and negatively correlated with C:N ratio . PAH degradation ability had no (for Euplotes sp.) or small (for Cyclidium sp.) effects on clearance rates . The models accounted for 63-75% of the variation in clearance rates on different bacteria . Only Euplotes sp . grazed on attached bacteria, at rates from 3 to 176 bacteria ciliate-1 h-1 . A regression model with only C:N ratio and protein content explained 45% of the variation in grazing rates . These models indicate that multiple properties of bacteria affect their susceptibility to predation by ciliates, but PAH-degradation ability per se has little effect.

Trans R Soc Trop Med Hyg, 1998 Jul-Aug, 92(4), 460 - 2
A randomized clinical trial to compare the efficacy of erythromycin, ampicillin and tetracycline for the treatment of cholera in children; Roy SK et al.; To compare the clinical outcome of treatment of cholera in children with ampicillin, erythromycin or tetracycline, a double-'blind' randomized four-cell trial was carried out in Bangladesh . Ampicillin was chosen as additional therapy for acute respiratory tract infection, present in many subjects with diarrhoea . One hundred and eighty-four children aged 1-5 years who were not wasted, with diarrhoea of duration < 48 h, signs of some or severe dehydration, dark-field stool microscopy demonstrating Vibrio cholerae, and a baseline purging rate > 4 mL/kg/h over 6 h were enrolled in the study . Ampicillin, tetracycline, erythromycin or placebo were given orally every 6 h for 3 d . After 3 d of antibiotic treatment, diarrhoeal stool volume was significantly reduced in all antibiotic groups, with mean volumes per kg body weight as follows: tetracycline, 318 mL (SEM = 50), ampicillin, 335 mL (SEM = 30); erythromycin, 323 mL (SEM = 25); placebo, 498 mL (SEM = 37) . Compared to tetracycline, the clinical recovery rates by 96 h were 75% with placebo, 91.3% with ampicillin, and 95.7% with eythromycin . Compared to tetracycline, the total mean times to recovery were increased by 66% with placebo (P < 0.001), 25% with ampicillin (P < 0.017), and 9% with erythromycin (P = 0.37) . These results indicated comparable clinical efficacy of tetracycline, ampicillin and erythromycin . We therefore recommend that, unless V . cholerae is resistant, ampicillin should be used as a cost-effective alternative to erythromycin for paediatric cholera, especially in children with concomitant acute respiratory infection.

J, Mar . Biotechnol. . 1998 Aug, 6(3), 193 - 7
Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta); Araki T et al.; A systematic method was designed for the isolation of a large number of protoplasts from an agarophyte alga Gracilaria verrucosa using agarase from a marine bacterium Vibrio sp . PO-303 and commercial enzymes (Cellulase Onozuka RS and Macerozyme R-10) . Pretreatment of the tissue with 5% papain at 22 degreesC for 30 min before digestion with polysaccharide-degrading enzymes increased the protoplast yield . Suitable pH and temperature for the polysaccharide-degrading enzyme reaction were 6.5 and 22 degreesC, respectively . Mannitol (0.7 M) was found to be an excellent osmotic stabilizer . When the tissue (1 g, fresh wt.) of G . verrucosa pretreated with 5% papain solution (20 mM MES buffer, pH 7.5, containing 0.7 M mannitol) was digested with an enzyme mixture consisting of 4 units of agarase, 4% Cellulase Onozuka, 2% Macerozyme, and 0.7 M mannitol in 20 mM MES buffer (pH 6.5) with gentle agitation for 150 min at 22 degreesC, 1.03 x 10(8) protoplasts were obtained.

J, Mar . Biotechnol. . 1998 Aug, 6(3), 178 - 82
Immunofluorescent detection of ice-ice disease-promoting bacterial strain Vibrio sp . P11 of the farmed macroalga, Kappaphycus alvarezii (Gigartinales, Rhodophyta); Largo DB et al.; A specific immunofluorescent probe consisting of polyclonal antibodies was developed to detect a marine bacterium, Vibrio sp . strain P11, which was found in a previous study to promote the ice-ice disease in the cultivated red macroalga, Kappaphycus alvarezii . The method involves a combined application of the fluorescent stains, 4',6-diamidino-2-phenylindole (DAPI) and the P11 PAbs, into a homogenized seaweed sample (<1 g wet wt), which is prediluted to make bacteria countable under an epifluorescence microscope without serious interference from autofluorescing algal debris . The algal tissue homogenate is then filtered through a 0.2-microm-pore size Nuclepore membrane filter, serving as a mounting pad, and viewed using alternating ultraviolet and IB excitation filters to detect total and specific bacteria, respectively, on the same microscopic field, at the same time . The immunofluorescent probe could be used as a valuable tool in studying the infection mechanism of the bacterium in the macroalga in vitro.

J, Mar . Biotechnol. . 1998 Aug, 6(3), 168 - 73
Characterization of lipolytic activity associated with a Vibrio species of bacterium isolated from fish intestines; Henderson RJ et al.; Cultures of a species of Vibrio isolated from fish intestines and known to synthesize the polyunsaturated fatty acid eicosapentaenoic acid (20:5n-3) were incubated with di-{1-14C}palmitoyl phosphatidylcholine to determine if lipolytic enzymes are produced by the bacteria . After two days of culture, most radioactivity was recovered in the phospholipids of the bacterial cells . When supernatants from cultures of the Vibrio were incubated with either di-{1-14C}palmitoyl phosphatidylcholine, 1-palmitoyl-2-{1-14C}linoleoyl phosphatidylcholine or 1-{1-14C}palmitoyl lysophosphatidylcholine almost all radioactivity was recovered in the free fatty acid fraction after 24 h . Only very small levels of radioactivity from di-{1-14C}palmitoyl phosphatidylcholine were recovered in diacylglycerols and phosphatidic acid . Over the same incubation period 61% and 5% of the radioactivity originally present in glycerol tri-{1-14C}oleate and cholesteroyl {1-14C}oleate, respectively, was released to free fatty acids . Soybean phosphatidylcholine and cod roe phosphatidylcholine, which differed in polyunsaturated fatty acid profile, were both hydrolyzed by culture supernatant . The results suggest that the Vibrio species examined produces a phospholipase B capable of hydrolyzing both intact phospholipids and lysophospholipids.

Microbiology, 1998 Nov, 144 ( Pt 11), 2987 - 3002
Internalization and cytotoxicity are important virulence mechanisms in Vibrio-fish epithelial cell interactions; Wang XH et al.; Vibrio anguillarum and Vibrio damselae are Gram-negative bacteria that cause systemic infections called vibriosis in fish . They can enter fish cells and survive as intracellular parasites . The host-pathogen interactions between these Vibrio species and the fish epithelial cell lines epithelioma papillosum of carp (EPC) and grunt-fin tissue (GF) cells, were examined using phase-contrast, scanning electron and confocal microscopy . In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors . Some Vibrio species induced morphological changes in fish cells and this allowed classification into a cytopathic group and a noncytopathic group . The cytopathic group could be subdivided into two invasive groups (I and II) and a cytotoxic group . Of the invasive strains V . anguillarum 811218-5W (group I) and G/Virus/5(3) (group II), genistein, a tyrosine kinase inhibitor, only inhibited internalization of V . anguillarum G/Virus/5(3) into EPC cells, whereas staurosporine, a protein kinase C inhibitor, accelerated internalization of both strains . Cytochalasin D, an inhibitor of microfilament polymerization, prevented internalization of both strains, whilst vincristin, a microtubule inhibitor, only inhibited internalization of V . anguillarum G/Virus/5(3) . For the cytotoxic strain V . damselae ATCC 33539, extracellular products (ECP) alone caused morphological changes in EPC and GF . Bacterial internalization may not be important in the pathogenesis of this group . The non-cytopathic strain V . anguillarum S2/5/93(2) did not enter cells or induce any changes in EPC and GF monolayers . This study has identified some major differences between Vibrio species in their interactions with fish cells in vitro and will thus facilitate future studies of the molecular basis of pathogenesis of vibriosis.

J Infect Dis, 1999 Jan, 179(1), 275 - 8
Survival of Vibrio vulnificus in whole blood from patients with chronic liver diseases: association with phagocytosis by neutrophils and serum ferritin levels; Hor LI et al.; Vibrio vulnificus causes severe wound infections and sepsis, mostly in persons with chronic liver diseases . Survival of this organism in the whole blood collected from healthy volunteers and patients with chronic hepatitis, liver cirrhosis, and hepatoma was analyzed as an indication of susceptibility . The bacterial numbers in the blood after 5 h of incubation tended to increase with the severity of the liver disease and differed significantly between hepatoma patients and healthy volunteers (P<.05) . Survival of V . vulnificus in the whole blood correlated positively with serum ferritin concentration (r=.266; P<.05) and percentage of transferrin iron saturation (r= . 200; P<.05) and correlated negatively with serum C4 concentration (r=-.198; P<.05) and phagocytosis by neutrophils (r=-.204; P<.05) . Among these parameters, low phagocytosis activity (P<.01) and high ferritin level (P<.01) in the blood were the independent predictors.

Curr Microbiol, 1999 Jan, 38(1), 1 - 8
Characterization of porins isolated from the outer membrane of Vibrio damsela; Nitzan Y et al.; A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells . This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline . The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm . This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass . After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation . The monomers of the 37-kDa protein were not active in the reconstitution assay . The effect of culture media on the composition of the outer membrane protein of V . damsela was examined . Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl-BHI broth and in 3% NaCl-nutrient broth with the addition of 2% glucose . Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl-nutrient broth . An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl-nutrient broth with the addition of 2% maltose . This protein was found to exhibit specificity to maltose derivatives . The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins.

Microbiol Mol Biol Rev, 1998 Dec, 62(4), 1301 - 14
Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae; Faruque SM et al.; Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation . The disease is characterized by a devastating watery diarrhea which leads to rapid dehydration, and death occurs in 50 to 70% of untreated patients . Cholera is a waterborne disease, and the importance of water ecology is suggested by the close association of V . cholerae with surface water and the population interacting with the water . Cholera toxin (CT), which is responsible for the profuse diarrhea, is encoded by a lysogenic bacteriophage designated CTXPhi . Although the mechanism by which CT causes diarrhea is known, it is not clear why V . cholerae should infect and elaborate the lethal toxin in the host . Molecular epidemiological surveillance has revealed clonal diversity among toxigenic V . cholerae strains and a continual emergence of new epidemic clones . In view of lysogenic conversion by CTXPhi as a possible mechanism of origination of new toxigenic clones of V . cholerae, it appears that the continual emergence of new toxigenic strains and their selective enrichment during cholera outbreaks constitute an essential component of the natural ecosystem for the evolution of epidemic V . cholerae strains and genetic elements that mediate the transfer of virulence genes . The ecosystem comprising V . cholerae, CTXPhi, the aquatic environment, and the mammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen.

J Biol Chem, 1998 Dec 11, 273(50), 33841 - 7
Characterization of the interaction between Fur and the iron transport promoter of the virulence plasmid in Vibrio anguillarum; Chai S et al.; The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur protein and the antisense RNA, RNAalpha . Here we report the identification of the promoter for the iron transport genes and studied the interaction between the V . anguillarum Fur protein and this promoter . The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1 mapping analysis . High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constitutive in the Fur-deficient strain . Gel retardation and DNase I footprint analysis showed that Fur binds specifically to two contiguous sites comprising the promoter region and the region downstream of the transcription start site . The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fur protein . DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hypersensitivity to the nuclease in the nontemplate strand . The periodicity of the hypersensitive sites suggested that the promoter DNA undergoes a structural change upon binding to Fur, which might play a role in the repression of gene expression.

Appl Environ Microbiol, 1998 Dec, 64(12), 4683 - 8
Effect of canavanine from alfalfa seeds on the population biology of bacillus cereus
Emmert EAB, Milner JL, Lee JC, Pulvermacher KL, Olivares HA, Clardy J, Handelsman J.
Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J . L . Milner, S . J . Raffel, B . J . Lethbridge, and J . Handelsman, Appl . Microbiol . Biotechnol . 43:685-691, 1995) . In this study, we determined the chemical basis for and biological role of the inhibitory activity . All of the alfalfa germ plasm tested included seeds that released inhibitory material . We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds . Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity . Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B . cereus, Bacillus thuringiensis, and Vibrio cholerae . The B . cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces . The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization . These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B . cereus.

Appl Environ Microbiol, 1998 Dec, 64(12), 4676 - 82
Heterogeneity among isolates of Vibrio vulnificus recovered from eels (Anguilla anguilla) in Denmark; Hoi L et al.; The findings of this study demonstrate that Vibrio vulnificus isolates recovered from diseased eels in Denmark are heterogeneous as shown by O serovars, capsule types, ribotyping, phage typing, and plasmid profiling . The study includes 85 V . vulnificus isolates isolated from the gills, intestinal contents, mucus, spleen, and kidneys of eels during five disease outbreaks on two Danish eel farms from 1995 to 1997, along with a collection of 12 V . vulnificus reference strains . The results showed that more than one serovar may be capable of causing disease in eels and that these isolates are genetically heterogenous as shown by ribotyping . Ribotyping also showed that the same isolates may persist in an eel farm and cause recurrent outbreaks . Phage typing did not correlate with ribotyping or serotyping . However, we observed that 26 of 28 isolates, which were not susceptible to any of the phages, showed the same ribotype, O serovar, and capsule type . This suggests that these isolates may possess features that make them resistant to lysis by the phages used in this study . Ninety-three of 97 isolates harbored between one and three high-molecular-weight plasmids which previously had been suggested to be associated with eel virulence . The subdivision of V . vulnificus into two biotypes based on the indole reaction can no longer be supported, since 82 of 97 isolates in this study were indole positive, and a subdivision into serovars appears to be more correct.

Neurosci Lett, 1998 Oct 23, 255(3), 139 - 42
Membrane carbohydrate conjugates desialylation does not alter {3H}-dopamine uptake in rat striatal slices; Page G et al.; Incubation of rat striatal slices induced a large decrease (about 50%) of DA uptake and a slight desialylation of polysialogangliosides (GT1b, GD1b, GD1a) with an increase of monosialogangliosides (GM1) . Moreover, a pretreatment of slices by exogenous added neuraminidase of Vibrio cholerae did not modify DA uptake, although the pattern of gangliosides was modified and there was considerable loss (about 45%) of sialic acid in gangliosides and glycoproteins . It was verified that neuraminidase activity occured in synaptic membrane . Thus, DA uptake was apparently not altered by desialylation of plasma membrane carbohydrate conjugates.

Chem Biol, 1998 Nov, 5(11), 631 - 45
Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin; Quadri LE et al.; BACKGROUND: Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron . Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion . The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively . RESULTS: A ten-gene cluster spanning 24 kilobases of the M . tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis . The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase . An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation . Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP . CONCLUSIONS: Mycobactins are produced in M . tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy . The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes . Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E . coli pathogens.

Mem Inst Oswaldo Cruz, 1998 Sep-Oct, 93(5), 601 - 7
The Amazonia variant of Vibrio cholerae: molecular identification and study of virulence genes; Baptista MA et al.; The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al . 1995a) . This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the E1 Tor and classical biotypes . The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found . As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains . This gene was not previously described in V . cholerae, but its sequence is present in the TIGR database specific for this species.

Mem Inst Oswaldo Cruz, 1998 Sep-Oct, 93(5), 595 - 9
Molecular basis of ribotype variation in the seventh pandemic clone and its O139 variant of Vibrio cholerae; Lan R et al.; Ribotyping has been widely used to characterise the seventh pandemic clone including South American and O139 variants which appeared in 1991 and 1992 respectively . To reveal the molecular basis of ribotype variation we analysed the rrn operons and their flanking regions . All but one variation detected by BglI, the most discriminatory enzyme, was found to be due to changes within the rrn operons, resulting from recombination between operons . The recombinants are detected because of the presence of a BglI site in the 16S gene in three of the nine rrn operons and/or changes of intergenic spacer types of which four variants were identified . As the frequency of rrn recombination is high, ribotyping becomes a less useful tool for evolutionary studies and long term monitoring of the pathogenic clones of Vibrio cholerae as variation could undergo precise reversion by the same recombination event.

Mem Inst Oswaldo Cruz, 1998 Sep-Oct, 93(5), 567 - 76
Evolutionary control of infectious disease: prospects for vectorborne and waterborne pathogens; Ewald PW et al.; Evolutionary theory may contribute to practical solutions for control of disease by identifying interventions that may cause pathogens to evolve to reduced virulence . Theory predicts, for example, that pathogens transmitted by water or arthropod vectors should evolve to relatively high levels of virulence because such pathogens can gain the evolutionary benefits of relatively high levels of host exploitation while paying little price from host illness . The entrance of Vibrio cholerae into South America in 1991 has generated a natural experiment that allows testing of this idea by determining whether geographic and temporal variations in toxigenicity correspond to variation in the potential for waterborne transmission . Preliminary studies show such correspondences: toxigenicity is negatively associated with access to uncontaminated water in Brazil; and in Chile, where the potential for waterborne transmission is particularly low, toxigenicity of strains declined between 1991 and 1998 . In theory vector-proofing of houses should be similarly associated with benignity of vectorborne pathogens, such as the agents of dengue, malaria, and Chagas' disease . These preliminary studies draw attention to the need for definitive prospective experiments to determine whether interventions such as provisioning of uncontaminated water and vector-proofing of houses cause evolutionary reductions in virulence.

Appl Microbiol Biotechnol, 1998 Oct, 50(4), 455 - 8
A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation; Elasri MO et al.; We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisheri lux operon . This recA-lux fusion (pMOE15) was introduced into wild-type P . aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production . The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence . RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment.

J Bacteriol, 1998 Dec, 180(23), 6101 - 6
Structural and functional characterization of IS1358 from Vibrio cholerae; Dumontier S et al.; The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment . This O139-specific DNA fragment contains an insertion sequence that was described previously (U . H . Stroeher, K . E . Jedani, B . K . Dredge, R . Morona, M . H . Brown, L . E . Karageorgos, J . M . Albert, and P . A . Manning, Proc . Natl . Acad . Sci . USA 92:10374-10378, 1995) and designated IS1358O139 . We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis . Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141 . Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155 . We cloned and sequenced four copies of IS1358 from V . cholerae O22 and one copy from V . cholerae O155 . A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical . We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element . Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 x 10(-8) . Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1357 - 62
Assignment of Vibrio sp . strain ABE-1 to Colwellia maris sp . nov., a new psychrophilic bacterium; Yumoto I et al.; A psychrophilic bacterium, previously described as Vibrio sp . strain ABE-1T, has been reassigned by phenotypic characterization, chemotaxonomic analysis and 16S rRNA phylogenetic analysis . The organism was curved rods and it could reduce nitrate to nitrite and hydrolyse gelatin and DNA, but not chitin . NaCl was required for growth . This strain was susceptible to the vibriostatic compound O/129 . The major isoprenoid quinone was ubiquinone-8 and the DNA G + C content was 39.4 mol% . The whole-cell fatty acids comprised saturated and monounsaturated fatty acids with 10-18 C atoms; saturated and monounsaturated C16 fatty acids were predominant . Strain ABE-1T contained the unique trans-unsaturated fatty acid, 9-trans-hexadecenoic acid . Although strain ABE-1T has been identified as a Vibrio species, the strain did not ferment glucose . Phylogenetic analysis based on 16S rRNA sequencing indicated that strain ABE-1T was more closely related to Colwellia species than to Vibrio species . However, strain ABE-1T differed from other reported Colwellia species in terms of phylogenetic position, some phenotypic characteristics, chemotaxonomic analysis and relatedness by DNA-DNA hybridization . Accordingly, the name Colwellia maris is proposed . The type strain is ABE-1T (= JCM 10085T).

Nucleic Acids Res, 1998 Dec 1, 26(23), 5409 - 16
Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore; Masuko M et al.; A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target . In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer . The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal . The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution . Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole . We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14464 - 9
The Vibrio cholerae genome contains two unique circular chromosomes; Trucksis M et al.; Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, is a Gram-negative bacterium that belongs to the gamma subdivision of the family Proteobacteriaceae . The physical map of the genome has been reported, and the genome has been described as a single 3.2-Mb chromosome {Majumder, R., et al . (1996) J . Bacteriol . 178, 1105-1112} . By using pulsed-field gel electrophoresis of genomic DNA immobilized in agarose plugs and digested with the restriction enzymes I-CeuI, SfiI, and NotI, we have also constructed the physical map of V . cholerae . Our analysis estimates the size of the genome at 4.0 Mb, 25% larger than the physical map reported by others . Our most notable finding is, however, that the V . cholerae chromosome appears to be not the single chromosome reported but two unique and separate circular megareplicons.

Infect Immun, 1998 Dec, 66(12), 5819 - 25
Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTXPhi: molecular basis for origination of new strains with epidemic potential; Faruque SM et al.; Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin . The receptor for CTXPhi for invading V . cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island . We analyzed 146 CTX-negative strains of V . cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V . cholerae . DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS . An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice . The phage genome integrated into the chromosome of infected V . cholerae O1 cells forming stable lysogens . Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V . cholerae . To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V . cholerae strains by CTXPhi . The results of this study further indicated that strains belonging to the O1 serogroup of V . cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.

Infect Immun, 1998 Dec, 66(12), 5659 - 68
The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice; Paranjpye RN et al.; Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts . We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V . vulnificus genomic library . One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P . aeruginosa mutant deficient in the expression of PilD . The other gene (vvpC) encodes a homolog of PilC from P . aeruginosa, where it is essential for assembly of type IV pili . Phenotypic characterization of a V . vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V . vulnificus are of the type IV class . This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway . Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model . Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V . vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.

Epidemiol Infect, 1998 Oct, 121(2), 269 - 73
Crayfish: a newly recognized vehicle for vibrio infections; Bean NH et al.; We conducted a 1-year case-control study of sporadic vibrio infections to identify risk factors related to consumption of seafood products in two coastal areas of Louisiana and Texas . Twenty-six persons with sporadic vibrio infections and 77 matched controls were enrolled . Multivariate analysis revealed that crayfish (P < 0.025) and raw oysters (P < 0.009) were independently associated with illness . Species-specific analysis revealed an association between consumption of cooked crayfish and Vibrio parahemolyticus infection (OR 9.24, P < 0.05) . No crayfish consumption was reported by persons with V . vulnificus infection . Although crayfish had been suspected as a vehicle for foodborne disease, this is the first time to our knowledge that consumption of cooked crayfish has been demonstrated to be associated with vibrio infection.

Epidemiol Infect, 1998 Oct, 121(2), 259 - 68
Phenotypic and molecular characterization of Vibrio cholerae O1 isolated in Samutsakorn, Thailand before, during and after the emergence of V . cholerae O139; Dalsgaard A et al.; Seventy clinical strains of Vibrio cholerae O1 isolated from 1982-96 in Samutsakorn, a port city 30 km southwest of Bangkok where cholera occurs at low levels with regular seasonality, were characterized to investigate if there were any differences among the O1 strains isolated before, during and after the 0139 epidemic . Pulsed-field gel electrophoresis (PFGE) typing, ribotyping and southern blot hybridization with a cholera toxin probe (CT genotyping) demonstrated several genotypes among O1 strains isolated before the emergence of V . cholerae 0139 . However, O1 strains isolated during and after the advent of 0139 showed identical ribotypes which were distinctly different from the types identified in strains isolated before the emergence of 0139 . Ribotypes identified in strains during and after the advent of 0139 were also demonstrated by O1 strains isolated immediately before the emergence of 0139 . Considering the seasonality of cholera in Samutsakorn, the identical ribotype and CT genotype and the closely related PFGE types shown by all O1 strains isolated during and after the appearance of 0139 is remarkable and suggest that the V . cholerae O1 strain may reemerge from an environmental source . A subgroup of V . cholerae O1 strains isolated before the emergence of the 0139 epidemic had a ribotype identical to a type demonstrated by 0139 strains isolated in Thailand . Our results support similar findings in Bangladesh and India that a distinct O1 strain appeared during the 0139 epidemic . However, compared with the apparent identical strain which replaced 0139 in Bangladesh and India, the emerged O1 strain in Samutsakorn showed a different ribotype and CT genotype.

Epidemiol Infect, 1998 Oct, 121(2), 253 - 8
Changes in Vibrio cholerae O1 strains isolated in Romania during 1977-95; Israil A et al.; Six hundred and twenty-four Vibrio cholerae O1 strains, 623 serotype Ogawa and one serotype Inaba, isolated in Romania between 1977-95 were tested to detect all changing traits concerning serogroup, serotype, biotype, phage type and resistotype patterns and subsequently, the possible epidemiological relationship among these strains . Biotyping revealed one classical, 580 eltor strains and 43 intermediary variants . When tested with Mukerjee phages, 546 (87%) strains were sensitive and 78 (13%) resistant . One phage type (M4) dominated during 1977-90, two phage types (M4 and M5) exhibited the same high frequencies during 1991, a diversity of types occurred during 1993-4 whereas in 1995, two phage types (M4 and M5) showed similar distributions again . Five patterns of drug susceptibility were successively described during 1977-95 . The most prominent changes in Vibrio cholerae O1 strains were noticed during 1993-4: the highest number of non-typable strains and intermediary variants, the widest spectrum of phage types and of multidrug resistance . In 1995, the strains reverted to the previous typable forms but a new drug resistance pattern was noticed.

Epidemiol Infect, 1998 Oct, 121(2), 245 - 51
Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India; Bag PK et al.; Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country . The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V . cholerae O1 which emerged in Calcutta in 1994 . Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1 . The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V . cholerae 0139 in Calcutta . However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of 0139 in Calcutta . The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V . cholerae O1 . None of the strains in this study carried plasmids . Molecular studies clearly show that the new expanded drug resistant clone of V . cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Sep-Oct, (5), 77 - 80
{Immunoenzyme test-system for the detection of Vibrio cholerae 0139}; Fedorova VA et al.; On the basis of antibodies to the lipopolysaccharide (LPS) and capsular antigen of V.cholerae O139 two variants of the enzyme immunoassay (EIA) system permitting the detection of the infective agent at a concentration of 1 x 10(6) microbial cells/ml were developed . Antibodies to V.cholerae O139 K-antigen and LPS were found to be highly active and specifically reacted only with homologous antigens without additional adsorption . Both variants of the EIA system, developed on the basis of antibodies to LPS or K-antigen, may be used for the detection of V.cholerae O139 . The use of the latter variant making it possible to evaluate the virulence of the isolated cultures.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Sep-Oct, (5), 30 - 3
{Factors of persistence and (or) pathogenicity in vibrios and aeromonads belonging to different ecotopes}; Bukharin OV et al.; Factors of persistence and/or pathogenicity in Vibrio parahaemolyticus and Aeromonas hydrophila (hemolytic, lipase, lecithin, DNAase, RNAase, antilysozyme, "anti-interferon", anticomplementary activities and capacity for absorbing Congo red) were studied . The study revealed the interspecific and subpopulation (hospital and extraorganismal parts of the population) differences in the activity of the manifestation of these factors . Strong dependence of the whole complex of persistence and pathogenicity factors of their belonging to the hostal part of Vibrio and Aeromonas populations was shown.

Mol Microbiol, 1998 Nov, 30(3), 499 - 511
Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene; Okuda J et al.; Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor . The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains . We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients . We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study . Only the tdh2 gene, one of the two tdh genes (tdh1 and tdh2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain . The tdh1- and tdh2-lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods . Two bases (positions -24 and -34) within the determined tdh2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh2 and tdh1 genes both in Escherichia coli and in V . parahaemolyticus backgrounds . Representative tdh promoters of KP-negative strains are close to the tdh2 promoter; they differ at position -34 but have the same base at position -24 as the tdh2 promoter . We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position -34 is sufficient to increase the expression of these genes to the KP-positive level . Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.

Biochemistry, 1998 Nov 17, 37(46), 16130 - 8
Tryptophan fluorescence of the lux-specific Vibrio harveyi acyl-ACP thioesterase and its tryptophan mutants: structural properties and ligand-induced conformational change; Li J et al.; The lux-specific myristoyl-ACP thioesterase from Vibrio harveyi contains four tryptophan residues, Trp23, Trp99, Trp186, and Trp213 . Replacement of each of these residues with tyrosine by site-directed mutagenesis coupled with fluorescence and quenching studies of the purified mutant and wild type thioesterases during catalysis has been used to probe ligand-induced conformational changes . Mutant W99Y retained high enzyme activity (80%) with W213Y and W23Y retaining intermediate activity and W186Y having the lowest activity (20%) . The sum of the differential fluorescence spectra of the individual tryptophans was identical to the fluorescence spectrum of the wild type thioesterase, showing that mutation had not caused a major conformational change and energy transfer did not occur between the tryptophans . Fluorescence emission maxima and quenching by acrylamide revealed that Trp213 and Trp23 are in a polar environment and/or exposed to solvent while Trp186 appeared to be buried inside the molecule, consistent with the crystal structure of the thioesterase . The fluorescence intensities of the wild type, W23Y, W99Y, and W186Y thioesterases increased in direct correlation to their degree of acylation with myristoyl-CoA, while the fluorescence of the acylated W213Y mutant remained constant, showing that the enhancement of fluorescence was entirely due to interaction of the acyl group with Trp213 . Acrylamide quenching of the acylated mutants showed that the accessibility of the tryptophans to solvent was differentially altered and that the quenching of W23Y was enhanced in contrast to the quenching of the other mutants, supporting a ligand-induced conformational change during enzyme turnover.

Arch Microbiol, 1998 Oct, 170(5), 339 - 44
Morphological and physical characterization of the capsular layer of Vibrio cholerae O139; Meno Y et al.; The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined . An electron microscopic study using the freeze-substitution technique showed that all of the V . cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane . In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer . In addition, the capsule layer could also not be observed on the surface of V . cholerae strain O1 . To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes . The O139 strains were more resistant to the serum killing activity than were the V . cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains . The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain . In addition, all the V . cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes . The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role . These data suggest that the capsule of V . cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae.

J Clin Microbiol, 1998 Dec, 36(12), 3595 - 600
Rapid diagnosis of cholera caused by Vibrio cholerae O139; Chaicumpa W et al.; Hybridomas secreting specific monoclonal antibodies (MAbs) to Vibrio cholerae serogroup O139 were produced . Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of V . cholerae O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained . These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8 . The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively . Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by V . cholerae O139, and this assay was compared to the conventional bacterial isolation method . Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea . These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children's Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India . The V . cholerae O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested . Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42 V . cholerae O139-positive samples and gave a result of positive for three samples which were culture negative for V . cholerae O139 . The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively . The ELISA is easy to perform and relatively inexpensive . It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste . Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min . The assay is recommended as a rapid screening test of cholera cases caused by V . cholerae O139.

Scand J Infect Dis, 1998, 30(4), 393 - 6
Aetiological, clinical and epidemiological characteristics of a seasonal peak of diarrhoea in Dhaka, Bangladesh; Faruque AS et al.; During the spring peak of diarrhoea in Bangladesh, 113 consecutive patients who represented a systematic 4% sample of all patients attending an urban diarrhoea treatment facility between 18 and 23 April 1995 were studied . The study was conducted to characterize enteric pathogens associated with the spring peak of the diarrhoea outbreak in Bangladesh and to describe clinical and epidemiological features of the patients . The spring peak is traditionally thought to be mostly due to V . cholerae O1 . However, the most common cause of diarrhoea among the study patients was enterotoxigenic Escherichia coli (36%) followed by Vibrio cholerae O1 (23%) . The V . cholerae O1 patients attended significantly (p < 0.01) sooner after onset of diarrhoea than enterotoxigenic E . coli (ETEC) patients . Studies of behavioural and environmental characteristics are important to determine risk factors for observed higher proportion of ETEC infection during seasonal diarrhoea peaks.

J Travel Med, 1996 Mar 1, 3(1), 37 - 39
Cholera-Brucella Cross-Reaction: A New Potential Diagnostic Problem for Travelers to Latin America; Goicochea CE et al.; Background: Brucellosis is an endemic disease in Latin America and other countries . Serologic cross-reaction between cholera and Brucella infection is well recognized . Since the introduction to cholera in 1991 in Latin America, interpreting serologic tests has become potentially problematic . This study attempts to evaluate this problem . Methods: Tube agglutination tests were performed to detect Brucella antibodies in 44 Peruvian adult patients with moderate to severe diarrhea due to Vibrio cholerae O1 El Tor infection . These patients had no prior history and no clinical evidence of brucellosis . Results: False positive reactions were observed in 43.2% and 15.9% of the patients when cut-off points of >= 1/80 and >= 1/160 titer, respectively, were selected . These false positive reactions occurred within 4 to 14 days after the onset of diarrhea . The cross-reactivity decreased at the end of the fifth week (only 8.33% had a positive value at the fifth week) . Conclusions: Physicians should be alert to the false positive reaction to Brucella in patients with diarrhea . This is relevant to the evaluation of febrile illness in patients coming from developing countries where they could have been exposed to cholera.

J Pak Med Assoc, 1998 Jun, 48(6), 171 - 3
Cholera in children in Karachi from 1990 through 1995: a study of cases admitted to a tertiary care hospital; Nizami SQ et al.; Although cholera is an endemic disease in Bangladesh, India and other countries, it was never a significant cause of gastroenteritis in Pakistan before 1988 . Since then, cases of cholera are identified each year, both in adults and children in Pakistan . In order to see the contribution of Vibrio cholerae as a cause of gastroenteritis in children, we reviewed the cases of cholera admitted in the pediatric ward of the Aga Khan University Hospital, Karachi, Pakistan . Of 4346 children hospitalized with gastroenteritis during 1990 through 1995, 348 children (8%) were confirmed to have cholera . The youngest child with cholera was seven days old . The mean age was 31 +/- 34 months . The cases of cholera were received from all over the city . Most cases were due to Vibrio cholerae Ogawa biotype ELTOR but the new strain, i.e., Vibrio cholerae 0139 was isolated in 14% cases in 1994 . The sensitivity of Vibrio cholerae has also changed . In 1994, the organisms were resistant to commonly recommended antibiotics, i.e., tetracycline, ampicillin and erythrocin but sensitive to ceftrioxone, cefixime, ofloxacin and nalidixic acid . Adequate measures to improve hygiene and sanitation and supply of safe potable water is needed to prevent any future epidemic of cholera in the city.

Gene, 1998 Nov 5, 222(1), 25 - 30
Cloning and sequencing of major capsid protein (mcp) gene of a vibriophage, KVP20, possibly related to T-even coliphages; Matsuzaki S et al.; A large, tailed, prolate-headed vibriophage designated KVP20 was isolated from seawater . KVP20 was morphologically very similar to the previously described vibriophage, KVP40 (Matsuzaki, S., Inoue, T., Tanaka, S., 1998 . Virology, 242, 314-318) . However, they showed entirely different host specificities and could easily be differentiated from each other by their patterns of DNA restriction fragments . The major capsid protein (mcp) gene of KVP20 encoding the precursor of major capsid protein (pro-Mcp) was cloned and sequenced . The deduced amino-acid (aa) sequence of KVP20 pro-Mcp was compared with the reported aa sequences of KVP40 pro-Mcp, as well as of the equivalent proteins (gp23s) of coliphages T4 and RB49 . There was 96.7, 57.5, and 55.2% homology to the corresponding proteins of KVP40, T4, and RB49, respectively . These data strongly suggest that the two vibriophages are closely related to each other and that they are both distantly, but definitely, related to coliphages T4 and RB49.

J Pharm Sci, 1998 Nov, 87(11), 1351 - 6
Novel approaches for oral delivery of macromolecules; Fasano A; Traditional forms of administrations of nonabsorbable drugs and peptides often rely on their parenteral injection, since the intestinal epithelium is poorly permeable to these therapeutical agents . A number of innovative drug delivery approaches have been recently developed, including the drug entrapment within small vesicles or their passage through the intestinal paracellular pathway . Zonula occludens toxin, a recently discovered protein elaborated by Vibrio cholerae, provided tools to gain more insights on the pathophysiology of the regulation of intestinal permeability through the paracellular pathway and to develop alternative approaches for the oral delivery of drugs and macromolecules normally not absorbed through the intestine.

Med Trop (Mars), 1998, 58(2 Suppl), 32 - 5
{Cholera update and vaccination problems}; Fournier JM et al.; Cholera remains an important public health problem . The long-term control of cholera depends on good personal hygiene, uncontaminated water supply and appropriate sewage disposal . However, the improvement of hygiene is distant goal for many countries . Thus the availability of an effective cholera vaccine is important for the prevention of cholera in these countries . Research on new cholera vaccines has mainly focused on oral formulations that stimulate the mucosal secretory immune system . Two oral cholera vaccines were experimented on large scale in human . The first vaccine, containing inactivated bacterial cells and B-subunit of cholera toxin, has been tested in Bangladesh from 1985 to 1989 . This vaccine, according to WHO, may prove useful in the stable phase of refugee/displaced person crises, especially when given preventively . The second vaccine is a live attenuated vaccine containing the genetically manipulated Vibrio cholerae O1 strain CVD 103-HgR . Despite its efficacy in adult volunteers, results of a large-scale field trial carried-out in Indonesia for 4 years have shown a surprisingly low protection . Moreover, one of the safety concerns associated with live cholera vaccine is a possible horizontal gene transfer and recombination event leading to reversion to virulence . A new vaccine development program for cholera is based upon the hypothesis that immunoglobulins G directed to the O-specific polysaccharide of Vibrio cholerae O1 could confer protective immunity to cholera by inactivating the inoculum on intestinal mucosal surface . This program may lead to the development of cholera conjugate vaccines to elicit protection in infants.

Zentralbl Bakteriol, 1995 Oct, 282(4), 436 - 41
Role of outer membrane proteins on the adherence of Vibrio parahaemolyticus to rabbit intestinal epithelial cell in vitro; Chakrabarti MK et al.; Antiserum against outer membrane preparation of a Kanagawa phenomenon-positive strain of Vibrio parahaemolyticus were raised in rabbits and absorbed with their lipopolysaccharide . The anti-outer membrane protein serum and its Fab (IgG) fragment inhibited the adherence of Kanagawa-positive strains to rabbit intestinal epithelial cells in vitro . Preincubation of rabbit intestinal epithelial cells with outer membrane preparation also inhibited the adherence of these bacteria . Anti-lipopolysaccharide serum or its Fab (IgG) fragment did not inhibit adherence of V . parahaemolyticus . Moreover, pre-treatment of rabbit intestinal epithelial cells with lipopolysaccharide did not inhibit adherence of these strains . These results suggest that outer membrane proteins of V . parahaemolyticus play an important role in the adherence of Kanagawa-positive strains to rabbit intestinal epithelial cells.

Zentralbl Bakteriol, 1995 Nov, 283(1), 43 - 8
Nontoxigenic Vibrio cholerae O1 intestinal pathology in adult mice; Moyenuddin M et al.; The intestinal pathology caused by nontoxigenic Vibrio cholerae O1 was examined in the sealed adult mouse (SAM) model . Histologic examination demonstrated that a nontoxigenic V . cholerae O1 strain that elicited maximum fluid accumulation (FA) in the small intestine of adult mice caused damages to the villi and necrosis of lymphoid elements within solitary submucosal lymphoid nodules in the Peyer's patches . Challenge of mice with a strain that did not elicit intestinal FA produced none of the above tissue responses . Increased FA activity, intestinal alterations, and tissue pathology caused by the nontoxigenic V . cholerae O1 indicate its pathogenic potential.

Zentralbl Bakteriol, 1995 Nov, 283(1), 14 - 28
Genomic and biochemical relatedness between Vibrio cholerae serovar O139 and serovar O1 eltor strains; Prager R et al.; Vibrio cholerae O139 (Bengal) the new pandemic cholera strain emerging on the Indian subcontinent has revealed considerable homology to Vibrio cholerae O1 EL Tor (strain of the seventh pandemic cholera) in terms of genetic and biochemical properties . Apart from capsule and O139 LPS formation, all strains of V . cholerae O139 were found to be identical to V . cholerae O1 EL Tor strains with respect to genomic restriction fragment length polymorphism, genomic distribution of the pathogenic island, pattern of OMP and multilocus enzymes . However, the analysis of a nonpathogenic V . cholerae O139 isolate from Sri Lanka with a totally different pattern of genetic properties underline that horizontal gene transfer of a piece of DNA encoding biosynthesis of the Vibrio cholerae O139-specific LPS and capsule formation to an O1 El Tor precursor strains must have occurred giving rise to a kind of hybrid V . cholerae O1 El Tor encoding the new serovar-specific O139 antigens.

Vet Microbiol, 1998 Aug 28, 63(1), 61 - 9
Neurotoxic effect on two fish species and a PC12 cell line of the supernate of Vibrio alginolyticus and Vibrio anguillarum; Balebona MC et al.; The biological effects of supernates obtained from different strains of Vibrio alginolyticus and Vibrio anguillarum isolated from diseased fish have been studied by inoculation on two fish species, eel and rainbow trout, and two fish cell lines . These supernates possess neuroexcitatory properties, and so, when they are injected into both fish species, they trigger convulsions, wriggling, contortive swimming and respiratory arrest coupled with increased respiratory reflex . Furthermore, after the application of the supernates on cultures of noradrenergic pheochromocytoma PC12 cells, an increase of acetylcholine, released from the cells was obtained . The amount of released acetylcholine depends on the source of assayed supernates and on the dose applied to the cells . On the basis of the results obtained with PC12 cells, we suggest that the supernates from pathogenic Vibrio strains injected into fish may elicit an increased release of acetylcholine in the motor endplate of some muscles related to locomotion and ventilation of the inoculated fish.

Exp Parasitol, 1998 Nov, 90(3), 262 - 9
Entamoeba histolytica: identification of functional Gs and Gi proteins as possible signal transduction elements in the interaction of trophozoites with fibronectin; Soid-Raggi LG et al.; Trophozoites of Entamoeba histolytica adhere to several components of the extracellular matrix . Binding is mediated by specific receptors identified in the parasite surface . Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation . The process requires activation of signaling pathways in which PLC, IP3, Ca2-, and PKC participate . These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins . We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated by Vibrio cholerae and Bordetella pertussis toxins . Three of them are also recognized by antibodies prepared against the alpha-subunit of Gs-and Gi-proteins . Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins . Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity . Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin . The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment . Our present data show the presence and the functionality of Gs- and Gi-like proteins and their apparent activation during in vitro interaction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms in E . histolytica .

J Diarrhoeal Dis Res, 1998 Jun, 16(2), 87 - 90
Detection of tdh and trh genes in a urea-hydrolysing environmental isolate of Vibrio parahaemolyticus from the Andamans; Ghosh AR et al.; Co-existence of trh gene and urea-hydrolysing property in one of 44 marine water isolates of Vibrio parahaemolyticus correlates strongly with both genotypic and phenotypic characteristics of the bacterium . Thus, urease-producing phenotype can be considered a marker of virulence for the production of thermostable direct haemolysin-related haemolysin (TRH) (i.e . possession of trh gene) . The same isolate also possessed the tdh gene . An environmental isolate possessing all the characteristics of a pathogenic V . parahaemolyticus in this marine environment suggest that there is a likelihood of the occurrence of clinical cases of gastroenteritis caused by V . parahaemolyticus in the Andamans.

J Diarrhoeal Dis Res, 1998 Jun, 16(2), 66 - 73
Endemic cholera in Delhi, 1995: analysis of data from a sentinel centre; Singh J et al.; Data on cholera cases admitted to the Delhi Infectious Diseases Hospital (IDH) are presented to describe the pattern of occurrence of cholera in Delhi in 1995 . Rectal swabs from 4082 cases of acute diarrhoea admitted to the IDH were examined for excretion of Vibrio cholerae . Of them, 2004(49%) and 4(0.1%) were positive for V . cholerae O1 biotype El Tor and V . cholerae O139 respectively . Most cholera cases occurred during May-September (summer and monsoon months) . The period from January to March (winter) was completely free from cholera . The urban areas were not affected uniformly . Of the 80 PIN (Postal Index Number) code areas, 10 contributed to 57% of the cases . The early cases were scattered in PIN code areas distant from one another . The hospitalisation rates for cholera were the highest in children aged less than five years and declined significantly with increasing patients' age . Males had significantly higher rates than females aged up to 20 years, whereas the situation was reversed in the 20 to 39 year age group . Four per cent of the affected families had multiple cases . An estimated 1% of the household contacts of hospitalised cases of cholera were themselves hospitalised for cholera within 2 days of the first admission . Of the 260 V . cholerae O1 isolates tested, 4%, 7%, 8%, 89%, 91% and 95% were resistant to tetracycline, nalidixic acid, chloramphenicol, co-trimoxazole, streptomycin, and furazolidone respectively . The study highlights the usefulness of surveillance data to identify groups, urban areas and seasons with increased risk for cholera and to allow control measures to be focussed on those in greatest need.

Rev Cubana Med Trop, 1996, 48(3), 204 - 8
{Cholera in a district of Peru}; Perez Rodriguez AE et al.; Taking to consideration the low report of cholera patients and with the main knowing the reality about the introduction of Vibrio cholerae (V . cholerae) in Peru, a sample of 101 cases with acute diarrheal disease (ADD) was taken at the Distrito Villa El Salvador . They were selected by a systematic randomized sampling defined for each health care unit in the District, according to the daily average occurrence of ADD cases attended a week before the beginning of the study . All of them took part in a epidemiological survey . A sample was taken by rectal swab in order to isolate V . cholerae . 53 positive cases were found (52.2% and a confidence interval from 42.29 to 62.5%) with significant differences (p < 0.01) between the frequency in adults (67.3%) and children (34.8%) . V . cholerae was isolated only in 13 (61.9%) of the 21 cases who had contact with cholera patients, for a relative risk of 1.24 (0.83 < RR < 1.85) . A high positivity was also found, 21 cases (72.4%) among those who had raw food . A significant difference (p < 0.01) was observed in connection with those who had cooked food . In the multivariate logistic regression analysis it was only found a significant relationship with age and with the ingestion of raw food as regards the occurrence of cholera.

Rev Cubana Med Trop, 1996, 48(3), 169 - 70
{The application of the hybridization in colonies technic for the identification of toxigenic Vibrio cholerae 01}; Bravo Farinas L et al.; By means of the polymerase chain reaction (PCR) it was obtained a probe for the gen that codifies the subunit B of cholerae toxin (CTxB), which carried a Vibrio cholerae 01 reference strain . The checking of the amplified product was performed by using the hybridization techniques in colonies . This product hybridized with the gen that codifies for the subunit B of cholerae toxin isolated from Peru and Ecuador, representing the present epidemics in Latin America, but it did not so with the phylogenetically related strains.

Rev Cubana Med Trop, 1996, 48(3), 167 - 8
Detección de Escherichia coli toxigénica (LT) mediante la reacción en cadena de la polimerasa {The detection of toxigenic Escherichia coli (LT) by the polymerase chain reaction}; Ramirez M et al.; In this paper it is described the detection enteroxigenic Escherichia coli LT (+) . This method is based on the amplification of a DNA fragment of 400 pairs of bases by polymerase chain reaction (PRC) . The oligonucleotides were designed by the authors and the characteristic patterns were observed when the samples were submitted to an electrophoresis in an Agarose gel at 2% . The PCR had positive results with the strains of Escherichia coli 0:149 K; 88 (LT+) collection and with 20 strains isolated from patients with acute diarrhea . Negative results were found in Escherichia coli 0:101 K:99 NM (ST+), Vibrio cholerae 01 and Aeromonas hydrophila.

J Food Prot, 1998 Oct, 61(10), 1317 - 20
Survival of Vibrio cholerae 01 strains in shrimp subjected to freezing and boiling; Nascumento DR et al.; This research was undertaken to assess the resistance of Vibrio cholerae 01 strains inoculated into white shrimp, Penaeus schimitti, to heating and freezing treatments . Shrimp samples with and without carapace were obtained from Sao Luis, Brazil . Microbial analysis revealed the presence of marine vibrios including Vibrio alginolyticus, Vibrio parahaemolyticus, and other vibrios and aerobic gram-negative and gram-positive bacteria that grew on selective medium, thiosulfate-citrate-bile salt-sucrose agar . Samples with and without carapaces were heated before inoculating with cells of V . cholerae and then one-half of the samples was stored frozen at -200 degrees C and the other one-half was heated to boiling temperatures . Viable cells of the test organism were recovered from samples without carapaces, stored under frozen conditions, after 36 days . In contrast, no living cells were recovered after 26 days from samples with carapaces . Boiling temperatures were very damaging to V . cholerae 01 in shrimp samples with and without carapaces . Total destruction of the cells occurred within 1 to 2 min of exposure to heating.

Clin Infect Dis, 1998 Oct, 27(4), 774 - 80
Infections due to non-O1 Vibrio cholerae in southern Taiwan: predominance in cirrhotic patients; Ko WC et al.; Although Taiwan is not an area where cholera is endemic, from October 1988 to October 1997 30 episodes of non-O1, non-O139 Vibrio cholerae infection were noted at the National Cheng Kung University Hospital in Taiwan . Infections generally occurred in hot seasons, and two episodes were concomitant with Vibrio vulnificus infection . Three major clinical presentations were found: bacteremia with concurrent spontaneous bacterial peritonitis or invasive soft-tissue infections that occurred solely in cirrhotic patients; self-limited acute febrile gastroenteritis that occurred in patients with no underlying medical disease; and necrotizing fasciitis or cellulitis that often resulted from a wound on extremities . Other manifestations included fatal pneumonitis in a drowned man and acute pyosalpinx . The differential diagnosis of invasive infections in cirrhotic patients should include infections due to non-O1 V . cholerae or V . vulnificus, and a third-generation cephalosporin and a tetracycline analogue or a fluoroquinolone alone is recommended for treatment of severe vibrio infections.

Appl Environ Microbiol, 1998 Nov, 64(11), 4433 - 8
Implications of rRNA operon copy number and ribosome content in the marine oligotrophic ultramicrobacterium Sphingomonas sp . strain RB2256; Fegatella F et al.; Sphingomonas sp . strain RB2256 is a representative of the dominant class of ultramicrobacteria that are present in marine oligotrophic waters . In this study we examined the rRNA copy number and ribosome content of RB2256 to identify factors that may be associated with the relatively low rate of growth exhibited by the organism . It was found that RB2256 contains a single copy of the rRNA operon, in contrast to Vibrio spp., which contain more than eight copies . The maximum number of ribosomes per cell was observed during mid-log phase; however, this maximum content was low compared to those of faster-growing, heterotrophic bacteria (approximately 8% of the maximum ribosome content of Escherichia coli with a growth rate of 1 . 5 h-1) . The low number of ribosomes per cell appears to correlate with the low rate of growth (0.16 to 0.18 h-1) and the presence of a single copy of the rRNA operon . However, on the basis of cell volume, RB2256 appears to have a higher concentration of ribosomes than E . coli (approximately double that of E . coli with a growth rate of 1.5 h-1) . Ribosome numbers reached maximum levels during mid-log-phase growth but decreased rapidly to 10% of maximum during late log phase through 7 days of starvation . The cells in late log phase and at the onset of starvation displayed an immediate response to a sudden addition of excess glucose (3 mM) . This result demonstrates that a ribosome content 10% of maximum is sufficient to allow cells to immediately respond to nutrient upshift and achieve maximum rates of growth . These data indicate that the bulk of the ribosome pool is not required for protein synthesis and that ribosomes are not the limiting factor contributing to a low rate of growth . Our findings show that the regulation of ribosome content, the number of ribosomes per cell, and growth rate responses in RB2256 are fundamentally different from those characteristics in fast-growing heterotrophs like E . coli and that they may be characteristics typical of oligotrophic ultramicrobacteria.

Appl Environ Microbiol, 1998 Nov, 64(11), 4346 - 52
Identification and quantification of toxic chemicals by use of Escherichia coli carrying lux genes fused to stress promoters; Ben-Israel O et al.; The luxCDABE bioluminescence genes of the Vibrio fischeri lux system have been used as a reporter system for different stress and regulatory promoters of Escherichia coli . Selected E . coli strains carrying lux genes fused to different promoters were exposed to various toxic chemicals, and the recorded luminescence was used for the characterization of the biologic signature of each compound . Analysis of these data with the aid of a proper algorithm allowed quantitative and qualitative assessment of toxic chemicals . Of the 25 tested chemicals, 23 were identified by this novel strategy in a 3-h procedure . This system can also be adapted for the identification of simple mixtures of toxic agents when the biologic signatures of the individual compounds are known . This biologic recognition strategy also provides a tool for evaluating the degree of similarity between the modes of action of different toxic agents.

Appl Environ Microbiol, 1998 Nov, 64(11), 4269 - 75
Pathogenicity of Vibrio alginolyticus for cultured gilt-head sea bream (Sparus aurata L.); Balebona MC et al.; The in vivo and in vitro pathogenic activities of whole cells and extracellular products of Vibrio alginolyticus for cultured gilt-head sea bream were evaluated . The 50% lethal doses ranged from 5.4 x 10(4) to 1.0 x 10(6) CFU/g of body weight . The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line . In addition, the in vitro ability of V . alginolyticus to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy . The biological activities of extracellular products of V . alginolyticus were hydrolytic activities; the products were able to degrade sea bream mucus . V . alginolyticus was cytotoxic for fish cell lines and lethal for sea bream . Moreover, the extracellular products could degrade sea bream tissues . However, experiments performed with the bath immersion inoculation technique demonstrated that V . alginolyticus should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.

Carbohydr Res, 1998 Aug, 310(1-2), 117 - 9
More on the structure of Vibrio cholerae O22 lipopolysaccharide; Knirel YA et al.; The structure of a short-chain lipopolysaccharide (LPS) of Vibrio cholerae O22 strain 169-68, that cross-reacts with V . cholerae O139 Bengal, was elucidated . The structure differs in detail from that reported on another strain of O22 {A.D . Cox, J-R . Brisson, P . Thibault and M.B . Perry, Carbohydr . Res., 304 (1997) 191-208} . The similarity and difference between the LPS structures of the two strains as well as between O22 and O139 are discussed.

Zentralbl Veterinarmed A, 1998 Sep, 45(6-7), 369 - 81
Regional differences in the effect of cholera toxin and enterotoxigenic Escherichia coli infection on electrolyte and fluid transport in the porcine small intestine; Grondahl ML et al.; The regional differences in secretory and absorptive responses to cholera toxin (CT) and to infection by enterotoxigenic Escherichia coli (ETEC), producing heat-stable enterotoxins, were studied in the porcine small intestine . Proximal, mid and distal small intestine from newly weaned piglets were used . Na+ and Cl- fluxes and electrical parameters in CT-stimulated and ETEC-infected intestine were measured by the Ussing chamber technique . In addition, CT-induced fluid accumulation in ligated loops was measured . CT induced fluid accumulation, which was highest in the proximal segment and decreased in the aboral direction of the small intestine . In addition, CT induced a net Cl- secretion in the proximal and mid segments, while net Na+ absorption was reduced only in the proximal segment . The ETEC-infected intestine showed a net increase in Cl- secretion in the proximal part and abolished the net Na+ absorption in the distal segment . These results demonstrate segmental differences in the epithelial transport responses to enterotoxin from Vibrio cholerae and to ETEC infection . This needs to be taken into consideration in relation to oral rehydration studies.

Am J Prev Med, 1998 Oct, 15(3), 243 - 5
Raw shellfish consumption among renal disease patients . A risk factor for severe Vibrio vulnificus infection; Gholami P et al.; BACKGROUND: Raw shellfish-associated Vibrio vulnificus septicemia, with a case-fatality rate of nearly 50%, occurs most commonly in immunocompromised patients or those with liver disease . METHODS: Sixty patients with renal disease treated with hemodialysis at The George Washington University and awaiting renal transplantation completed an initial survey that assessed their raw shellfish eating habits and knowledge regarding the pathogen V . vulnificus . Patients were then given educational materials describing the risks of eating raw shellfish and, one month later, completed a second survey that assessed their knowledge retention and intent to eat or not eat raw shellfish in the future . RESULTS: Sixty of 68 (88%) eligible patients completed the survey . Forty-eight percent of patients reported having eaten raw shellfish after being diagnosed with kidney disease, with the highest rates reported among subjects < or = 49 years old and subjects with more than a high school education . Prior to receiving the educational materials, no patient had heard of the pathogen V . vulnificus . Three quarters of patients reported never having been advised by a physician to avoid eating raw shellfish . One month after reading the educational materials, 75% of patients said they would refrain from eating raw shellfish in the future . CONCLUSIONS: In view of their immunocompromised status, patients with end-stage renal disease should be counseled to abstain from eating raw shellfish.

J Bacteriol, 1998 Nov, 180(21), 5591 - 600
Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae; Connell TD et al.; The chiA gene of Vibrio cholerae encodes a polypeptide which degrades chitin, a homopolymer of N-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans . chiA has a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa . A 52-bp region with promoter activity was found immediately upstream of the chiA open reading frame . Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity by V . cholerae required chiA . Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for beta,1-4 glycosidic bonds located between GlcNAc monomers in chitin . Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein . Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes of V . cholerae and by the closely related bacterium Aeromonas hydrophila . Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by the eps system . The eps system is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA . These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential for eps-dependent secretion.

Biochem Biophys Res Commun, 1998 Oct 29, 251(3), 894 - 7
Molecular cloning and functional characterization of SecE of a marine bacterium, Vibrio alginolyticus; Nishiyama K et al.; SecE is an essential membrane component of the Escherichia coli protein translocation machinery, which utilizes ATP and the proton motive force as energy sources . The secE homologue of marine Vibrio alginolyticus, in which protein translocation requires ATP and the sodium motive force, was cloned, sequenced, and characterized . Unlike most SecE homologues found in various organisms, SecE of V . alginolyticus (Va-SecE) possesses three transmembrane sequences like E . coli SecE (Ec-SecE) and complements the DeltasecE mutation of E . coli . Alignment of the Ec-SecE and Va-SecE sequences revealed that the non-essential N-terminal half was less homologous than the essential C-terminal half between the two SecEs . The E . coli strain was able to grow and translocate the secretory protein in the complete absence of Ec-SecE when Va-SecE was expressed . The stabilization of SecY overproduction by Va-SecE was as effective as that by Ec-SecE, indicating that Va-SecE interacts with E . coli SecY despite the difference in energy requirement .

Jpn J Med Sci Biol, 1997 Dec, 50(6), 227 - 32
Isolation of a new variant of Vibrio cholerae O1: V . cholerae O1 ribotype B27 toxinogenotype TB31 during the last cholera epidemic in Senegal; Aidara-Kane A et al.; A total of 205 Vibrio cholerae O1 isolates from recent cholera epidemic in Senegal were analyzed by conventional methods, polymerase chain reaction (PCR) for genes encoding cholera toxin (ctx A), zonula occludens toxin (zot) and accessory cholera enterotoxin (ace), ribotyping and toxinogenotyping . Ribotyping after Bg1 I digestion of total DNA revealed that ribotype B5a, the predominant ribotype of the seventh pandemic in Africa and Asia, was not isolated . A new ribotype designated B27 in our database is predominant and was associated with a new toxinogenotype designated TB31.

Microbios, 1998, 94(377), 23 - 34
Characterization of the phytopathogen Pseudomonas syringae pathovar ribicola NCPPB 963; Charnock C; In 1939, a bacterial spot caused severe defoliation of Ribes aureum (Golden Currant) The causal agent is now recognized as Pseudomonas syringae pathovar ribicola . This communication extends the phenotype of the only identified strain of P . syringae pv . ribicola, which is reminiscent of those of other pathovars, and provides a molecular biological characterization . A minimum size of 5.55 Mb for the bacterial genome was obtained using pulsed-field electrophoresis . The SDS-PAGE outer-membrane profile contained seven major bands, and has obvious similarities to that of P . aeruginosa . SDS-PAGE of concentrated mid-log phase culture supernatants revealed large amounts of a single, cryptic 24.0 kD protein . The amino acid composition and 57 residues in the N-terminus of this protein . were determined . The protein sequence was nearly identical to the translation of a region of unknown function in the P . aeruginosa genome . Extensive similarity in N-terminal sequence, composition and subunit size to a secreted hydrophilic Vibrio cholerae protein of unknown function was also found . Neither protein has been directly associated with disease development.

Infect Immun, 1998 Nov, 66(11), 5485 - 93
Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages; Godfroid F et al.; Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans . The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described . Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined . A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella . One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet . Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7 . The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp . The survival of B . melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined . The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Jul-Aug, (4), 9 - 12
{A monoclonal antibody-based study of the antigenic interrelations of typical and R forms of Vibrio cholerae}; Alekseeva LP et al.; Monoclonal antibodies to surface determinants of V . cholerae R forms (R-McA) were obtained . R-McA and monoclonal antibodies to lipopolysaccharide (LPS) of V . cholerae S forms (S-McA) were used to show that the LPS of deeply altered vibrios, agglutinating only with RO serum, completely lost its O-side chain . Some common O determinants on the basis of S-McA were detected in typical cultures of V . cholerae O1 and RO vibrios which agglutinated to 1/4 T with O serum and, in low titers, with RO serum . V . cholerae O1 were not capable of specifically binding with R-McA . Not all R strains under study were identified with the use of available R-McA due to essential differences of their terminal monosaccharides responsible for serological specificity.

Microbiology, 1998 Sep, 144 ( Pt 9), 2517 - 23
Close proximity of the tdh, trh and ure genes on the chromosome of Vibrio parahaemolyticus; Iida T et al.; The distribution and location of the virulence-factor genes of Vibrio parahaemolyticus, tdh and trh, and the structural gene of urease, ureC, were examined on the genomic DNAs of 115 clinical isolates of V . parahaemolyticus . The majority of strains (81%) had two copies of tdh on the chromosome, and no copies of trh or ure . Southern hybridization with a tdh probe, after pulsed-field gel electrophoresis of Notl-digested genomic DNA of each strain revealed only single bands, suggesting that the two copies of the exist on single Notl fragments in each strain . Of the 115 strains, 7% had the tdh, trh and ure genes on chromosomal DNA . The three genes were also detected on single Notl fragments in these strains . More detailed analysis revealed that the three genes were localized within 40 kb . By long and accurate polymerase chain reactions (LA-PCR) the distance between trh and ure was shown to be less than 8.5 kb . These results reveal a close proximity of the tdh, trh and ure genes on the chromosome of pathogenic V . parahaemolyticus strains.

Mol Microbiol, 1998 Sep, 29(6), 1493 - 507
Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes; Occhino DA et al.; Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins . The first set (tonB1, exbB1, exbD1) was obtained by complementation of a V . cholerae tonB mutant . In the mutant, a plasmid containing these genes permitted transport via the known V . cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome . When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V . cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background . Another region of the V . cholerae chromosome was cloned that encoded a second functional TonB/Exb system (tonB2, exbB2, exbD2) . A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V . cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant . The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes (hutBCD), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E . coli strain expressing the V . cholerae haem receptor, HutA . Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V . cholerae haem transport system in E . coli . In V . cholerae, the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V . cholerae.

Mol Microbiol, 1998 Sep, 29(6), 1481 - 92
Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation; Marsh JW et al.; Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae . Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified . This report describes the identification of a prepilin peptidase from the V . cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa . Plasmid disruption or deletion of this peptidase gene in either EI Tor or classical V . cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion . In the case of the EI Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished . The cloned V . cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E . coli . Mutation of the V . cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU . The V . cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V . vulnificus . Accordingly, the V . cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.

Arch Med Res, 1998 Autumn, 29(3), 231 - 4
Intestinal colonization of the infant mouse model by attenuated and virulent Vibrio cholerae strains; Cedre Marrero B et al.; BACKGROUND: Intestinal colonization of humans with virulent Vibrio cholerae stimulates substantial, lasting immunity against reinfection . The purpose of this study was to evaluate the colonizing capability of various Vibrio cholerae strains which are promising candidates to oral vaccine . METHODS: Infant mouse model modification was used . In order to standardize the method, several parameters were tested, such as culture medium and optimal time of incubation and appropriate number of cells to be inoculated . The following were tested: Vibrio cholerae strain 81, 413, and 251A, which were obtained at the Molecular Biology Department of the National Center for Scientific Research, Havana, Cuba . Their virulence cassettes which code for the main virulence factors were deleted . RESULTS: Good variance coefficient (VC) was obtained in repeated experiments . The colonizing properties of attenuated Vibrio cholerae strains evaluated by this method correlated well with those observed for parental strains . CONCLUSIONS: Genetically attenuated Vibrio cholera strains have the same intestinal colonization level as their parental strains in the infant mouse model; thus, genetic manipulation does not affect genes that encode for the synthesis of colonization factors.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1995 Nov, 28(4), 291 - 9
Molecular epidemiological studies of Vibrio cholerae in Taiwan: genotyping by polymerase chain reaction and DNA sequencing; Liu DP et al.; To type the Vibrio cholerae strains isolated from sporadic and epidemic cases in Taiwan, 28 toxigenic isolates were studied by sequencing polymerase chain reaction-amplified cholera toxin gene (ctx) fragments . Based on specific base substitutions on positions 115 and 203 of ctxB and comparison with previously published typing system from Centers for Disease Control and Prevention (Olsvik theta et al., J Clin Microbiol 1993; 31:22-5, Ref.1), two genotypes were identified . Cholera strains from imported seafood and sporadic cases in Taiwan had ctxB polymorphism of genotype 1; strains from patients in the 1962 Taiwan epidemic and Taiwan raised soft-shell turtles had ctxB polymorphism of genotype 3 . Moreover, one toxigenic non-O1 strain was found to have other 11 different nucleotides in ctxB compared with those of the O1 and O139 strains . Therefore, DNA sequencing is a useful method for obtaining more complete genetic information . The approach could be improved by applying it to other more polymorphic regions of bacterial genome to obtain better epidemiological information among infected cases.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1995 Nov, 28(4), 270 - 9
Ribotyping of clinical Vibrio vulnificus isolates; Yang SF et al.; Restriction fragment length polymorphism analysis of rRNA genes (ribotyping) was used to differentiate Vibrio vulnificus isolates . Among the 10 restriction enzymes tested, HindIII was shown to provide the most discriminatory patterns . Stul was used for further analysis of strains that were indistinguishable with HindIII . Thirteen clinical V . vulnificus strains were analyzed for their ribotypes with HindIII, as well as Stul when necessary . Four of the clinical strains were isolated from different samples collected from the same patient, and were shown to have identical ribotypes . All the others gave unique ribotypes, indicating the large genetic divergence in V . vulnificus clinical isolates . The ribotype of V . vulnificus by HindIII remained unchanged after successive in vitro and in vivo passages . HindIII gave rise to five bands which were shown in every V . vulnificus strain but not in other vibrio species tested, suggesting that ribotyping with this restriction enzyme may be useful for confirming the identification of this bacterium.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1995 Nov, 28(4), 256 - 69
Characterization of the manganese-resistant mutants derived from Vibrio parahaemolyticus; Weng JH et al.; The virulence and some related factors of Vibrio parahaemolyticus are regulated by the level of iron . In this study, five Mn-resistant mutants were selected after N-methyl-N'-nitrosoguanidine treatment and two transfers in medium containing high levels of manganese chloride . Production of siderophores and the 77-kDa iron-regulated outer-membrane protein and the bacterial growth in these Mn-resistants were deregulated, as compared with the wild-type strain . In addition, the regulation of these phenomena was partially or completely restored by the introduction of Escherichia coli fur gene . Also, the total cellular protein profiles of the wild-type and mutants showed that production of some proteins were positively or negatively regulated by iron, and expression of some of these proteins remained unaffected in these mutants . These results suggested the presence of a complicated iron regulation system, similar to the Fur system of E . coli, in this pathogen.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1995 Feb, 28(1), 70 - 8
Differentiation of Vibrio vulnificus strains by an arbitrarily primed polymerase chain reaction; Wu JJ et al.; A synthetic 17 mer oligonucleotide (5'-GTTGGGTAACGCCAGGG-3') was used as a primer for the arbitrarily primed polymerase chain reaction (AP-PCR) to differentiate various strains of Vibrio vulnificus . A total of 37 genomic DNAs that were extracted from the clinical and environmental strains were successfully differentiated . Among them, 32 profiles of the 37 strains were characterized . None of the environmental and clinical strains had the same amplification profile, suggesting the highly heterogeneous population existed in the strains of V . vulnificus . The size of the amplified sequences ranged from 0.3 to 2.0 Kb and the DNAs were separated to 12 to 20 bands by the 1.2% agarose gel . The clinical isolates from two independent episodes of V . vulnificus infections in a patient were shown to have the same profile, indicating that the second episode was due to recurrence rather than reinfection . The profiles of amplification were reproducible with different preparations of genomic DNA . Arbitrarily primed polymerase chain reaction can therefore be a useful tool for epidemiological study of V . vulnificus infection.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1995 Feb, 28(1), 59 - 69
{Survey of disease of cultural shrimp in Taiwan}; Liu WY et al.; The large number of hemocytes infiltrated several abnormal tissues of kuruma shrimp (Penaeus japonicus), including musculature, hepatopancreas, lymphoid organ, gill filament and sponge tissue . In addition, there were many denatured hemocytes existing inside acidophilic particles and forming granules . Futhermore, in hepatopancreas of kuruma shrimp, a white spot baculovirus (WSBV; 40-50 x 50-300 nm) was discovered in UH (undifferential haemocyte) . The epithelium cells, which including stomach cuticle and underlying epidermis of exoskeletal cuticle, could also be infected by WSBV in another main cultural species--grass shrimp (P . monodon) . During a period of high water temperature, with pond shrimp in normal condition, the CFU/ml of water bacteria rose from 10(5) to 10(7), but this number had decreased to 10(5) CFU/ml by the time moribund shrimp began to appear . Coincidentally, the total bacterial number isolated from hepatopancreas and musculature of moribound shrimp was over 10(5) (CFU/g) and 10(3)-10(5), respectively . The fauna of bacteria was taken over by the active metabolitic species which were represented by Vibrio species causing the pond shrimp to undergo either behavioral changes, such as swimming on the water surface, or histological changes, such as having whitish muscle color, hemocyte infiltration and granuloma formation etc . Pathogenetic species of Vibrio including V . parahaemolyticus, V . alginoly ticus V . anguillarum, V . fischery and V . damsela were isolated from those tissues of moribund shrimp . The main pathogens, isolated from musculature and hepatopancreas, were V . parahaemolyticus and V . alginolyticus . On the other hand there, was no bacterium could be isolated from the musculature of healthy shrimp and only a single species of Gram (+) coccus--Micrococcus--was isolated from the tissue of hepatopancreas.

Am J Public Health, 1998 Oct, 88(10), 1545 - 53
A rivalry of foulness: official and unofficial investigations of the London cholera epidemic of 1854; Paneth N et al.; Contemporaneous with John Snow's famous study of the 1854 London cholera epidemic were 2 other investigations: a local study of the Broad Street outbreak and an investigation of the entire epidemic, undertaken by England's General Board of Health . More than a quarter-century prior to Koch's description of Vibrio comma, a Board of Health investigator saw microscopic "vibriones" in the rice-water stools of cholera patients that, in his later life, he concluded had been cholera bacilli . Although this finding was potential evidence for Snow's view that cholera was due to a contagious and probably live agent transmitted in the water supply, the Board of Health rejected Snow's conclusions . The Board of Health amassed a huge amount of information which it interpreted as supportive of its conclusion that the epidemic was attributable not so much to water as to air . Snow, by contrast, systematically tested his hypothesis that cholera was water-borne by exploring evidence that at first glance ran contrary to his expectations . Snow's success provides support for using a hypothetico-deductive approach in epidemiology, based on tightly focused hypotheses strongly grounded in pathophysiology.

MMWR Morb Mortal Wkly Rep, 1998 Sep 25, 47(37), 782 - 6
Incidence of foodborne illnesses--FoodNet, 1997.
{Characterization of monoclonal antibodies that recognize the thermolabile toxin of Vibrio cholerae}
Gonzalez Garriga M, Garcia Sanchez HM, Hermida Diaz C, Marcet Sanchez R, Monte Boada R.

Instituto de Medicina Tropical Pedro KouriThe obtention of two monoclonal antibodies which recognize a single epitope present in the subunit B of the thermolabile toxin of Vibrio cholerae and another which shows a cross-reaction with those produced by certain enteropathogenic toxins, is reported . The standardization of a solid phase indirect immunoenzymatic assay (ELISA) for the primary screening and selection of hybrids was performed; in addition, the isotype was determined.

Rev Cubana Med Trop, 1991, 43(3), 186 - 8
{Isolation and identification of Vibrio genus microorganisms in the Quibu River}; Bravo Farinas L et al.; The Quibu River sewages were studied during 9 weeks, in order to isolate and characterize Vibrio genus microorganisms . Twenty Moore's hyssops were placed 2 or 3 times a week on the banks of the river, where each of them was kept in a passive capture stay for 24 hours . In all the hyssops placed, Vibrio cholerae non-01 were isolated.

Biochim Biophys Acta, 1998 Oct 2, 1394(1), 85 - 9
Cloning and identification of a phospholipase gene from vibrio mimicus; Kang JH et al.; The phospholipase gene phl was identified from Vibrio mimicus (ATCC33653) and sequenced . The entire open reading frame (ORF) was composed of 1410 nucleotides and encoding 470 amino acids . The phl was placed upstream of hemolysin gene (vmhA) with opposite direction of transcription . From the BLAST search program, the deduced amino acids sequence showed 74.4% identity with phospholipase gene (lec) from V . cholerae El Tor . The entire ORF of phospholipase gene was amplified by PCR and inserted into an Escherichia coli expression vector, pET22b(+) and introduced E . coli BL21(DE3) . SDS-PAGE demonstrated that a protein corresponding to the phospholipase was overexpressed and migrated at a molecular mass of 53 kDa.

Trans R Soc Trop Med Hyg, 1998 Mar-Apr, 92(2), 164 - 5
Aetiology of cholera in Tamil Nadu: recent observations; Sundaram SP et al.; Vibrio cholerae was isolated from 1008 of 3496 stool samples (28.8%) examined in Tamil Nadu State, India, between November 1992 and December 1995 . During November and December 1992, 363 of the 370 isolates serotyped (98%) were V . cholerae O139 (Bengal) . The epidemic predominantly affected adults (91%; 597/656) . Both V . cholerae O1 and O139 serotypes were sometimes isolated in the same locality from different individuals . From January 1993 onwards, the rate of isolation of V . cholerae O139 declined, and in 1995 V . cholerae E1 Tor (serotype O1) was isolated from most of the cases (85.6%; 131/153) . V . cholerae E1 Tor has clearly not been replaced by serotype O139, but can survive during inter-epidemic periods and reappear at an opportune moment . The decline of serotype O139 may be due to the development of immunity as a result of repeated exposure.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 462 - 5
Identification and analysis of the regulatory region R&R* with the cnf1 gene encoding the cytotoxic necrotizing factor type 1 that closely links to the lux regulon of Vibrio fischeri; Lin JW et al.; Nucleotide sequence of the regulatory region R&R* and the partial 5'-end of the cnf1 gene (GenBank Accession No . AF023157) of Vibrio fischeri ATCC 7744 has been determined, and the cytotoxic necrotizing factor 1 (CNF1) encoded by the cnf1 gene is deduced . Alignment and comparison of the cytotoxic necrotizing factor 1s of V . fischeri and E . coli show that they are homologous . Nucleotide sequence reveals that the cnf1 gene is closely linked to the lux regulon in genome; the gene order of the cnf1 gene and the lux regulon is <--cnf1-R&R*<--rrn-<--luxR-R&R-luxI-luxC-luxD -luxA-luxB-luxE-luxG-omega-->, whereas R&R is the regulatory region of the lux regulon, and R&R* is the regulatory region of the cnf1 gene; the sequence approximately 2 kb lay between the luxR gene of the lux regulon and the cnf1 gene is an rrn-like operon . It is unexpected to find the cnf1 gene in V . fischeri, since the CNF1 protein enables necrosis; the marine luminous bacterium V . fischeri is never to be identified as a pathogenic microbe . The cnf1 gene might be concerned with symbosis of the luminous bacteria and host fishes.

Trop Gastroenterol, 1998 Apr-Jun, 19(2), 59 - 61
Typing and antibiotic susceptibility patterns of Vibrio cholerae during six consecutive cholera seasons in north India; Kaur H et al.; A total of 10,427 diarrhoeal stool specimens were cultured for Vibrio cholerae between 1992 and 1997 . The isolation rates were 2%, 2.6%, 6.7%, 7.08%, 0.9% and 2.6% in the years from 1992 to 1997 respectively . Till 1992, Vibrio cholerae 01 ogawa was the predominant strain . In 1993, 81.3% of the isolates were of 0139 Bengal strain and the rest were V . cholerae 01 . From 1994 to 1997, V . cholerae 01 ogawa was the predominant strain and there were no isolation of 0139 strain . The predominant phage type in 1992 and 1993 were T2 and T27 thereafter . Most Vibrio cholerae strains were sensitive to tetracycline, gentamycin, netromycin, norfloxacin and furazolidine . Strains were resistant to cotrimaxozole till 1996, but were 100% sensitive in 1997 . Strains were sensitive to chloramphenicol till 1993 but acquired resistance thereafter.

Lett Appl Microbiol, 1998 Aug, 27(2), 116 - 20
The use of alkaline phosphatase-labelled oligonucleotide probes as culture confirmation reagents for identification of commercially important bacteria; Glover DJ et al.; A range of rRNA-targeted alkaline phosphatase labelled oligonucleotide probes was tested for use as culture confirmation reagents for the rapid identification of micro-organisms . The probes were specific to clinically important bacteria (Helicobacter pylori and Mycobacterium tuberculosis), fish and shellfish pathogens (Renibacterium salmoninarum and Vibrio vulnificus), food spoilage bacteria (Listeria spp . and L . monocytogenes), for bacteria of biotechnological importance (Streptomyces spp.) and for bacteria associated with the oil industry (Sulphate-reducing bacteria, SRB) . A universal bacterial probe and a eukaryotic probe were included in the study as positive and negative controls, respectively . A total of 93 bacterial strains was screened . With the exception of a large number of cross-reactions of the SRB probe (specificity value of 29.4%) and a single cross-reaction of the R . salmoninarum probe (specificity value of 97.7%), dot blot analysis indicated that each probe hybridized 100% specifically to the organisms tested . A simple culture confirmation method was then developed using these probes to enable the identification of bacterial colonies using a simple hybridization procedure.

J Bacteriol, 1998 Oct, 180(19), 5256 - 9
Identification of multiple sigma54-dependent transcriptional activators in Vibrio cholerae; Klose KE et al.; In the pathogenic bacterium Vibrio cholerae, the alternate sigma factor sigma54 is required for expression of multiple sets of genes, including an unidentified gene(s) necessary for enhanced colonization within the host . To identify sigma54-dependent transcriptional activators involved in colonization, PCR was performed with V . cholerae chromosomal DNA and degenerate primers, revealing six novel and distinct coding sequences with homology to sigma54-dependent activators . One sequence had high homology to the luxO gene of V . harveyi, which in that organism is involved in quorum sensing . Phenotypes of V . cholerae strains containing mutations in each of the six putative sigma54-dependent activator genes identified one as a probable ntrC homologue . None of the mutant strains exhibited a defect in the ability to colonize infant mice, suggesting the presence of additional sigma54-dependent activators not identified by this technique.

J Bacteriol, 1998 Oct, 180(19), 5094 - 101
Filamentous bacteriophages of Vibrio parahaemolyticus as a possible clue to genetic transmission; Chang B et al.; We have previously reported the isolation and characterization of two filamentous bacteriophages of Vibrio parahaemolyticus, designated Vf12 and Vf33 . In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs . The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined . An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions . The gene organization of their conserved regions was similar to that of CTX phage of Vibrio cholerae and coliphage Ff of Escherichia coli, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes . Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of V . parahaemolyticus at the distinctive region of the phage genome and were also distributed on some plasmids of V . parahaemolyticus and total cellular DNAs of one Vibrio damsela and one nonagglutinable Vibrio strain tested . These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of V . parahaemolyticus strains and of genetic transmission among strains through these filamentous phages.

Epidemiol Infect, 1998 Aug, 121(1), 67 - 76
The prevalence of Vibrio spp . in drinking water and environmental samples in Vellore South India; Thomson CJ et al.; The prevalence of Vibrio cholerae in drinking water, lakes and sewage outfalls during July and August 1996 in Vellore, India was determined . Drinking water samples were collected on single occasions from 12 sites in different geographic areas of the town where cholera had been reported . Samples of water, plankton and sediment were collected from fixed sites at three lakes on three occasions separated by at least 3 days during the course of the study . Samples from open sewers were taken from two representative sites in four areas of the town . Bacteria isolated from samples were identified by standard biochemical tests and isolated strains of V . cholerae tested for their ability to agglutinate O1 and O139 antisera . Water samples from lakes were also tested for the presence of V . cholerae O1 and O139 by fluorescent antibody staining . Non-O1, non-O139 strains of V . cholerae were detected in 41% of drinking water samples and 100% of water, sediment and plankton samples from the test lakes . Eighty-seven per cent of open sewers sampled contained viable non-O1, non-O139 V . cholerae . Fluorescent antibody staining gave positive results for V . cholerae O1 and O139 for all water samples from the three lake sites . Strains of Aeromonas spp . were isolated from 58% of drinking water samples and from 66% of sediment, 77% of plankton and 55% of water samples from lakes . All open sewers sampled contained Aeromonas spp . PCR amplification employing specific primers demonstrated that none of the non-agglutinating V . cholerae isolates contained the ctx operon . The non-O1, non-O139 V . cholerae isolates showed different patterns of antibiotic resistance to ampicillin, ciprofloxacin, chloramphenicol, tetracycline and trimethoprim.

Epidemiol Infect, 1998 Aug, 121(1), 15 - 29
Investigation of the 1994-5 Ukrainian Vibrio cholerae epidemic using molecular methods; Clark CG et al.; Thirty-seven Vibrio cholerae and four non-cholera Vibrio isolates from Ukraine, including strains from the epidemic of 1994-5, were analysed by molecular methods . Results from PFGE and ribotyping indicated that all Ukrainian toxigenic V . cholerae were closely related to each other and to an isolate from a patient from Pakistan . A non-toxigenic river water strain obtained during the height of the epidemic was more distantly related to these V . cholerae strains, while the Vibrio parahaemolyticus isolates and Vibrio alginolyticus isolate were not closely related to V . cholerae or each other . ERIC- and REP-PCR allowed the differentiation of strains identical by other methods . The results obtained confirm that the epidemic Ukrainian strains are most closely related to seventh pandemic strains from Asia and support a hypothesis that the Ukrainian epidemic of 1994-5 was caused by toxigenic environmental strains surviving since the time of the 1991 Ukrainian epidemic or before.

Epidemiol Infect, 1998 Aug, 121(1), 1 - 13
Microbiological and epidemiological investigation of cholera epidemic in Ukraine during 1994 and 1995; Clark CG et al.; The Ukraine cholera epidemic of 1994 and 1995 was caused by Vibrio cholerae O1, serotype Ogawa, biotype El Tor . This epidemic was centred in the area around Respublika Krim (Crimea) and Mykolajiv, and spread to include parts of southern Ukraine . Cases of cholera occurred between September and November of 1994 and between June and October of 1995 . The 32 fatalities among 1370 recorded cases (case fatality ratio, 2.3%) occurred throughout the course of the epidemic . V . cholerae from patients with cholera produced cholera toxin and were resistant to multiple antibiotics, though no resistance plasmids were found . Conjugation experiments suggested that resistance to multiple antibiotics may be present on a self-transmissible genetic element . Environmental sources of V . cholerae O1 El Tor included sewage, sea and surface water, and fresh water and marine fish . All but one of the environmental V . cholerae isolated during the epidemic were very similar to selected isolates from patients at the same time, supporting the role of these environmental sources in the spread of disease.

Mol Gen Genet, 1998 Aug, 259(2), 179 - 89
Cloning and characterization of the dnaK heat shock operon of the marine bacterium Vibrio harveyi; Klein G et al.; We cloned the DNA region of the Vibrio harveyi chromosome containing the heat shock genes dnaK and dnaJ and sequenced them . These genes are arranged in the chromosome in the order dnaK-dnaJ, as in other proteobacteria of the alpha and gamma subdivisions . The dnaK gene is 1923 nucleotides in length and codes for a protein of 640 amino acid residues, with a predicted molecular mass of 69,076 Da and 81.2% similarity to the DnaK protein of Escherichia coli . The V . harveyi dnaJ gene has a coding sequence of 1158 nucleotides . The predicted DnaJ protein contains 385 amino acids, its calculated molecular mass is 41,619 Da and it has 74.7% similarity to the DnaJ protein of E . coli . Northern hybridization experiments with RNA from V . harveyi cells and a DNA probe carrying both the dnaK and dnaJ genes showed a single, heat-inducible transcript, indicating that these genes form an operon . Primer extension analysis revealed five heat-inducible transcriptional start sites upstream of the dnaK gene, two of which (T1 and T4) are preceded by sequences typical of the E . coli heat shock promoters recognized by the sigma 32 (sigma32) factor . Location of these promoters is highly similar to that of the E . coli dnaK promoters . No transcriptional start sites were detected upstream of the dnaJ gene . The V . harveyi dnaKJ operon cloned in a plasmid in E . coli cells was transcribed in a sigma32 dependent manner and the size of the transcript, the kinetics of transcription, and the transcriptional start sites were as in V . harveyi cells . This indicates a high conservation of the transcriptional heat shock regulatory elements between E . coli and V . harveyi, both belonging to the gamma subdivision of proteobacteria . We tested the ability of the cloned dnaKJ genes to complement E . coli dnaK and dnaJ mutants and found that V . harveyi DnaJ restored a thermoresistant phenotype to dnaJ mutants and enabled lambda phage to grow in the mutant cells . V . harveyi DnaK did not suppress the thermosensitivity of dnaK mutants but complemented the dnaK deletion mutant with respect to growth of lambda phage . V . harveyi DnaK, in contrast to DnaJ, failed to modulate the heat shock response in E . coli . Our results suggest that the DnaK chaperone may be more species specific than the DnaJ chaperone.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1994 Nov, 27(4), 173 - 85
Identification and characterization of a protease produced by Vibrio parahaemolyticus in iron-limited medium; Wong HC et al.; Two proteolytic proteins (about 43 and 90 kDa) were produced by clinical strains of Vibrio parahaemolyticus cultured in iron-limited medium . The 43 kDa-protease was partially purified by ammonium sulfate precipitation, ultrafiltration fractionation and DEAE-Sephacel chromatography . This protease had an optimum pH range of 7 to 8, and an optimum reaction temperature of about 40 degrees C . It was heat-labile, being partially inactivated by heat-treatment at 60 or 90 degrees C for 10 min . The protease hydrolyzed casein, gelatin, elastin, collagen and hemoglobin . As a chymotrypsin-like protease, it was inhibited only by the chymostatin among seven protease inhibitors tested . Activity of this protease was partially inhibited by 1 mM of Co2+, Cu2+, Zn2+ and Hg2+ and slightly enhanced by Ca2+ and Ba2+ . It was completely inactivated by orthophenanthroline (OPA), and the OPA-inactivated sample was partially reactivated by Ca2+ and Fe2+ . In conclusion, this 43-kDa protease of V . parahaemolyticus was an unstable neutral chymotrypsin-like metalloprotease; Ca2+ and/or Fe2+ was essential for its activity or stability.

Dis Aquat Organ, 1998 Jul 30, 33(3), 157 - 66
Route of vaccine administration: effects on the specific humoral response in rainbow trout Oncorhynchus mykiss; Palm RC Jr et al.; The specific humoral response of teleost fish to extracellular bacteria was examined using a rainbow trout-Vibrio anguillarum model . Treatment groups were immunized by oral, immersion, and injection routes . All 3 delivery methods conferred full protection in controlled laboratory challenges (p < 0.01) . Prior to boosting, serum antibody titers did not correlate with protection in the orally and immersion-vaccinated groups, but, contrary to previous studies, titers measured 10 and 17 d after boosting correlated positively with protection in all 3 vaccinated groups . The route of administration strongly affected the magnitude of the antibody response as measured by enzyme-linked immunosorbent assay (ELISA) and Western blots; however, the antigenic epitopes recognized were not substantially altered by delivery method as evidenced in immunoblot patterns . Given that the primary and booster vaccination protocols were identical, the data suggest that all 3 vaccinated groups may have had a specific humoral response following initial immunization but that specific serum antibody levels before boosting were too low to be detected by ELISA in fish vaccinated by oral and immersion routes . An anamnestic response was evident in all 3 groups . The data support the possibility that teleosts, like higher vertebrates, have a protective immune response to extracellular bacteria that is predominantly humoral . Route of delivery may primarily affect the efficiency with which the immunogenic constituents of the vaccine are presented to the relevant recognition and effector components of the immune system.

Infect Immun, 1998 Oct, 66(10), 4851 - 5
Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the thermolysin family; Miyoshi S et al.; Vibrio vulnificus is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin . This human pathogen secretes a metalloprotease (V . vulnificus protease {VVP}) as an important virulence determinant . When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity . The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel . Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP . Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP . Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane . Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.

Infect Immun, 1998 Oct, 66(10), 4726 - 8
Characterization of outer membrane protein OmpU of Vibrio cholerae O1; Nakasone N et al.; The outer membrane protein OmpU of Vibrio cholerae O1 strain 86B3 was characterized with reference to colonization of the intestine by the organism . The purified OmpU exhibited a pI of 3.6 . Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated to 38, 32, and 110 kDa when the sample was heated at 100 degrees C for 2 min, 50 degrees C for 15 min, and room temperature for 30 min, respectively . The purified OmpU was not hemagglutinative . Anti-OmpU serum did not agglutinate strain 86B3 or other V . cholerae organisms . OmpU adhered to the brush border of the rabbit small intestine; adhesion of the organisms to the intestine treated in advance with OmpU was not inhibited . Treating the organisms in advance with anti-OmpU Fab did not inhibit adhesion to the intestine . These results obtained in vitro suggest that OmpU is not involved in the adhesion of V . cholerae to the intestinal epithelium.

Indian J Med Res, 1998 Jul, 108, 1 - 2
Re-emergence of Vibrio cholerae serogroup O139 during June-August, 1997 in Yavatmal (Maharashtra); Jalgaonkar SV et al.; A clone of V . cholerae serogroup O139 which emerged as a novel epidemic strain, was reported from this region in 1993 as from many other parts of India and adjoining countries . The decline in the isolation rate of this organism in subsequent years was followed by a sudden increase in 1997, this requires careful monitoring.

J Biolumin Chemilumin, 1998 Jul-Aug, 13(4), 185 - 8
H-NS controls the transcription of three promoters of Vibrio fischeri lux cloned in Escherichia coli; Ulitzur S; We have recently proposed that the expression of V . fischeri right lux operon is controlled by two promoters; the first one located upstream of the luxl gene, while the second one seems to be located upstream of the luxC gene . The transcription from both promoters is negatively controlled by H-NS protein . Escherichia coli MC4100 rpoS hns mutant that carried the V . fischeri lux system with a deletion in either the luxl or luxR gene showed a constitutive mode and more than 10,000-fold higher luminescence than the control cells . The present study shows that neither luxR nor luxl are required for the transcription of the luxCDABE genes in an H-NS deficient strain of E . coli . The MC4100 rpoS hns mutant harbouring the luxCDABE-carrying plasmid showed constitutive mode and 70,000-fold higher luminescence than the wild-type cells . The question whether both the left and the right operons of V . fischeri lux system are controlled by H-NS was addressed with the aid of plasmids harbouring the lacZ gene fused with luxR or luxl . In MC4100 hns rpoS background, luxR and luxl genes were very early and actively transcribed, as judged by the strong beta-galactosidase activity that was developed at early stage of growth . The beta-galactosidase activity in the wild-type cells was 20-40 times lower and occurred mainly during the second half of the growth cycle . It thus appears that H-NS inhibits the transcription of three promoters of the lux system of V . fischeri; the left operon that codes for LuxR protein and two promoters located upstream and downstream to luxl gene.

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 373 - 8
A proposal to transfer Vibrio marinus (Russell 1891) to a new genus Moritella gen . nov . as Moritella marina comb . nov; Urakawa H et al.; The taxonomic positions of Vibrio marinus and 11 related natural isolates were examined . Their phylogenetic positions on the basis of almost complete 16S rRNA sequences were determined by maximum likelihood, maximum parsimony and neighbor-joining analyses . V . marinus and the 11 isolates fell into a single cluster that was clearly distinct from other genera . There is now strong evidence that V . marinus should be reclassified as Moritella marina gen . nov., comb . nov.

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 239 - 46
Cloning, sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum; Das M et al.; Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum . Motility is an important virulence factor for this non-invasive pathogen . We cloned and sequenced a locus in V . cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum . The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria . All of these genes of V . cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction . These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum . Our data indicated the presence of more than one flagellar locus in V . cholerae which could provide a means of immunoavoidance during infection . When compared with homologs in other bacteria, the flagellin core protein of V . cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.

Syst Appl Microbiol, 1998 Mar, 21(1), 128 - 34
A comparison of strategies for the detection and recovery of Vibrio vulnificus from marine samples of the western Mediterranean coast; Arias CR et al.; We have compared the effectiveness of culture-based methods and a DNA-based method for the detection, of Vibrio vulnificus from a seawater and three types of shellfish collected from the costal waters of Valencia, Spain . For culture-based method, we used two selective media, thiosulphate-citrate-salts-sucrose (TCBS), and cellobiose-polymyxin B-colistin (CPC) agars with and without previous enrichment in alkaline-saline-peptone-water (APWS) . Presumptive colonies were confirmed as V . vulnificus by the polymerase chain reaction (PCR) using previously described 23S rRNA V . vulficus-specific sequences as primers (Dvu 9V and Dvu 45R) . Direct detection was accomplished by a nested-PCR procedure developed for environmental samples, with the above mentioned primers for the second amplification . Of 32 seawater samples, only one yielded positive results by direct detection by PCR, whereas five were positive by culture methods . Of the 32 bivalve samples, two were positive by PCR and five by culture methods . From a total of 675 presumptive colonies selected on the two media, only 48 (20 from seawater and 28 from bivalves) were confirmed as V . vulnificus by PCR . Forty-six V . vulnificus isolates were obtained after enrichment and only two after direct inoculation of CPC . Except for one sampling, positive results by direct detection did not correlate with confirmed strains obtained from culture media . API 20E profiles were recorded for all isolates previously identified as V . vulnificus, revealing that around 20% of the strains were sucrose-positive . For our samples, the best strategy consisted in the combination of culture based methods (3 h enrichment in APWs at 40 degrees C followed by CPC at the same temperature) and DNA-based procedures (specific PCR amplification of the presumptive colonies with primers Dvu 9V and Dvu 45 R), which allowed the detection and accurate identification of V . vulnificus in less than 48h . This is the first report on the detection of cells of V . vulnificus naturally present in seawater and edible shellfish in the Spanish Mediterranean coast.

J Clin Microbiol, 1998 Oct, 36(10), 2887 - 92
Direct identification of Vibrio vulnificus in clinical specimens by nested PCR; Lee SE et al.; This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification of Vibrio vulnificus in clinical specimens . We designed two sets of primers targeting the V . vulnificus hemolysin/cytolysin gene . The target of the first primer set (P1-P2; sense, 5'-GAC-TAT-CGC-ATC-AAC-AAC-CG-3', and antisense, 5'-AGG-TAG-CGA-GTA-TTA-CTG-CC-3', respectively) is a 704-bp DNA fragment . The second set (P3-P4; sense, 5'-GCT-ATT-TCA-CCG-CCG-CTC-AC-3', and antisense, 5'-CCG-CAG-AGC-CGT-AAA-CCG-AA-3', respectively) amplifies an internal 222-bp DNA fragment . We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA-0.5% Triton X-100 solution . The new DNA extraction method was more sensitive and reproducible than other conventional methods . The DNA extraction method guaranteed sensitivity as well, even when V . vulnificus cells were mixed with other bacteria such as Escherichia coli or Staphylococcus aureus . The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V . vulnificus . We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients . Seventeen (94.4%) of the 18 V . vulnificus culture-positive specimens were positive by the nested PCR . Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Mar, 119(3), 557 - 62
Comparative tyrosine degradation in Vibrio cholerae strains . The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell; Sanchez-Amat A et al.; The relationship between L-tyrosine catabolism and melanin formation was studied in the Vibrio cholerae strains ATCC 14035 and CECT 557 . It is shown that both strains degrade L-tyrosine by the same pathway as eukaryotic cells, giving homogentisate as intermediate . ATCC 14035, an O1 strain, which is not able to grow using L-tyrosine as sole carbon and energy source, but it forms pyomelanin from homogentisate . The second strain, which is non-O1, is able to grow using L-tyrosine as sole carbon and energy source, but it does not form any pigment . Both strains contain all the enzymes involved in the L-tyrosine catabolism . The three late enzymes of the pathway, homogentisate oxygenase, maleylacetoacetate isomerase and fumarylacetoacetate hydrolase, are induced by L-tyrosine, but the degree of induction is much lower in the ATCC 14035 strain . Thus, the distal part of the pathway becomes the rate-limiting steps in the L-tyrosine catabolism, explaining homogentisate accumulation and pyomelanogenesis in this strain . It is proposed that V . cholerae might be a useful prokaryotic model to show that alkaptonuria and other diseases related to L-tyrosine metabolism could occur in animals even when no particular enzyme involved in that pathway is lacking.

Curr Microbiol, 1998 Oct, 37(4), 231 - 5
Overexpression of a mutant B subunit in toxigenic Vibrio cholerae diminishes production of active cholera toxin in vivo; Silva A et al.; A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V . cholerae . The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium . The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo . Overexpression of BG33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 573 - 80
Vibrio halioticoli sp . nov., a non-motile alginolytic marine bacterium isolated from the gut of the abalone Haliotis discus hannai; Sawabe T et al.; Six alginolytic, facultatively anaerobic, non-motile marine bacteria were isolated from the gut of abalone Haliotis discus hannai . DNA-DNA hybridization data showed that the six strains constituted a single genospecies . Phylogenetic analyses of 16S rDNA sequences indicated that the isolates should be assigned to the genus Vibrio . The phenotypic features of the isolates were closely related to Vibrio fischeri and Vibrio pelagius biovar I, but 13 traits (motility, luminescence, alginase production, lipase production, lysine decarboxylase, indole production, growth in 1 and 6% NaCl and assimilation of five carbon compounds) distinguished these strains from V . fischeri, and 17 traits (motility, growth at 37 degrees C, lipase production, indole production, growth in 1 and 6% NaCl, acid from sucrose and D-sorbitol, and assimilation of nine carbon compounds) distinguished these strains from V . pelagius . The G + C content of the isolates was 41.6-43.1 mol% . According to DNA-DNA hybridization data and 16S rDNA phylogenetic analyses, it was concluded that the six isolates constitute a new species different from any other Vibrio species . The name Vibrio halioticoli sp . nov . (type strain IAM 14596T) is proposed . A set of phenotypic features which enables differentiation of the new species from other species of the Vibrionaceae family is described.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 481 - 7
Vibrio pectenicida sp . nov., a pathogen of scallop (Pecten maximus) larvae; Lambert C et al.; Five strains were isolated from moribund scallop (Pecten maximus) larvae over 5 years (1990-1995) during outbreaks of disease in a hatchery (Argenton, Brittany, France) . Their pathogenic activity on scallop larvae was previously demonstrated by experimental exposure . The phenotypic and genotypic features of the strains were identical . The G + C content of the strains was in the range 39-41 mol% . DNA-DNA hybridization showed a minimum of 73% intragroup relatedness . Phylogenetic analysis of small-subunit rRNA sequences confirmed that these strains should be affiliated within the family Vibrionaceae and that they are closely related to Vibrio tapetis and Vibrio splendidus . Phenotypic and genotypic analyses revealed that the isolates were distinct from these two vibrios and so constitute a new species in the genus Vibrio . They utilized only a limited number of organic substrates as sole carbon sources, including betaine and rhamnose, but did not utilize glucose and fructose . In addition, their responses were negative for indole, acetoin, decarboxylase and dihydrolase production . The name Vibrio pectenicida is proposed for the new species; strain A365 is the type strain (= CIP 105190T).

Scand J Infect Dis, 1998, 30(2), 192 - 3
Necrotizing fasciitis due to Vibrio alginolyticus following an injury inflicted by a stingray; Ho PL et al.; We describe the case of a patient with necrotizing fasciitis due to Vibrio alginolyticus in a patient with cirrhosis following an injury inflicted by a stingray . The patient was successfully treated with aggressive surgical debridement and a combination of ciprofloxacin and amoxicillin-clavulanate . Cases of invasive V . alginolyticus reported in the literature were reviewed.

Gene, 1998 Aug 31, 216(2), 303 - 9
Cloning and expression of the secA gene of a marine bacterium, Vibrio alginolyticus, and analysis of its function in Escherichia coli; Kunioka E et al.; We report the cloning, sequencing and functional characterization of the secA gene of a marine bacterium, Vibrio alginolyticus, which has been suggested to utilize ATP and the sodium motive force for protein translocation . Oligodeoxynucleotides corresponding to highly conserved regions of Escherichia coli secA located in the high affinity ATP binding site were utilized as PCR primers to clone the secA gene of V . alginolyticus . It was shown to encode a 103.3-kDa protein . The deduced amino acid sequence of V . alginolyticus SecA (VaSecA) exhibits a high degree of identity (72.7%) to SecA of E . coli (EcSecA) . The secA gene of E . coli forms an operon with upstream orfX, whereas no counterpart is present upstream of V . alginolyticus secA . Azide derepresses the EcSecA translation, whereas the level of VaSecA was unaffected by azide . Expression of VaSecA in E . coli carrying a temperature-sensitive secA mutation restored both growth and protein translocation at a non-permissive temperature . VaSecA was thus able to substitute for EcSecA despite the fact that the energy requirement for protein translocation differs between the two organisms . VaSecA was overproduced in V . alginolyticus and purified to homogeneity for N-terminal sequencing . The endogenous ATPase activity of the purified VaSecA was comparable with that of EcSecA . 1998 Elsevier Science B.V.

Int J Food Microbiol, 1998 Jul 21, 42(3), 167 - 73
Survival of Vibrio spp . including inoculated V . cholerae 0139 during heat-treatment of cockles (Anadara granosa); Liew WS et al.; The effect of heat-treatment on the internal temperature of raw cockles (Anadara granosa) and survival of their intrinsic flora of Vibrio spp . as well as of inoculated V . cholerae 0139 was examined . The cockles were purchased from markets in Malaysia and had an average weight including shells of 8.90+/-2.45 g . In one experiment heatpenetration of individual cockles was examined . Cockles weighing < 8 g (including shell) exhibited maximum internal temperatures of between 50 and 75 degrees C when heated in water at 99 degrees C for 10 s and 71-93 degrees C when heated for 30 s . Cockles weighing > 12 g exhibited maximum internal temperatures between 42 and 58 degrees C when heated in water at 99 degrees C for 10 s and 56-69 degrees C when heated for 30 s . In another experiment, heat-treatment of 10 cockles treated as a group at 99 degrees C for 10 or 30 s resulted in reduction of levels of intrinsic Vibrio spp . (enumerated directly on thiosulphate-citrate-bile salt sucrose agar; TCBS) from 5.73 to 3.15 log cfu g(-1) or below 1 log cfu g(-1), respectively . The levels of Vibrio spp . after heat-treatment decreased with an increase in numbers of cockles grouped together during treatment . In a third experiment V . cholerae 0139 was inoculated into cockles and subjected to heat-treatment at 99 degrees C for 0, 10, 15, 20, 25 or 30 s . The levels of Vibrio spp . in uninoculated, non-heat-treated cockles was 4.89 log cfu g(-1) on TCBS, and the predominant species were V . parahaemolyticus and V . alginolyticus . V . cholerae 0139 inoculated into cockles with an average weight of 13.5+/-1.90 g (including shell) decreased for samples examined immediately after heat-treatment from 6 log cfu g(-1) initially to 3.5 log cfu g(-1) after 25 s and < 1 log cfu g(-1) (TCBS) after 30 s of heat-treatment . The most probable number method by enrichment in alkaline peptone water gave in general within 1 log unit higher counts than TCBS direct enumeration . TCBS direct enumeration and MPN counts were up to 2.38 or 1.30 log units higher, respectively, for samples heat-treated for 20 s or longer and stored for 6 h at 30 degrees C before examination, than for samples heat-treated for same periods of time and examined immediately . This study shows that a mild heat-treatment of cockles for up to 25 s is inadequate to ensure a large reduction in numbers of Vibrio spp., including V . cholerae 0139.

J Infect Dis, 1998 Sep, 178(3), 752 - 9
The role of Gulf Coast oysters harvested in warmer months in Vibrio vulnificus infections in the United States, 1988-1996 . Vibrio Working Group; Shapiro RL et al.; Vibrio vulnificus infections are highly lethal and associated with consumption of raw shellfish and exposure of wounds to seawater . V . vulnificus infections were reported to the Centers for Disease Control and Prevention from 23 states . For primary septicemia infections, oyster trace-backs were performed and water temperature data obtained at harvesting sites . Between 1988 and 1996, 422 infections were reported; 45% were wound infections, 43% primary septicemia, 5% gastroenteritis, and 7% from undetermined exposure . Eighty-six percent of patients were male, and 96% with primary septicemia consumed raw oysters . Sixty-one percent with primary septicemia died; underlying liver disease was associated with fatal outcome . All trace-backs with complete information implicated oysters harvested in the Gulf of Mexico; 89% were harvested in water >22 degrees C, the mean annual temperature at the harvesting sites (P < .0001) . Control measures should focus on the increased risk from oysters harvested from the Gulf of Mexico during warm months as well as education about host susceptibility factors.

Appl Environ Microbiol, 1998 Sep, 64(9), 3403 - 10
Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR; Arias CR et al.; Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR . Results were validated by ribotyping . For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene . Random amplification of DNA was performed with M13 and T3 universal primers . The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates . The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters . Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates . Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V . vulnificus . On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V . vulnificus isolates.

Appl Environ Microbiol, 1998 Sep, 64(9), 3209 - 13
Competitive dominance among strains of luminous bacteria provides an unusual form of evidence for parallel evolution in Sepiolid squid-vibrio symbioses; Nishiguchi MK et al.; One of the principal assumptions in symbiosis research is that associated partners have evolved in parallel . We report here experimental evidence for parallel speciation patterns among several partners of the sepiolid squid-luminous bacterial symbioses . Molecular phylogenies for 14 species of host squids were derived from sequences of both the nuclear internal transcribed spacer region and the mitochondrial cytochrome oxidase subunit I; the glyceraldehyde phosphate dehydrogenase locus was sequenced for phylogenetic determinations of 7 strains of bacterial symbionts . Comparisons of trees constructed for each of the three loci revealed a parallel phylogeny between the sepiolids and their respective symbionts . Because both the squids and their bacterial partners can be easily cultured independently in the laboratory, we were able to couple these phylogenetic analyses with experiments to examine the ability of the different symbiont strains to compete with each other during the colonization of one of the host species . Our results not only indicate a pronounced dominance of native symbiont strains over nonnative strains, but also reveal a hierarchy of symbiont competency that reflects the phylogenetic relationships of the partners . For the first time, molecular systematics has been coupled with experimental colonization assays to provide evidence for the existence of parallel speciation among a set of animal-bacterial associations.

Arch Virol, 1998, 143(7), 1277 - 94
Characterisation of G serotype dependent non-antibody inhibitors of rotavirus in normal mouse serum; Beisner B et al.; Serotype specific (non-immunoglobulin) inhibitors of rotavirus have been identified in normal mouse serum obtained from BALB/c, CBA, and BL10 mice . Sialic acid was essential for the neutralising activity sera treated with the neuraminidase from Vibrio cholerae failed to neutralise rotavirus . G serotypes 4, 5, 7, 8, 9, and 10 were unaffected by the inhibitor(s) while G serotypes 1, 2, 6 and two G3 strains were neutralised to significant titres . Assessment of neutralisation of reassortants suggested that VP7 is the virus protein involved in the interaction although it remains possible that VP7 is influencing VP4 binding . Analysis of the sera by Western blot followed by virus overlay confirmed that binding is dependent on the presence of sialic acid . The human strain tested, Wa, bound to two (glyco) proteins (50 and 80 kDa) while the bovine strains tested, NCDV and UK bound to one (55 kDa) and two (36 and 55 kDa) proteins respectively . This indicates that while the bovine rotaviruses may bind to a common element, the human strain binds to clearly distinct proteins . We propose that these inhibitors interact with animal rotaviruses in a manner analogous to that by which they attach to target cells . The glycoprotein to which NCDV bound was purified and identified by N-terminal sequencing as murine alpha-1-anti-trypsin (MuAAT) and was confirmed to possess both neutralisation and anti-trypsin activity . Since MuAAT is known to possess only three N-linked glycans, identification and analysis of the actual virus-binding structure should now be possible.

J Bacteriol, 1998 Sep, 180(17), 4724 - 33
Mutations in toxR and toxS that separate transcriptional activation from DNA binding at the cholera toxin gene promoter; Pfau JD et al.; ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in Vibrio cholerae . ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain . ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction . By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12 toxR and toxS positive control mutants producing variant ToxR proteins from the toxRS operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated . One mutation in toxR specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation . Seven toxR mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm . Deletion of toxS did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription . However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR . Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function . These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS . This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.

J Bacteriol, 1998 Sep, 180(17), 4516 - 22
Vibrio cholerae O139 Bengal: combined physical and genetic map and comparative analysis with the genome of V . cholerae O1; Khetawat G et al.; A combined physical and genetic map of the genome of strain SG24 of Vibrio cholerae O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes NotI, SfiI, and CeuI . The genome of SG24 is circular, and the genome size is about 3 . 57 Mb . The linkages between 47 NotI and 32 SfiI fragments of V . cholerae SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B . The linkages between nine CeuI fragments were determined primarily by analyses of partial fragments of the CeuI-digested genome . More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map . The map of the SG24 genome represents the second map of a V . cholerae genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions . Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.

Carbohydr Res, 1998 Jun, 309(1), 103 - 8
Structure of the acidic polysaccharide chain of the lipopolysaccharide of Shewanella alga 48055; Shashkov AS et al.; A lipopolysaccharide (LPS) with an acidic polysaccharide chain was isolated from the bacterium Shewanella alga strain 48055 and cleaved selectively at the glycosidic linkage of N-acetylneuraminic acid to give a tetrasaccharide . Studies of the tetrasaccharide and the O-deacylated LPS by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, rotating-frame NOE spectroscopy (ROESY), and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, revealed the following structure of the polysaccharide repeating unit: -->3)-beta-D-GalpA6GroN-(1-->3)-beta-D-GlcpNAc-(1-->3)-alpha-D- GalpA6GroN- (1-->4)-alpha-Neup5Ac-(2--> where GroN is an amidically linked residue of 2-amino-1,3-propanediol (2-amino-2-deoxyglycerol) . A similar structure, but with 2-acetamido-2,6-dideoxy-D-glucose instead of 2-acetamido-2-deoxy-D-glucose, has been reported previously for the polysaccharide chain of a non-O1 Vibrio cholerae H11 LPS {E . V . Vinogradov, O . Holst, J.E . Thomas-Oates, K.W . Broady, and H . Brade, Eur . J . Biochem., 210 (1992) 491-498}.

Carbohydr Res, 1998 Jun, 309(1), 65 - 76
Structure of a muramic acid containing capsular polysaccharide from the pathogenic strain of Vibrio vulnificus ATCC 27562; Gunawardena S et al.; Vibrio vulnificus strains isolated from septicemia cases and from the environment show a wide variety of capsular types . In an attempt to find common structural features which can be correlated with pathogenicity and toxicity, we have determined structures of the capsular polysaccharides (CPS) from several pathogenic strains . We report the complete structure of the polysaccharide from the pathogenic V . vulnificus strain ATCC 27562 using a combination of homonuclear and heteronuclear one-dimensional and two dimensional NMR experiments . The 13C and 1H NMR spectra, including the exchangeable amide proton resonances, have been completely assigned . The amide linkage between Ser and C6 of GalA has been unambiguously determined by water-suppressed 2D NOESY . To verify the structure established by NMR, we have fragmented the polymer employing the Smith degradation procedure . The Smith product identified by NMR and matrix-assisted laser desorption mass spectrometry is consistent with the proposed structure for the CPS, which is composed of D-GlcNAc, MurNAc, D-GalA, L-Rha and is serine-linked as shown: {formula: see text}

Microbiology, 1998 Aug, 144 ( Pt 8), 2281 - 9
Cloning of the trkAH gene cluster and characterization of the Trk K(+)-uptake system of Vibrio alginolyticus; Nakamura T et al.; K(+)-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake . A 4.1 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E . coli trkH (V . alginolyticus trkH) and a new gene, orf1, whose function is not clear . Products of V . alginolyticus trkA and orf1 were detected in E . coli minicells . trkA and trkH from V . alginolyticus restored growth at low K+ concentrations of an E . coli delta trkA and an E . coli delta trkG delta trkH strain, respectively, suggesting that these V . alginolyticus genes can functionally replace their E . coli counterparts . In addition, a plasmid containing V . alginolyticus trkAH permitted growth of an E . coli delta sapABCDF (delta trkE) strain at low K+ concentrations . This effect was mainly due to V . alginolyticus trkH and was enhanced by trkA from this organism . Measurements of net K(+)-uptake rates indicated that the presence of these genes in E . coli renders the Trk systems independent of products from the E . coli sapABCDF (trkE) operon.

J Appl Microbiol, 1998 Jun, 84(6), 1175 - 8
Note: molecular cloning of chitinase genes from Vibrio anguillarum and V . parahaemolyticus; Hirono I et al.; Chitinase genes from Vibrio anguillarum KV9001 and V . parahaemolyticus ATCC17802 were cloned into Escherichia coli . Open reading frames of chitinase genes from V . anguillarum (vac) and V . parahaemolyticus (vpc) are 1755 bp and 1890 bp, respectively . The deduced amino acid sequences of these genes have 71.6% identity . There are two consensus sequence regions in the VAC and VPC proteins . The vac gene was highly prevalent in V . anguillarum, and the DNA probe of the vac gene hybridized to V . alginolyticus and Beneckea proteolytica DNA . The DNA probe of the upc gene hybridized to V . alginolyticus, V . harveyi and V . ordalii DNA.

Dev Genes Evol, 1998 Aug, 208(6), 295 - 303
Induction of apoptosis by cooperative bacteria in the morphogenesis of host epithelial tissues; Foster JS et al.; Associations with pathogenic bacteria have recently been shown to initiate apoptotic programs in the cells of their animal hosts, where host cell death is hypothesized to be a response of the immune system, either initiated as a mechanism of host defense or bacterial offense . In this study, we present evidence that bacterial initiation of apoptosis is neither restricted to pathogenesis nor to the initation of an immune response . In the cooperative association between the sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri, the bacteria induce a dramatic morphogenesis of the host tissues during the first few days of interaction between these partners . The most striking change is the bacteria-triggered loss of an extensive superficial epithelium that potentiates the infection process . Our analyses of these tissues revealed that the bacteria induce apoptosis in the cells that comprise this epithelium within hours of the interaction with bacteria . Ultrastructural analysis revealed that after 24 h the integrity of the epithelium had been lost, i.e., the basement membrane had degenerated and the majority of the cells exhibited signs of apoptosis, most notably chromatin condensation . Analysis of these tissues with probes that reveal intracellular acidification showed that the cells first undergo an initial acidification beginning about 6-8 h after exposure to V . fischeri . As determined by end-labeling of DNA fragments, extensive endonuclease activity was detected at approximately 16-20 h post-infection . These data provide evidence that cooperative bacteria can participate in the remodeling of host tissues through the induction of host apoptotic programs.

Infect Immun, 1998 Sep, 66(9), 4025 - 9
Vibrio cholerae hemagglutinin/protease inactivates CTXphi; Kimsey HH et al.; Pathogenic strains of Vibrio cholerae are lysogens of the filamentous phage CTXphi, which carries the genes for cholera toxin (ctxAB) . We found that the titers of infective CTXphi in culture supernatants of El Tor CTXphi lysogens increased rapidly during exponential growth but dropped to undetectable levels late in stationary-phase growth . When CTXphi transducing particles were mixed with stationary-phase culture supernatants of El Tor strains, CTXphi infectivity was destroyed . Our data indicate that this growth phase-regulated factor, designated CDF (CTXphi-destroying factor), is the secreted hemagglutinin/protease (HA/P) of V . cholerae . A strain containing a disrupted hap gene, which encodes HA/P of V . cholerae, did not produce CDF activity in culture supernatants . Introduction of the HA/P-expressing plasmid pCH2 restored CDF activity . Also, CDF activity in culture supernatants of a variety of pathogenic V . cholerae isolates varied widely but correlated with the levels of secreted HA/P, as measured by immunoblotting with anti-HA/P antibody . CDF was purified from V . cholerae culture supernatants and shown to contain a 45-kDa polypeptide which bound anti-HA/P antibodies and which comigrated with HA/P in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The production of high levels of secreted HA/P by certain V . cholerae strains may be a factor in preventing CTXphi reinfection in natural environments and in the human host.

FEMS Microbiol Lett, 1998 Aug 1, 165(1), 139 - 43
Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): antimicrobial resistance, plasmid profiles and random amplification of polymorphic DNA analysis; Radu S et al.; Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa) . Thirty-six isolates were analyzed . The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%) . Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa . Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found . In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility . Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V . vulnificus strains investigated . RAPD identity across biotypes was also observed among Vibrio vulnificus strains.

Artif Organs, 1998 Aug, 22(8), 705 - 7
Endotoxin removal column containing polymyxin B immobilized fiber is useful for the treatment of the patient with Vibrio vulnificus septicemia; Sato T et al.; A case of primary septicemia due to Vibrio vulnificus infection is reported . The patient was successfully treated with appropriate antibiotic therapy, drainage, and debridement of the necrotic tissues and direct hemoperfusion (DHP) using polymyxin B immobilized fiber (PMX-F) . The effectiveness of DHP using PMX-F, which removes endotoxin in the circulating blood for the treatment of septic shock and multiple organ dysfunction occurring due to this fulminant infectious disease, is discussed.

Mol Microbiol, 1998 Jul, 29(1), 235 - 46
Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae; Crawford JA et al.; The membrane proteins ToxR and ToxS regulate a variety of genes associated with the virulence of Vibrio cholerae, the agent of human cholera . One of the ToxRS-regulated genes is the ompU gene, which encodes a porin that may also act as an adhesin . To begin to understand the mechanism of ompU transcription activation by ToxRS, we performed genetic and biochemical studies on the ompU promoter . Deletions with a 5' end-point at or downstream of -128, relative to the start site for transcription, did not direct expression of a lacZ reporter gene in wild-type V . cholerae, although the -128 promoter fragment did direct ToxRS-dependent reporter gene activity under conditions of ToxR overexpression in E . coli . Consistent with the activation data is that membranes containing ToxR and ToxS caused a gel electrophoretic mobility shift when mixed at low concentrations with deletion fragments whose end-point is at -211, but not with -128 or -68 fragments . ToxRS membranes did shift the -128 fragment when added at higher concentrations . DNase I footprinting analysis of ompU promoter DNA complexed with ToxRS membranes demonstrated protection of three sites: an upstream site ranging from -238 to -139, and two downstream sites ranging from -116 to -58 and -53 to -24 . Within the DNA protected from DNase I digestion by ToxRS membranes, there are no elements bearing similarity to those identified previously within the promoters of two other ToxR-dependent genes, ctxA and toxT . We suggest a model for transcription activation that involves sequential ToxR-binding events to distinct regions in the ompU promoter.

Acta Biochim Pol, 1998, 45(1), 261 - 70
Cloning of the groE operon of the marine bacterium Vibrio harveyi using a lambda vector; Kuchanny D et al.; groES and groEL genes encode two co-operating proteins GroES and GroEL, belonging to a class of chaperone proteins highly conserved during evolution . The GroE chaperones are indispensable for the growth of bacteriophage lambda in Escherichia coli cells . In order to clone the groEL and groES genes of the marine bacterium Vibrio harveyi, we constructed the V . harveyi genomic library in the lambdaEMBL1 vector, and selected clones which were able to complement mutations in both groE genes of E . coli for bacteriophage lambda growth . Using Southern hybridization, in one of these clones we identified a DNA fragment homologous to the E . coli groE region . Analysis of the nucleotide sequence of this fragment showed that the cloned region contained a sequence in 71.7% homologous to the 3' end of the groEL gene of E . coli . This confirmed that the lambda clone indeed carries the groE region of V . harveyi . The positive result of our strategy of cloning with the use of the genomic library in lambda vector suggests that the same method might be useful in the isolation of the groE homologues from other bacteria . The V . harveyi cloned groE genes did not suppress thermosensitivity of the E . coli groE mutants.

Zhonghua Yi Xue Za Zhi (Taipei), 1998 Jul, 61(7), 421 - 6
Recurrent spontaneous bacterial empyema in cirrhosis: a case report; Hsieh YH et al.; Spontaneous bacterial empyema occurs in about 0.4% of cirrhotic patients, but recurrent attack has rarely been reported . Herein we report a case of repeat episodes of spontaneous bacterial empyema . The patient presented with fever, shortness of breath and three episodes of spontaneous bacterial empyema (accompanied by septic shock in two episodes) within six months . Chest roentgenography showed massive right-side pleural effusion . Thoracentesis yielded pleural effusion containing a neutrophil count of more than 500/microliter . A blood culture grew non-O1 Vibrio cholerae in one episode and the pleural effusion culture grew Aeromonas hydrophila . A chest-tube was inserted during the latest episode due to a low pH and glucose concentration in the pleural fluid . The patient recovered well after medical treatment . The etiology, diagnosis and management of spontaneous bacterial empyema are discussed.

Mem Inst Oswaldo Cruz, 1998 Jan-Feb, 93(1), 17 - 22
Serotypes of Vibrio cholerae non-O1 isolated from water supplies for human consumption in Campeche, México and their antibiotic susceptibility pattern; Isaac-Marquez AP et al.; The presence of Vibrio cholerae non-O1 in water supplies for human consumption in the city of Campeche and rural locality of Becal was investigated . V . cholerae non-O1 was detected in 5.9% of the samples obtained in deep pools of Campeche . Studies conducted in Becal and neighbourhood of Morelos in Campeche indicated that collected samples harbored V . cholerae non-O1 in 31.5% and 8.7% respectively . There was a particular pattern of distribution of V . cholerae non-O1 serotypes among different studied regions . Accordingly, V . cholerae non-O1 serotype O14 predominated in the deep pools of Campeche and together with V . cholerae non-O1, O155 were preferentially founds in samples taken from intradomiciliary faucets in the neighbourhood of Morelos . Samples from Becal predominantly presented the serotype O112 . 60% and 53.8% of all studied strains of V . cholerae non-O1 proved to be resistant to ampicillin and carbenicillin . 3.1%, 7.7% and 6.2% presented resistant to doxycycline, trimethoprim-sulfamethoxazole and erythromycin respectively . The study showed the necessity of performing a strong epidemiologic surveillance for emergence and distribution of V . cholerae non-O1.

Immunol Lett, 1998 Jun, 62(2), 117 - 20
Immunostimulatory activity of LT-IIa, a type II heat-labile enterotoxin of Escherichia coli; Connell TD et al.; Certain bacterial molecules potentiate immune responses to parenterally administered antigens . One such molecule that has been intensely investigated is cholera toxin, a type I heat-labile enterotoxin produced by the Gram-negative bacterium Vibrio cholerae . Immunization with a mixture of a foreign antigen and cholera toxin enhances the immune response to the antigen . Similar adjuvant activity is associated with LT-I, a closely related type I heat-labile enterotoxin produced by Escherichia coli . The adjuvant activities of LT-IIa, a member of the type II heat-labile enterotoxins produced by E . coli, have not been described . LT-IIa and CT differ significantly in amino acid sequence of the B polypeptides and in receptor binding affinity . In this study, rats were subcutaneously immunized with fimbrillin, a protein isolated from the bacterium Porphyromonas gingivalis, and with fimbrillin in combination with LT-IIa, the prototypical type II enterotoxin . Previous studies documented that fimbrillin administered alone is a poor immunogen . Animals immunized with the mixture of fimbrillin and LT-IIa produced high titers of specific IgG antibody directed against fimbrillin . Anti-fimbrillin antibody titers in sera from animals receiving the combination of LT-IIa + fimbrillin were comparable to those obtained from sera of animals immunized with cholera toxin + fimbrillin . The results of these experiments demonstrate that LT-IIa exhibits an adjuvant activity that is equal to that of cholera toxin . Recombinant methods have been established for producing large amounts of LT-IIa, an advantage that will likely provide an economic impetus to consider incorporating the enterotoxin as an immunostimulatory agent in future vaccines.

Microb Comp Genomics, 1998, 3(2), 119 - 32
Horizontal transfer of chromosomal DNA between the marine bacterium Vibrio furnissii and Escherichia coli revealed by sequence analysis; Charbit A et al.; Previous in silico analysis of the 67.4-76.0 minutes region of the Escherichia coli genome led to the identification of a gene cluster (named aga) comprising five genes encoding homologs of the mannose transporter of E . coli, a member of the sugar-specific phosphoenolypyruvate/sugar phosphotransferase system (PTS) . In the present work, we compared the aga gene cluster of E . coli, which has been considered to be involved in N-acetylgalactosamine or N-acetylmannosamine transport and metabolism, to the region comprising the recently identified mannose transporter of the marine bacterium Vibrio furnissii . Our analysis revealed that the proteins encoded by three genes (agaV, agaW, and agaA), located in the proximal portion of the aga gene cluster, shared striking similarities with the proteins encoded by the manX (IIBMan), manY (IICMan), and manD (a putative deacetylase) genes of V . furnissii, respectively (70%-82.3% identity among the three pairs of proteins) . Moreover, we found that the two following aga genes (agaS and agaY) were homologous to the sequences flanking the mannose operon of V . furnissii . These observations strongly support the idea of a horizontal transfer of the chromosomally encoded man operon of V . furnissii into the E . coli genome.

Dis Aquat Organ, 1998 Mar 5, 32(2), 91 - 7
Growth of Atlantic salmon Salmo salar after intraperitoneal administration of vaccines containing adjuvants; Midtlyng PJ et al.; Growth of Atlantic salmon after intraperitoneal (i.p.) administration of adjuvanted vaccines was studied using groups of individually tagged fish held together in one tank or pen under commercial farming conditions . Parallel experiments were initiated at 2 freshwater sites and 1 marine site . Trivalent (vibriosis, cold water vibriosis and furunculosis) vaccines containing oil or beta-1, 3 glucan as adjuvants were used for immunisation of pre-smolts, whereas identical formulations containing furunculosis antigens only were used in growers . Control fish remained unvaccinated . No outbreak of bacterial or viral disease was experienced at any of the sites . At all sites, the highest daily growth rate was recorded in unvaccinated fish . At one site, the average weight of post-smolts that had received oil-adjuvant vaccine was significantly reduced by 345 g (23%) after 15 mo . Impaired growth rate was associated with increasing severity of intra-abdominal lesions as determined during necropsy . At the second post-smolt site and in growers, weight development and growth rates were non-significant between groups throughout the study . The results indicate that intraperitoneal administration of oil-adjuvanted vaccines may retard growth of farmed Atlantic salmon, although the extent of this effect may vary between sites . Unidentified factors coinciding with vaccination are thought to have caused the highly variable results seen between parallel sites in this study.

Microbiology, 1998 Jul, 144 ( Pt 7), 1901 - 6
A novel filamentous phage, fs-2, of Vibrio cholerae O139; Ikema M et al.; A novel filamentous bacteriophage, fs-2, was isolated from Vibrio cholerae O139 strain MDO14 . The fs-2 phage was a long filamentous particle 1200 nm long and 7 nm wide . The purified phage formed a turbid plaque when spotted on a lawn of the host organisms . The plaque-formation activity was stable following heating to 70 degrees C but was inhibited by treatment with chloroform . fs-2 had a single-stranded DNA genome and was converted to a double-stranded replicative form in the host cell . Almost all V . cholerae O139 and O1 El Tor biotype strains tested were sensitive to the phage, but most O1 classical strains and non-O1 non-O139 strains were resistant . The fs-2 genome comprised 8651 nucleotides containing nine open reading frames, five of which had predicted protein products partially homologous to the reported protein products of other filamentous phages . Although the extent of the homology was not particularly high, the genetic organization of other filamentous phages appears to be preserved in fs-2 . The phage was not integrated into the chromosome of its host, but a 715 nucleotide fragment located in the large intergenic region of fs-2 was highly homologous to a part of region RS2 (repetitive sequence 2) of the V . cholerae CTX phi sequence which is speculated to be required for integration of the phage into the V . cholerae chromosome at a specific site.

Kansenshogaku Zasshi, 1998 Jun, 72(6), 575 - 84
{Analysis of Vibrio cholerae O1 isolated in Japan by pulsed-field gel electrophoresis}; Gyobu T et al.; Vibrio cholerae O1 strains isolated mostly in Japan between 1977 and 1995 were typed according to restriction fragment patterns by cleavage of genomic DNA with Sfi I and Not I and separation by Pulsed-field gel electrophoresis (PFGE) . Two hundred sixty five strains from human were divided into 60 PFGE patterns (provisional types) . Strains of type 2-3, 3-4, 4-5 and 51-54 were dominant in the Philippines, Thailand, India and Indonesia, respectively . Types 1-1, 2-3, 2-53, and 3-4 were detected over a long period of time in contrast to the other types . Strains of the same type (Types 1-1, 2-3, 2-53 and others) isolated from the Japanese who had never been outside Japan were often found among strains from Southeast Asia . Most strains from humans were cholera-toxin (CT)-positive, while those from the environment and the sea were generally not . 23 strains from the environment and the sea in Japan were divided into 12 types . Strains of the same types as CT-positive strains from humans could not be found in the environment and sea . These results suggest that cholera in Japan is closely related with cholera in Southeast Asia and PFGE is useful for epidemiological analysis of cholera in Japan.

Eur J Biochem, 1998 Jul 1, 255(1), 279 - 88
Structure determination of the capsular polysaccharide from Vibrio vulnificus strain 6353; Reddy GP et al.; Vibrio vulnificus is a pathogenic gram-negative bacterium, endemic to brackish waters, which is often isolated from sediments, from the water column or from shellfish . It is associated with wound infections and septicemia in humans and the virulence of V . vulnificus has been strongly associated with encapsulation . The capsular polysaccharide purified from a virulent strain of V . vulnificus 6353 did not show cross reactivity with antibodies to the capsular polysaccharide of a related pathogenic strain of V . vulnificus (MO6-24) the structure of which was recently reported . NMR spectroscopic analysis of the purified polysaccharide from strain 6353 showed that the polymer is composed of four sugar residues per repeating subunit including 2,6-dideoxy-2-N-acetylamino-alpha-D-glucose (QuiNAc), 2-deoxy-2-N-acetylamino-alpha-D-galactose (alpha-D-GalNAc), 2-deoxy-2-N-acetylamino-alpha-D-galcturonic acid (alpha-D-GalNAcA) and 2-N-acetylamino-alpha-D-glucuronamide (alpha-D-GlcNAcANH2) . The 1H- and 13C-NMR spectra were completely assigned by homonuclear and heteronuclear NMR spectroscopy . Sugar types and anomeric configurations were determined from proton homonuclear coupling constants and glycosidic linkages were determined from 1H-13C heteronuclear multiple bond correlation spectra . Sugar identities were confirmed by high performance anion-exchange chromatography and absolute configurations were determined by gas chromatography in combination with molecular modeling and NMR spectroscopy . The structure of the polysaccharide repeating unit is: {-->4)-alpha-D-GalpNAc-(1-->3)-alpha-D-GalpNAcA-(1-->3)-alpha-D-++ +QuipNAc-(1-->}n alpha-D-GlcpNAcANH2 (1-->4)- --> . While there are some common features shared among the structures of the capsular polysaccharides of pathogenic strains of V . vulnificus, there are distinct differences in the detailed structures.

Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1157 - 60
Hemagglutinating activity of extracellular alkaline metalloendopeptidases from Vibrio sp . NUF-BPP1; Fukuda K et al.; Alkaline metalloendopeptidase (metalloprotease) AP1 (48 kDa) from Vibrio sp . isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) was reported in our previous paper to produce AP2 (36 kDa) by releasing a peptide fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1 by autodigestion . AP1 strongly agglutinated fish (flounder, Paralichthys olivaceus) and rabbit erythrocytes, and weakly chicken erythrocytes . In contrast, AP2 had no significant hemagglutinating activity toward any erythrocytes tested, except for weak activity on flounder erythrocytes, suggesting that the C-terminal region of AP1 may be required for the strong hemagglutinating activity . The optimum temperature for the hemagglutinating activity of AP1 was found to be lower than that for the proteolytic activity . At acidic pHs (below pH 7.5), the hemagglutinating activity of AP1 decreased, and its pH profile resembled that of the proteolytic activity . The hemagglutinating activity of AP1 was not observed in the presence of o-phenanthroline or synthetic and proteinous substrates, but different kinds of saccharides and lipids had no effect . While the proteolytic activity of AP1 was not affected by CaCl2, the hemagglutinating activity of AP1 decreased with increases in CaCl2 concentrations . These results suggested that the hemagglutinating activity of these proteases (AP1 and AP2) was most likely caused by their proteolytic action on erythrocyte cell surfaces.

Carbohydr Res, 1998 Jan, 306(1-2), 163 - 70
Synthesis of terminal disaccharide elements corresponding to the Ogawa and Inaba antigenic determinant from Vibrio cholerae O1; Arencibia-Mohar A et al.; Vibrio cholerae O1 LPS terminal mono- and disaccharide elements were synthesized by reduction of the azido group in several 4-amino-4,6-dideoxy-D-mannose mono- and disaccharide derivatives, followed by coupling with 2, 4-di-O-acetyl-3-deoxy-L-glycero-tetronic acid in the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline . This compound represents a useful model in order to elucidate the size of the epitopes which define Ogawa and Inaba serotypes from Vibrio cholerae O1.

Toxicon, 1998 Jul, 36(7), 1001 - 5
Nicking sites in a subunit of cholera toxin and Escherichia coli heat-labile enterotoxin for Vibrio cholerae hemagglutinin/protease; Naka A et al.; We analyzed the nicking site of the A subunit of Escherichia coli heat-labile enterotoxin for hemagglutinin/protease produced by Vibrio cholerae non-O1 (NAG-HA/P) . The determined nicking site was the Thr193-Ile194 junction, which was distinct from that for a protease of V . cholerae (Ichinose et al., European Journal of Epidemiology 8, 743-747, 1992) . We further analyzed proteolytic cleavage by NAG-HA/P of a synthetic peptide corresponding to the nicking region of cholera toxin A subunit and determined the cleavage site to be preferentially between Ser194 and Met195, and in addition between Ser193 and Ser194.

Appl Environ Microbiol, 1998 Aug, 64(8), 3025 - 8
Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnificus cells; Warner JM et al.; Vibrio vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans . Because of the low nutrient levels and temperature fluctuations found in the organism's natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest . A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of V . vulnificus strains grown in rich media and has been applied to starved and VBNC cells of V . vulnificus in the present study . As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period . Most noticeable was a uniform loss of RAPD amplification products . By 4 h of starvation, the cells were undetectable by the RAPD method . Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm . The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state . VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method . The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states . This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify V . vulnificus DNA in these states.

Dis Aquat Organ, 1998 Jun 19, 33(2), 111 - 8
Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae; Sugumar G et al.; Recurrent outbreaks of a disease leading to mass mortalities in an oyster (Crassostrea gigas) hatchery located in western Japan were investigated . The disease occurred regularly in 2- to 8-d-old larvae and has been experimentally controlled in the hatchery by treating the larval rearing water with streptomycin, without ascertaining the etiological agent . The signs of the disease and the course of infection resembled bacillary necrosis reported in oysters and other bivalve molluscs in the USA and Europe . Quantitative and qualitative examinations of the bacterial flora of hatchery samples including source water, broodstock, larval feed and larvae revealed a very high total bacterial load and presumptive vibrios in diseased larvae . Further, the bacterial profile revealed that Vibrio spp . constituted approximately 60 to 95% of the bacteria isolated from infected larvae and most isolates were identified as V . splendidus biovar II and V . harveyi, suggesting their possible role in the disease . However, experimental challenges proved the pathogenicity of V . splendidus II . Several isolates of V . splendidus II from infected larvae were highly pathogenic, producing 100% mortality at levels of 10(5) cfu ml-1 in 24 h, while isolates from other sources demonstrated a low degree of virulence . Detection of V . splendidus II from broodstock, especially in the gonad of a few breeders, suggests the probability that broodstock could be the source and route of transmission of this pathogen.

Immunol Cell Biol, 1998 Jun, 76(3), 270 - 9
The role of ADP-ribosylation and G(M1)-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin; de Haan L et al.; The mucosal route of vaccination has attracted a great deal of attention recently . Not only is mucosal application of vaccines, for example, orally or intranasally, particularly convenient, it also offers the possibility to induce locally produced and secreted S-IgA antibodies in addition to systemic IgG antibodies . These IgA antibodies are known to play a key role in protection against pathogens that invade the host through mucosal surfaces . Induction of such responses is not readily achieved by currently used vaccination strategies, which generally involve intramuscular or subcutaneous injection with inactivated pathogens or antigens thereof . For the induction of a mucosal immune response, the vaccine needs to be applied locally . However, local vaccination with non-replicating antigens is usually ineffective and may result in tolerance unless a mucosal immunoadjuvant is included . The most potent mucosal immunoadjuvants known to date are probably cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT) . Although CT and LT have become standard adjuvants for experimental mucosal vaccines, the intrinsic toxicity has thus far precluded their use as adjuvants for human vaccine formulations . In the present review, the mucosal immunogenic and adjuvant properties of LT and CT are described, with special emphasis on the functional role of the individual subunits on their immune-stimulatory properties.

FEMS Microbiol Lett, 1998 Jul 15, 164(2), 353 - 61
Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable; Chung S et al.; Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327 . Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable . The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant . The doubling time of the csmC mutant was approximately twice that of the wild-type strain . Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800-804 nm, was less efficient in the csmC mutant cells than in wild-type cells . These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp . and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles.

Mol Genet Metab, 1998 May, 64(1), 12 - 8
Modulation of intestinal permeability: an innovative method of oral drug delivery for the treatment of inherited and acquired human diseases; Fasano A; Conventional forms of administrations of nonabsorbable drugs and peptides rely on their parenteral injection . The intestinal epithelium represents the major barrier to the oral absorption of these therapeutical agents into the systemic circulation . Recently, a number of innovative drug delivery approaches have been developed, including the drug entrapment within small vesicles or their passage through the intestinal paracellular pathway . Zonula occludens toxin, a recently discovered protein elaborated by Vibrio cholerae, provided tools for gaining more insights on the pathophysiology of the regulation of intestinal permeability and to developing alternative approaches for the oral delivery of drugs and macromolecules normally not absorbed through the intestine.

Mol Microbiol, 1998 Jun, 28(6), 1247 - 54
Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage; Rubin EJ et al.; We identified a 4.7kb cryptic plasmid in all ctxAB+ Vibrio cholerae strains we tested . An isolate of the V . cholerae classical biotype strain 0395 that harbours the cryptic plasmid at high copy number was found . Hybridization analysis demonstrated that sequences highly related or identical to this plasmid exist in all toxigenic strains of V . cholerae but were notably absent in all non-toxigenic environmental isolates that lacked the genes for toxin-co-regulated pili and the filamentous CTX prophage . Accordingly, we have named the cryptic plasmid pTLC for toxin-linked cryptic . The complete nucleotide sequence of pTLC from the high-copy-number isolate was determined . The largest open reading frame in the plasmid is predicted to encode a protein similar to the replication initiation protein (pII) of Escherichia coli F-specific filamentous phages . The nucleotide sequence of pTLC also facilitated the structural characterization of the DNA homologous to pTLC in other strains of V . cholerae . pTLC-related DNA exists in these strains as both low-copy-number, covalently closed circular DNA and tandemly duplicated, chromosomally integrated DNA . Remarkably, the chromosomally integrated form of pTLC is adjacent to the CTX prophage . The strain distribution, chromosomal location and DNA sequence of pTLC suggests that it may be a genetic element that plays some role in the biology of CTXphi, perhaps facilitating either its acquisition or its replication.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 141 - 7
Replicating function of the RS1 element associated with Vibrio cholerae CTX phi prophage; Campos J et al.; The RS1 element associated with Vibrio cholerae CTX phi prophage was cloned from an E1 Tor biotype Vibrio cholerae strain . We used the recA- vaccine strain Peru-15, that lacks the target for RS-mediated site-specific integration, to show that RS1 promotes autonomous replication of a suicide vector . A linker insertion in the rstR open reading frame abolished autonomous replication in Peru-15 but not in a strain containing an RS1 in the chromosome . An AT-rich region containing cis-acting elements involved in autonomous replication was identified by deletion . This region was sufficient to support autonomous replication in a strain containing an RS1 in the chromosome . DNA sequence analysis of a region present in RS1 and not RS2 revealed the presence of putative binding sites for host proteins involved in plasmid replication . These results indicate that RS1 contains a replicon distinct from RS2 which could be involved in replicative recombination events associated with tandem amplification of the CTX element.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 91 - 8
Vibrio cholerae O22 might be a putative source of exogenous DNA resulting in the emergence of the new strain of Vibrio cholerae O139; Dumontier S et al.; The new epidemic strain O139 of Vibrio cholerae, the etiologic agent of cholera, has probably emerged from the pandemic strain O1 E1 Tor through a genetic rearrangement involving the horizontal transfer of exogenous O-antigen- and capsule-encoding genes of unknown origin . In V . cholerae O139, these genes are associated with an insertion sequence designated IS1358O139 . In this work, we studied the distribution of seven genes flanking the IS1358O139 element in 13 serovars of V . cholerae strains . All these O139 genes and an IS1358 element designated IS1358O22-1 were only found in V . cholerae O22 with a similar genetic organization . Sequence analysis of a 4.5-kb fragment containing IS1358O22-1 and the adjacent genes revealed that these genes are highly homologous to those of V . cholerae O139 . These results suggest that strains of V . cholerae O22 from the environment might have been the source of the exogenous DNA resulting in the emergence of the new epidemic strain O139.

J Appl Microbiol, 1998 May, 84(5), 747 - 51
Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood; Moreno ML et al.; The virulence factors of Vibrio vulnificus are not yet well understood . So far, many hydrolytic enzymes have been implicated in the pathogenesis of this micro-organism . The present research was carried out in order to study the presence of some of these enzymes in 133 V . vulnificus strains isolated from 45 seafood samples . The results showed that 100% of these strains were positive for the production of lecithinase and lipase (Tween-80), 99.2% for caseinolytic protease, 96.9% for DNase, 65.4% for mucinase and 46.6% for elastase . None of the strains was positive for the production of collagenase and 96% were haemolytic against sheep blood cells . In relation to colony morphology on brain heart infusion (BHI) agar and nutrient agar, 59.4% of strains showed opaque morphology on BHI agar and 57.9% on nutrient agar, 10.5% presented translucent morphology on both agars and 30.1 and 31.6% of strains showed a mixture of opaque and translucent morphology on BHI agar and nutrient agar, respectively . None of the translucent colonies was virulent to mice . Therefore, opacity was a useful marker for potential virulence . Of 45 food samples contaminated with V . vulnificus, 29 (64.4%) presented strains lethal to adult mice.

Infect Immun, 1998 Aug, 66(8), 3995 - 9
Immune responses in ileostomy fluid and serum after oral cholera vaccination of patients colectomized because of ulcerative colitis; Kilhamn J et al.; The capacity of an oral inactivated B-subunit-whole-cell cholera vaccine to induce immune responses in patients colectomized due to ulcerative colitis was studied . Two doses of vaccine induced significant mucosal immunoglobulin A (IgA) antibody responses in ileostomy fluid against cholera toxin in 14 of 15 (93%) patients and against whole vibrios in 9 of 15 (60%) cases . The serological responses were lower (but not significantly) than those observed in healthy Swedish volunteers . Increased IgA antitoxin levels were found in ileostomy fluid as late as 2 years after vaccination.

Infect Immun, 1998 Aug, 66(8), 3974 - 7
Use of monoclonal antibodies to identify phospholipase C as the enterotoxic factor of the bifunctional hemolysin-phospholipase C molecule of Vibrio cholerae O139; Pal S et al.; Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities . Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected . In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule . The enterotoxicity of the purified PC molecule isolated from an Hly- spontaneous mutant of the HlyPC-producing parent strain further confirms this contention . The Hly molecule isolated from a PC- mutant was not diarrheagenic.

Infect Immun, 1998 Aug, 66(8), 3752 - 7
Induction of the lysogenic phage encoding cholera toxin in naturally occurring strains of toxigenic Vibrio cholerae O1 and O139; Faruque SM et al.; In toxigenic Vibrio cholerae, the CTX genetic element which carries the genes for cholera toxin (CT) is the genome of a lysogenic bacteriophage (CTXPhi) . Clinical and environmental strains of V . cholerae O1 or O139 and stools that were culture positive for cholera were analyzed to study the induction and transmission of CTXPhi . To our knowledge, this is the first report of the examination of CTXPhi in clinical materials and in naturally occurring strains . DNA probe analysis revealed that 4.25% (6 of 141) of the isolated V . cholerae strains spontaneously produced a detectable level of extracellular CTXPhi particles in the culture supernatants whereas another 34.04% (48 of 141) produced CTXPhi particles when induced with mitomycin C . CTXPhi isolated from 10 clinical or environmental strains infected a CT-negative recipient strain, CVD103, both inside the intestines of infant mice and under laboratory conditions . All culture-positive stools analyzed were negative for the presence of CTXPhi both in the DNA probe assay and by in vivo assay for the infection of the recipient strain in infant mice . These results suggested that naturally occurring strains of toxigenic V . cholerae are inducible lysogens of CTXPhi but that cholera pathogenesis in humans is not associated with the excretion of CTXPhi particles in stools, indicating that induction of the phage may not occur efficiently inside the human intestine . However, in view of the efficient transmission of the phage under conditions conducive to the expression of toxin-coregulated pili, it appears that propagation of CTXPhi in the natural habitat may involve both environmental and host factors.

Indian J Med Res, 1998 May, 107, 199 - 203
Molecular analysis of the cholera toxin gene & antibiotic sensitivity profile of Vibrio cholerae O1 & O139 associated with mixed infection; Sharma C et al.; In the context of the reemergence of V . cholerae O1 in India and the recent evidence that O139 strains could have evolved from O1 E1 Tor strains, restriction fragment length polymorphism (RFLP) of the rRNA and the ctx genes and the antibiotic sensitivity profile of the two strains of V . cholerae, one an O1 and the other an O139, associated with mixed infection, were examined to determine their relatedness . Our results demonstrate that although the strains belonged to different clones of V . cholerae, they showed similar antibiotic sensitivity, profile indicating some exchange of genetic elements.

Microbios, 1997, 92(372-373), 209 - 17
Effects of extracellular products of Vibrio vulnificus on Acanthopagrus schlegeli serum components in vitro and in vivo; Lee KK et al.; A Vibrio strain Ls001, originally isolated from a body surface lesion of a moribund black porgy (Acanthopagrus schlegeli) in 1994 in Taiwan, was identified as Vibrio vulnificus . The extracellular products (ECP) of the strain were lethal to the fish, and its effects on fish serum in vitro and in vivo are described in the present study . Nine major precipitation arcs were visualized in normal fish serum in a crossed immunoelectrophoresis (CIE) gel using rabbit antiserum to the fish normal serum and staining with Coomassie brilliant blue . Only four and six of the nine major arcs could be tentatively identified by CIE following in vivo and in vitro ECP treatment, respectively . The same two major arcs were both missing following either in vivo or in vitro treatment with ECP . These complex events may significantly contribute to the pathogenesis of V . vulnificus in A . schlegeli.

Antimicrob Agents Chemother, 1998 Jul, 42(7), 1778 - 82
NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli; Morita Y et al.; We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin . We cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA of V . parahaemolyticus by using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced the gene (norM) . Cells of E . coli transformed with a plasmid carrying the norM gene showed elevated energy-dependent efflux of norfloxacin . The transformants showed elevated resistance not only to norfloxacin and ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin . These results suggest that this is a multidrug efflux system . The hydropathy pattern of the deduced amino acid sequence of NorM suggested the presence of 12 transmembrane domains . The deduced primary structure of NorM showed 57% identity and 88% similarity with that of a hypothetical E . coli membrane protein, YdhE . No reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM . Thus, NorM seems to be a novel type of multidrug efflux protein . We cloned the ydhE gene from E . coli . Cells of E . coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin, acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured . Thus, it seems that NorM and YdhE differ somehow in substrate specificity.

Cad Saude Publica, 1998 Apr, 14(2), 319 - 25
Prevalence of vibrio cholerae O(1) infection in manacapuru, amazonas state, brazil (1992)
Goncalves EGR, Sabroza PC, Hofer E.
This study focused on the prevalence of V . cholerae O(1) infection in 1,196 individuals living in Manacapuru, Amazonas State, through microtitering of vibriocidal antibody and somatic agglutination test . The role of living conditions and individual characteristics as possible risk factors for infection was also assessed . Vibriocidal titers >/= 1: 40 and/or agglutinating titers >/= 1: 80 were considered indicators of V . cholerae O(1) infection . Infection prevalence was 25.7% . There was no significant statistical difference (p=0.05) when analyzed against housing patterns, sanitary facilities, source and treatment of water, destination of domestic waste, sex, or profession . Household location, number of occupants/household, age, and schooling showed significant statistical differences in infection prevalence (p=0.05).

Vet Immunol Immunopathol, 1998 Jun 30, 64(1), 59 - 68
Influence of the sequence of administration of beta-glucans and a Vibrio damsela vaccine on the immune response of turbot (Scophthalmus maximus L.); Figueras A et al.; Yeast (Saccharomyces cerevisae) beta-1,3 glucans were used as adjuvant in a Vibrio damsela vaccine for turbot (Scophthalmus maximus L.) . Turbot were injected with the adjuvant prior, at the same time and after the vaccine . Several immune parameters (index and rate of phagocytosis, passive haemolytic plaque numbers, and agglutinating antibody titers) were determined at different times postinoculation . The highest activity of all the immune parameters was obtained when glucans were injected after the bacterin . It is concluded that the sequence of glucan administration is critical when used as a vaccine adjuvant.

Eur J Biochem, 1998 May 15, 254(1), 58 - 62
Structure of the O-antigen of Vibrio cholerae O155 that shares a putative D-galactose 4,6-cyclophosphate-associated epitope with V . cholerae O139 Bengal; Senchenkova SN et al.; The O-specific polysaccharide of Vibrio cholerae 0155 was studied by sugar and methylation analyses, dephosphorylation with 48% hydrofluoric acid, 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and heteronuclear single-quantum coherence (HSQC) experiments . The following structure of the pentasaccharide repeating unit of the polysaccharide was established: carbohydrate sequence {see text} . An unusual component, D-galactose 4,6-cyclophosphate, has been reported previously as a component of the capsular polysaccharide and O-antigen of V . cholerae O139 Bengal and appears to be responsible for the known serological cross-reactivity between V . cholerae O139 and O155.

J Clin Microbiol, 1998 Jul, 36(7), 2149 - 52
Molecular epidemiology of reemergent Vibrio cholerae O139 Bengal in India; Mukhopadhyay AK et al.; We report the prevalence of the O139 serogroup in Calcutta, India, after its reemergence in August 1996 and the spread of the reemerged clone to other parts of the country by using previously established molecular markers . Phenotypically, the reemerged Vibrio cholerae O139 displayed a difference compared to those that appeared in late 1992 and 1993 in that the current O139 strains are sensitive to co-trimoxazole . Ribotyping with the enzyme BglI produced two rRNA restriction patterns in the O139 strains isolated after August 1996, and these patterns were identical to those exhibited by strains of O139 isolated in 1992 . Three clones of V . cholerae O139 are currently prevailing in the country, with strains exhibiting three bands after HindIII digestion and hybridization with a ctxA probe being dominant . The reemergence of V . cholerae O139 in Calcutta after a 32-month quiescent period reestablishes the O139 serogroup as an entity which is likely to play a crucial role in the temporal antigenic variations among the serogroups of V . cholerae causing cholera.

Appl Environ Microbiol, 1998 Jul, 64(7), 2701 - 4
Enhanced broth media for selective growth of Vibrio vulnificus; Hsu WY et al.; Rapid detection of Vibrio vulnificus can be enhanced by optimizing the components of enrichment broth . PNC (5% peptone, 1% NaCl, and 0.08% cellobiose {pH 8.0}) enhanced the growth of V . vulnificus compared to alkaline peptone broth . PNCC (PNC with 1.0 to 4.1 U of colistin methanesulfonate per ml) increased the growth of low levels of V . vulnificus while suppressing non-target bacteria.

Sci Total Environ, 1998 May 27, 216(3), 205 - 15
QSAR study of the toxicity of benzoic acids to Vibrio fischeri, Daphnia magna and carp; Zhao YH et al.; The toxicities of benzoic acids to Vibrio fischeri, Daphnia magna and carp were measured . The results showed that the toxicity to V . fischeri and Daphnia decreased in the order of bromo > chloro > fluoro approximately equal to aminobenzoic acids . The toxicity of substituted benzoic acids to carp and Daphnia was much lower that to V . fischeri . The results also showed that the toxicity of benzoic acids to Daphnia decreased as the pH increased . It is suggested that ionized and non-ionized forms have different toxic responses . The non-ionized form may play an important role in toxicity because the toxicity of benzoic acids to Daphnia greatly decreases as the pH increases . The toxicity of benzoic acids to Daphnia may operate through non-polar narcosis, based on the regression results between the toxicities and partition coefficients (log P) and apparent partition coefficients (log D) . However, toxicity cannot be predicted from non-polar baseline models because the ionized and non-ionized form of benzoic acids have different contributions to toxicity . Compared with the single descriptors, the prediction of toxicity of the benzoic acids was improved remarkably by using log P with pKa and log P with ELUMO . For the toxicity of benzoic acids to V . fischeri, it is suggested that the toxic mechanism may be different from the mechanism in Daphnia and carp . A probable reason is that V . fischeri is a unicellular organism with low lipid content, and hence both ionized and non-ionized forms of benzoic acids can easily cross the cell membrane and contribute to toxicity.

J Bacteriol, 1998 Jul, 180(13), 3491 - 4
KtrAB, a new type of bacterial K(+)-uptake system from Vibrio alginolyticus; Nakamura T et al.; Vibrio alginolyticus contained two adjacent genes, ktrA and ktrB, which encode a new type of bacterial K(+)-uptake system . KtrA and KtrB are peripheral and integral membrane proteins, respectively . Six of the nine sequenced bacterial genomes contain homologs to both ktrA and ktrB, suggesting that KtrAB is widespread.

Biochim Biophys Acta, 1998 Jul 1, 1407(1), 21 - 30
Role of intracellular second messengers and reactive oxygen species in the pathophysiology of V . cholera O139 treated rabbit ileum; Gorowara S et al.; Vibrio cholerae O139 has pandemic potential and it produces copious amounts of fluid secretion . The levels of various second messengers (intracellular Ca2+, cAMP, IP3, PKC) were measured to determine the cause of fluid secretion produced by this strain of V . cholerae . There was a significant increase in the levels of these second messengers in V . cholerae O139 treated ileum as compared to control ileum (enterocytes) . Levels of these second messengers were also assessed in V . cholerae 569B induced fluid secretion in rabbit ileum and it was found that the levels were raised more in V . cholerae O139 treated ileum than in V . cholerae 569B treated rabbit ileum . The intestinal damage was assessed by measuring changes in the extent of lipid peroxidation of the enterocytes . Intracellular second messengers are known to raise the extent of lipid peroxidation . In V . cholerae O139 treated loops calcium ionophore A23187 enhanced the extent of lipid peroxidation whereas l-verapamil could only marginally decrease the lipid peroxidation . Dantrolene and H7 significantly decreased the extent of lipid peroxidation of enterocytes in V . cholerae O139 treated rabbit ileum . However, PMA could not enhance further the extent of lipid peroxidation in V . cholerae O139 treated rabbit ileum . So intracellular calcium and protein kinase C appear to be involved in intestinal damage caused by V . cholerae O139 . Reactive oxygen species are responsible for causing tissue damage and the extent of oxidative damage depends on the balance between the pro-oxidants and the anti-oxidants . So the changes in the enterocytes' antioxidant level during V . cholerae O139 mediated intestinal infection was estimated . There was a significant decrease in the enterocyte level of the antioxidant enzymes SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase and glucose-6-phosphate dehydrogenase in V . cholerae O139 mediated intestinal infection . So a significant decrease in the levels of antioxidant defenses and a significant increase in the levels of second messengers appear to be important in mediating V . cholerae O139 induced lipid peroxidation which contributes to the changes in membrane permeability and thus to fluid secretion.

MMWR Morb Mortal Wkly Rep, 1998 Jun 12, 47(22), 457 - 62
Outbreak of Vibrio parahaemolyticus infections associated with eating raw oysters--Pacific Northwest, 1997; Sialic acid-binding lectin with antibacterial activity from the horse mussel: further characterization and immunolocalization; Department of Marine Biochemistry, The Norwegian College of Fishery Science, University of TromsoA heterogeneous sialic acid-binding lectin with affinity for bacterial LPS was isolated and partially characterized from hemolymph of the horse mussel Modiolus modiolus.(1) Using two-dimensional electrophoresis with immobilized pH gradients, the lectin revealed three subunits with different molecular weight and isoelectric points (pI); Mr14 (pI approximately 5.1 and approximately 5.5), 17.5 (pI approximately 5.5) and 20 (pI approximately 4.9) kDa . The affinity purified lectin existed in its native state as aggregates, and by stepwise centrifugation it could be fractionated into molecular entities with distinct specificities towards human and/or horse erythrocytes (modiolin H and/or E activity, respectively) . While the medium size entities (range < or = 30 and < 100 kDa) exhibited only modiolin E activity and the lowest size entities (range < or = 5 and < 10 kDa) demonstrated only modiolin H activity, the largest aggregates (> or = 100 kDa-)expressed both activities . Antibacterial activity of the lectin has been observed against various marine bacteria, whereas the whole hemolymph was less effective . The lectin exhibited strong antibacterial effect against all tested strains of Vibrio anguillarum, Vibrio salmonicida, Vibrio viscosus, Vibrio wodanis, and Vibrio ordalii, slight effect on Aeromonas salmonicida salmonicida and Shewanella putrefaciens, and no inhibitory effect with Alteromonas sp . Hemolymph of the horse mussel demonstrated no antibacterial effect against A . salmonicida salmonicida, Alteromonas sp., Sh . putrefaciens and some strains of V . anguillarum, but slight effects against some strains of V . anguillarum and both strains of V . ordalii, and more predominantly against V . wodanis, V . salmonicida and V . viscosus . These results indicate that the lectin plays a role in elimination of bacteria . Circulating hemocytes were demonstrated to be the source of the lectins since granules of the hemocytes were immunoreactive to anti-hemolymph lectin antibody and protein A/gold labelling.

Infect Immun, 1998 Jul, 66(7), 3095 - 9
Phase 1 evaluation of Vibrio cholerae O1, serotype Inaba, polysaccharide-cholera toxin conjugates in adult volunteers; Gupta RK et al.; Conjugate vaccines were prepared by binding hydrazine-treated lipopolysaccharide (DeALPS) from Vibrio cholerae O1, serotype Inaba, to cholera toxin (CT) variants CT-1 and CT-2 . Volunteers (n = 75) were injected with either 25 microg of DeALPS, alone or as a conjugate, or the licensed cellular vaccine containing 4 x 10(9) organisms each of serotypes Inaba and Ogawa per ml . No serious adverse reactions were observed . DeALPS alone did not elicit serum LPS or vibriocidal antibodies in mice and only low levels of immunoglobulin M (IgM) anti-LPS in the volunteers . Recipients of the cellular vaccine had the highest IgM anti-LPS levels, but the difference was not statistically significant from that elicited by the conjugates . The conjugates elicited the highest levels of IgG anti-LPS (DeALPS-CT-2 > DeALPS-CT-1 > cellular vaccine) . Both conjugates and the cellular vaccine elicited vibriocidal antibodies: after 8 months, recipients of cellular vaccine had the highest geometric mean titer (1,249), followed by DeALPS-CT-2 (588) and DeALPS-CT-1 (330) . The correlation coefficient between IgG anti-LPS and 2-mercaptoethanol (2-ME)-resistant vibriocidal antibodies was 0 . 81 (P = 0.0004) . Convalescent sera from cholera patients had a mean vibriocidal titer of 2,525 that was removed by treatment with 2-ME . The vibriocidal activities of sera from all vaccine groups and from the patients were absorbed (>75%) by LPS but not by either CT-1 or CT-2 . Conjugate-induced IgG vibriocidal antibodies persisted longer than those elicited by the whole-cell vaccine . Both conjugates, but not the cellular vaccine, elicited IgG anti-CT.

Infect Immun, 1998 Jul, 66(7), 3066 - 71
Effect of mild acid treatment on the survival, enteropathogenicity, and protein production in vibrio parahaemolyticus; Wong HC et al.; Vibrio parahaemolyticus is an important food-borne enteropathogen that encounters various adverse conditions in its native environment or during infection . Effects of mild acid treatment on survival under stress conditions, enteropathogenicity, and protein production in this pathogen were investigated . Logarithmically grown cells, at pH 7.5 shifted to pH 5.0 for 30 min, were more resistant to subsequent acid challenge at pH 4.4 . A two-phase adaptive procedure (pH 5.8 for 30 min; pH 5.0 for 30 min) was better than a single-phase procedure for enhancing the acid tolerance of this pathogen . The acid-adapted cells were cross-protected against the challenges of low salinity and thermal inactivation . One-dimensional polyacrylamide gel electrophoresis revealed that proteins with molecular masses of 6.4, 9.0, 13.6, 16.3, 18.9, 22.9, 24.4, 28.3, 33 . 9, 36.9, 41.2, 47.6, 58.1, 65.6, 80.5, 88.2, and 96.9 kDa were induced or significantly enhanced, while proteins of 25.3, 30.1, 30 . 7, and 91.7 kDa were significantly inhibited . Two-dimensional polyacrylamide gel electrophoresis revealed that 20 species of proteins were induced or significantly enhanced, while 26 species were inhibited . In assays conducted using the suckling mouse model, enteropathogenicity of the acid-adapted cells was significantly enhanced in terms of intestine/body weight ratio and in vivo recovery of infected cells.

Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7687 - 92
A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp . stewartii; von Bodman SB et al.; Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems . For Pantoea stewartii subsp . stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner . Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing . EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator . esaI, DeltaesaR, and DeltaesaI-esaR mutations were constructed to establish the regulatory role of EsaR . We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal . Our data indicate that quorum-sensing regulation in P . s . subsp . stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL . In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent . This finding suggests that quorum sensing in P . s . subsp . stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

Cell Tissue Res, 1998 Jul, 293(1), 133 - 41
Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy; Esteban MA et al.; In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed . Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry . Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied . Attached and interiorized bacteria were distinguished . Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min . The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes . Cytochalasin B or colchicine was used to inhibit phagocytosis . Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy . In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes . Our ultrastructural results demonstrate that V . anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.

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