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 m