Vibriosis

Indian J Med Res, 1997 Jun, 105, 254 - 7
Isolation of Vibrio cholerae 0139 phages to develop a phage typing scheme; Chakrabarti AK et al.; Five V . cholerae 0139 phages isolated from different parts of India have been used for phage typing study . A strain isolated from Nagpur city (NPR-4) was used as the host for phage propagation . All but one of the 260 strains of V . cholerae 0139 were found to be typeable and could be clustered into 8 distinct phage types as revealed by lytic patterns . Phage type 1 was the predominant type (61.15%) followed by type 2 (18.46%) . The strains isolated from Madras exhibited 7 out of 8 phage types . These newly isolated phages could be adopted for phage typing of V . cholerae 0139 strains as an epidemiological tool.

Glycoconj J, 1997 Jun, 14(4), 433 - 8
Synthesis of the dodecasaccharide fragment representing the O-polysaccharide of Vibrio cholerae O:1, serotype Ogawa, bearing an aglycon offering flexibility for chemical linking to proteins; Ogawa Y et al.; Two azidohexasaccharide building blocks, of which the glycosyl acceptor was the 5-(methoxycarbonyl)pentyl glycoside, were coupled using the trichloroacetimidate technology . The 12 azido functions present in the dodecasaccharide thus formed were then converted to amino groups using hydrogen sulfide as a reducing reagent . Subsequent N-acylation with 4-O-benzyl-L-glycero-tetronic acid, followed by catalytic debenzylation yielded the desired spacer-equipped, title dodecasaccharide.

Immunobiology, 1997 Jun, 197(1), 82 - 96
Leukosialin (CD43) is proteolytically cleaved from stimulated HMC-1 cells; Weber S et al.; Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA) . The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA . Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA . In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6) . PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation . The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane . Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43 . In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors . Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant . Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced . In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells . Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells.

Vet Immunol Immunopathol, 1997 Jun, 57(1-2), 147 - 52
Immunostimulating effect of growth hormone: in-vivo administration of growth hormone in rainbow trout enhances resistance to Vibrio anguillarum infection; Sakai M et al.; In-vivo administration of purified (nGH) and recombinant (rGH) chum salmon, Oncorhynchus keta, growth hormone enhanced the survival of rainbow trout, O . mykiss, against virulent Vibrio anguillarum . Macrophages obtained from rainbow trout that were treated in vivo with either GH preparation showed enhanced stimulation of the chemiluminescent responses induced by opsonized V . anguillarum cells and increased phagocytic activities . However, the serum bactericidal activity against V . anguillarum was not enhanced in fish injected with nGH or rGH . These findings show that in-vivo administration of either GH preparation can effectively prime macrophages and increase the resistance to V . anguillarum in rainbow trout.

Comp Biochem Physiol B Biochem Mol Biol, 1997 Jun, 117(2), 273 - 86
A heterogeneous sialic acid-binding lectin with affinity for bacterial LPS from horse mussel (Modiolus modiolus) hemolymph; Tunkijjanukij S et al.; A sialic acid-binding lectin that agglutinates a variety of erythrocytes and bacteria and react with sialoconjugates and purified lipopolysaccharides from marine vibrios has been affinity purified from hemolymph of the horse mussel Modiolus modiolus using Bovine submaxillary mucin conjugated to CNBr-activated Sepharose 4B . The lectin demonstrated heterogeneous activity, and at least two main entities were partially characterized, and are referred to as modiolin H and modiolin E activities for the agglutination of human and horse (equine) erythrocytes, respectively . Only modiolin E activity required calcium ions for hemagglutination . The M . modiolus lectin was mainly specific for NeuAc, although the lectin demonstrated a broader range of specificity, similarly to the Limulus polyphemus lectin . The purified lectin was a glycoprotein, and in the native state existed as aggregates with M(r) in the range of 100-1,300 kDa as observed by gradient-gel electrophoresis and gel filtration on Biogel and Superose . SDS-PAGE under reducing conditions revealed three subunits of M(r) 14, 17.5 and 20 kDa . Various marine bacteria adsorbed the hemagglutinating activities of the M . modiolus lectin . Purified LPS preparations from various pathogenic marine vibrios were also effective inhibitors, in particular for modiolin E activity . These results indicate that the lectin play a role in recognition of bacteria.

Mol Microbiol, 1997 Jun, 24(5), 917 - 26
Regulation, replication, and integration functions of the Vibrio cholerae CTXphi are encoded by region RS2; Waldor MK et al.; CTXphi is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae . CTXphi is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V . cholerae chromosome at a specific site . The CTXphi genome has two regions, the 'core' and RS2 . Integrated CTXphi is frequently flanked by an element known as RS1 which is related to RS2 . The nucleotide sequences of RS2 and RS1 were determined . These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2 . RS1 contains an additional ORF designated rstC . Functional analyses indicate that rstA2 is required for CTXphi replication and rstB2 is required for CTXphi integration . The amino terminus of RstR is similar to the amino termini of other phage-encoded repressors, and RstR represses the expression of rstA2 . Although genes with related functions are clustered in the genome of CTXphi in a way similar to those for other filamentous phages, the CTXphi RS2-encoded gene products mediating replication, integration and repression appear to be novel.

Epidemiol Infect, 1997 Jun, 118(3), 221 - 5
Vibrio vulnificus infection reporting on death certificates: the invisible impact of an often fatal infection; Banatvala N et al.; This study assessed accuracy of (a) recording Vibrio vulnificus infection on death certificates and (b) International Classification of Disease (ICD)-9 codes for V . vulnificus . Patients with microbiologically confirmed V . vulnificus infection were identified as part of co-ordinated surveillance in four USA Gulf Coast states between 1989 and 1993 . Of 60 deaths, 51 death certificates were reviewed and V . vulnificus was recorded as the immediate cause of death on 11 (22%) . There was no ICD-9 code for V . vulnificus infection, thus no patients had an ICD-9 code indicating V . vulnificus infection . Of 23 certificates where V . vulnificus was recorded on the death certificate, only 5 (22%) were coded for Gram-negative, septicaemia . This study highlights the importance of teaching physicians how to provide epidemiologically meaningful data on death certificates and the need for accurate ICD mortality codes.

Epidemiol Infect, 1997 Jun, 118(3), 207 - 14
Epidemic cholera among refugees in Malawi, Africa: treatment and transmission; Swerdlow DL et al.; Between 23 August and 15 December 1990 an epidemic of cholera affected Mozambican refugees in Malawi causing 1931 cases (attack rate = 2.4%); 86% of patients had arrived in Malawi < 3 months before illness onset . There were 68 deaths (case-fatality rate = 3.5%); most deaths (63%) occurred within 24 h of hospital admission which may have indicated delayed presentation to health facilities and inadequate early rehydration . Mortality was higher in children < 4 years old and febrile deaths may have been associated with prolonged i.v . use . Significant risk factors for illness (P < 0.05) in two case-control studies included drinking river water (odds ratio {OR} = 3.0); placing hands into stored household drinking water (OR = 6.0); and among those without adequate firewood to reheat food, eating leftover cooked peas (OR = 8.0) . Toxigenic V . cholerae O1, serotype Inaba, was isolated from patients and stored household water . The rapidity with which newly arrived refugees became infected precluded effective use of a cholera vaccine to prevent cases unless vaccination had occurred immediately upon camp arrival . Improved access to treatment and care of paediatric patients, and increased use of oral rehydration therapy, could decrease mortality . Preventing future cholera outbreaks in Africa will depend on interrupting both waterborne and foodborne transmission of this pathogenPIP: An epidemiologic investigation of a 1990 cholera outbreak among Mozambican refugees in the Nyamithuthu camp in Malawi highlighted the challenges of providing adequate treatment and prevention in this setting . Between August 23 and December 15, 1990, 1931 cholera patients were admitted to the camp's intravenous (IV) treatment tent (attack rate, 2.4%); 28% were under 6 years of age . There were 68 deaths among these patients, for a case-fatality rate of 3.5% . 84% of patients for whom data were available had come to Malawi less than 3 months before the onset of illness and 52% were admitted for treatment within 16 days of camp arrival . 60% of the 40 cholera deaths investigated in detail involved children under 4 years of age (17% of total cases) . Acute dehydration was the most common cause of death among the 63% who died within 24 hours of IV tent admission, suggesting delayed presentation and inadequate early rehydration . The remaining patients died from complications (e.g., infections with fever caused by prolonged IV use) . In 2 case-control studies, cholera was significantly associated with placing hands into the storage container holding household drinking water (odds ratio, 6.0), obtaining drinking water from the river (odds ratio, 3.0), and eating leftover unheated cooked peas (odds ratio, 8.0) . Toxigenic Vibrio cholerae O1, serotype Inaba, was isolated from patients and stored household water . Increased water rations and running water during cholera outbreaks are recommended to reduce contamination of stored drinking water during washing . More rapid referral to IV tents, administration of oral rehydration solution in addition to IV, quick removal or replacement of IV lines to prevent infection, and more attention to child cases also would reduce cholera mortality .

Microbiology, 1997 Jun, 143 ( Pt 6), 1805 - 13
VlpA of Vibrio cholerae O1: the first bacterial member of the alpha 2-microglobulin lipocalin superfamily; Barker A et al.; We have identified a gene, vlpA, which is closely linked to the mfrA,B locus associated with mannose-fucose-resistant haemagglutination . VlpA is an outer-membrane protein which can be labelled with {3H}palmitate and whose processing is globomycin-sensitive, suggesting that it is a lipoprotein . Homology searches revealed that VlpA belongs to the group of lipocalins of the alpha 2-microglobulin superfamily which function as small hydrophobic molecule transporters, and is the first identified bacterial member of this group . Multiple copies of this gene are present in Vibrio cholerae O1 and O139 and Southern hybridization reveals a biotype-specific pattern of fragment sizes . Construction of strains capable of hyperproducing VlpA suggested that it is able to bind haemin with low affinity but this may be due to a simple hydrophobic interaction . Attempts to construct specific mutants in vlpA have been unsuccessful, presumably because of the multiple copies of vlpA genes and their linkage to the VCR element.

J Bacteriol, 1997 Jun, 179(12), 4043 - 5
Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi; Bassler BL et al.; Different species of bacteria were tested for production of extracellular autoinducer-like activities that could stimulate the expression of the luminescence genes in Vibrio harveyi . Several species of bacteria, including the pathogens Vibrio cholerae and Vibrio parahaemolyticus, were found to produce such activities . Possible physiological roles for the two V . harveyi detection-response systems and their joint regulation are discussed.

Appl Environ Microbiol, 1997 Jun, 63(6), 2464 - 7
Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats; DePaola A et al.; Phages lytic to Vibrio vulnificus were found in estuarine waters, sediments, plankton, crustacea, molluscan shellfish, and the intestines of finfish of the U.S . Gulf Coast, but no apparent relationship between densities of V . vulnificus and its phages was observed . Phage diversity and abundance in molluscan shellfish were much greater than in other habitats . V . vulnificus phages isolated from oysters did not lyse other mesophilic bacteria also isolated from oysters . Both V . vulnificus and its phages were found in a variety of oyster tissues and fluids with lowest densities in the hemolymph and mantle fluid . These findings suggest a close ecological relationship between V . vulnificus phages and molluscan shellfish.

Appl Environ Microbiol, 1997 Jun, 63(6), 2200 - 5
Photoreactivation compensates for UV damage and restores infectivity to natural marine virus communities; Weinbauer MG et al.; We investigated the potential for photoreactivation to restore infectivity to sunlight-damaged natural viral communities in offshore (chlorophyll a, < 0.1 microgram liter-1), coastal (chlorophyll a, ca . 0.2 microgram liter-1), and estuarine (chlorophyll a, ca . 1 to 5 micrograms liter-1) waters of the Gulf of Mexico . In 67% of samples, the light-dependent repair mechanisms of the bacterium Vibrio natriegens restored infectivity to natural viral communities which could not be repaired by light-independent mechanisms . Similarly, exposure of sunlight-damaged natural viral communities to > 312-nm-wavelength sunlight in the presence of the natural bacterial communities restored infectivity to 21 to 26% of sunlight-damaged viruses in oceanic waters and 41 to 52% of the damaged viruses in coastal and estuarine waters . Wavelengths between 370 and 550 nm were responsible for restoring infectivity to the damaged viruses . These results indicate that light-dependent repair, probably photoreactivation, compensated for a large fraction of sunlight-induced DNA damage in natural viral communities and is potentially essential for the maintenance of high concentrations of viruses in surface waters.

J Bacteriol, 1997 Jun, 179(11), 3808 - 12
Identification of a second endogenous Porphyromonas gingivalis insertion element; Wang CY et al.; In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P . gingivalis was identified . Nucleotide sequence analysis of the Tn4351 insertion site in a P . gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351 . PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence . Results of Southern hybridization of chromosomal DNA isolated from several P . gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P . gingivalis strains . Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12.

J Bacteriol, 1997 Jun, 179(11), 3594 - 603
The plasmid R64 thin pilus identified as a type IV pilus; Kim SR et al.; The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined . Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus . Protein products of eight pil genes were identified by the maxicell procedure . The pilN product was shown to be a lipoprotein by an experiment using globomycin . A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence . The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase . These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili . The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations . Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.

Infect Immun, 1997 Jun, 65(6), 2475 - 9
The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels; Amaro C et al.; Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans . Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E) . In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 strains (of serogroup E) are resistant to NIS, and (iii) rough mutants of biotype 2 lacking the O polysaccharide side chain are sensitive to NIS and avirulent for eels.

Infect Immun, 1997 Jun, 65(6), 2127 - 35
Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibrio cholerae vaccine strains; Butterton JR et al.; A promoterless gene for the Shiga toxin 1 B subunit (stxB1) has been placed under transcriptional control of the Vibrio cholerae heat shock gene htpG . A chromosomal enterohemorrhagic Escherichia coli fragment containing eaeA and 400 bp of upstream DNA was added to the construct, downstream of stxB1; no transcription terminators were located between the two genes . The plasmid construct was confirmed by DNA sequencing; in vitro transcription-translation studies demonstrated expression of EaeA from the plasmid . The htpGp-->stxB1, eaeA construct was inserted into lacZ on the chromosome of Peru2, an El Tor V . cholerae strain with both attRS1 sequences and the entire cholera toxin genetic element deleted, and into lacZ in JRB10, a Peru2 derivative that has a second copy of htpGp-->stxB1 also inserted in the V . cholerae virulence gene irgA . Two plasmid constructs, one containing stxB1 under the control of the tac promoter and another containing htpGp-->stxB1,eaeA, were transformed into Peru2 . Expression of StxB1 by these constructs was quantified by enzyme-linked immunosorbent assay and was highest in the plasmid construct with stxB1 under the control of the tac promoter . Localization of EaeA to the outer membrane of the vector strains was demonstrated both by Western blotting and by immunofluorescence with an anti-EaeA antibody . A rabbit model for colonization by V . cholerae was used to compare the immune responses to the two heterologous antigens, StxB1 and EaeA, expressed by these strains . Rabbits immunized with Peru2 transformed with a plasmid carrying tac-->stxB1 developed neutralizing serum anti-StxB1 immunoglobulin G antibody responses . One of two rabbits immunized with a strain carrying a chromosomal copy of eaeA developed a marked immune response against EaeA . The plasmid construct containing htpGp-->stxB1,eaeA was unstable, producing low levels of StxB1 in vitro and not evoking anti-EaeA antibody responses in vivo following oral immunization . Chromosomal insertion of eaeA may be preferred for future expression of this antigen in V . cholerae vaccine constructs.

Infect Immun, 1997 Jun, 65(6), 2107 - 11
Comparison of alternative buffers for use with a new live oral cholera vaccine, Peru-15, in outpatient volunteers; Sack DA et al.; During development of Peru-15, a new live oral vaccine for cholera, the role of buffer needed to be evaluated . Generally, oral bacterial vaccines are acid labile and need to be administered by using a formulation which protects them from gastric acid . We compared three different buffers for use with Peru-15, including a standard bicarbonate-ascorbic acid buffer, Alka-Seltzer, and a new electrolyte-rice buffer, CeraVacx . Saline served as the control . Thirty-nine healthy adult volunteers received Peru-15 (10(8) CFU) with one of the three buffers or saline in a double-masked study . The volunteers were monitored for symptoms for 7 days after the dose, serum was tested for antibody responses by vibriocidal antibody and immunoglobulin G antitoxin enzyme-linked immunosorbent assays, and stool samples were tested for excretion of the vaccine strain . Side effects were minimal in all groups . All 30 volunteers who took Peru-15 with a buffer showed significant rises in vibriocidal antibody titer . The magnitude of the rises was higher in the CeraVacx group than in the other two buffer groups . Four of nine volunteers who took the vaccine with saline also showed increased titers, but they were lower than those in any of the three buffer groups . Excretion of the vaccine strain was similar in the buffer groups, but excretion was not associated with the magnitude of the vibriocidal responses . Excretion of Peru-15 was not detected in the saline group . We conclude that buffer does amplify the serological response to Peru-15 and that CeraVacx may provide benefits not provided by other buffers.

J Clin Microbiol, 1997 Jun, 35(6), 1633 - 5
Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR; Albert MJ et al.; In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V . cholerae O139 Bengal . PCR with the same primer pair was used to screen 180 diarrheal stool specimens . All the 67 V . cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR.

Biochim Biophys Acta, 1997 May 24, 1360(3), 277 - 82
A mutant cell line resistant to Vibrio parahaemolyticus thermostable direct hemolysin (TDH): its potential in identification of putative receptor for TDH; Tang G et al.; Thermostable direct hemolysin (TDH), a pore-forming toxin produced by Vibrio parahaemolyticus, is cytotoxic to Rat-1, a fibroblast cell line derived from rat embryo . Through mutagenesis of Rat-1 with nitrosoguanidine, we established a mutant cell line, MR-T1 . MR-T1 was over 200 times more resistant to the cytotoxic activity of TDH than Rat-1 . TDH increased membrane permeability of Rat-1 but not of MR-T1 . Binding analysis showed that, while being able to bind to Rat-1 . TDH failed to bind to MR-T1, indicating that MR-T1 is deficient in the putative receptor for TDH . Somatic hybrid cells between Rat-1 and MR-T1 were similarly sensitive to TDH as Rat-1 . Moreover, TDH could bind to the hybrid cells as well as to Rat-1 cells . These results indicate that MR-T1 is promising for complementation cloning of a gene related to the putative receptor for TDH.

Biochim Biophys Acta, 1997 May 23, 1339(2), 247 - 52
Structure of membrane glutamate carboxypeptidase; Rawlings ND et al.; Membrane glutamate carboxypeptidase (mGCP) hydrolyses pteroylpoly-gamma-glutamates, methotrexate tri-gamma-glutamate and N-acetyl-aspartyl-alpha-glutamate . The enzyme is thought to be required for intestinal uptake of folate, for the resistance of some tumours to methotrexate, and for the metabolism of N-acetyl-aspartyl-glutamate, an abundant neuropeptide . It has recently been reported that mGCP is a protein also known as prostate-specific membrane antigen, homologous with transferrin receptor . This allows us to predict the domain structure of mGCP . Moreover, we have been able to assign the catalytic domain of mGCP to peptidase family M28, which contains cocatalytic zinc metallopeptidases . On the basis of the known structure of an aminopeptidase in family M28, we predict that Asp377, Asp387, Glu425, Asp453 and His553 are ligands of two atoms of zinc bound in the catalytic site of mGCP, and suggest that the aminopeptidases of Vibrio and Streptomyces can serve as valuable models in the design of inhibitors for this medically important enzyme.

Carbohydr Res, 1997 May 19, 300(4), 329 - 39
Synthesis of methyl alpha-glycosides of some higher oligosaccharide fragments of the O-antigen of Vibrio cholerae O1, serotype Inaba and Ogawa; Zhang J et al.; The title oligosaccharides, the tri- through the hexasaccharide in the Inaba series and the penta- and the hexasaccharide in the Ogawa series, have been synthesized using 1-thioglycosides of precursors to 3-O-benzyl-perosamine (4-amino-4,6-dideoxy-D-mannose) as building blocks and N-iodosuccinimide/silver triflate as a promoter . The azido groups in the assembled oligosaccharides were reduced to amino groups, which were then acylated using 2,4-O-benzylidene-3-deoxy-L-glycero-tetronic acid as the derivatizing reagent . Catalytic hydrogenolysis, simultaneously of the benzyl and benzylidene groups, gave the desired products that were characterized by 1H and 13C NMR spectroscopy.

FEMS Microbiol Lett, 1997 May 15, 150(2), 289 - 96
Analysis of functional domains of Vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies; Tang G et al.; Neutralizing monoclonal antibodies (mAbs) against Vibrio parahaemolyticus thermostable direct hemolysin (TDH) were used in probing the functional domains of this toxin . While pre-incubation of TDH with mAb 2A-13C inhibited further binding of TDH to erythrocytes, pre-incubation with another mAb 1-24 did not, indicating that mAb 1-24 epitope resides in a domain which is not involved in binding of TDH to erythrocytes . On the other hand after binding to erythrocytes, TDH could react with mAb 1-24 but poorly with mAb 2A-13C, indicating that the mAb 2A-13C epitope is masked, possibly by erythrocyte surface . As both antibodies are TDH-specific and do not react with TRH (TDH-related hemolysin), we used TDH/ TRH chimeric proteins to identify location of the epitopes for mAbs by inhibition ELISA as well as Western blotting . The results showed that the mAb 1-24 epitope resides on a region near the C-terminal of TDH (residues 99-139), while the mAb 2A-13C epitope resides on the N-terminal (residues 1-31) . All these results suggested that, in TDH, the N-terminal region may be involved in binding process while the region near C-terminal may be involved in postbinding process.

FEMS Microbiol Lett, 1997 May 15, 150(2), 249 - 54
El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes; Ikigai H et al.; We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers . The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent . The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation . The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes . These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis.

Kansenshogaku Zasshi, 1997 May, 71(5), 417 - 20
{Incidence of Kanagawa phenomenon-positive and -negative Vibrio parahaemolyticus strains isolated from traveller's diarrhea and their relation to tdh and trh genes}; Suzuki N et al.; A total of 1,319 strains of Vibrio parahaemolyticus isolated from traveller's diarrhea were analysed for Kanagawa phenomenon (KP) with the Wagatsuma blood agar test and the results were also compared with those of analyses of tdh and trh genes which encode thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) . The majority of the strains (1,152 strains) counting 87.3% had positive KP, among which 1,049 and 103 strains were only tdh and both tdh and trh-positive ones, respectively . However, 167 strains counting 12.7%, which is quite high compared to the previous report, were found to have negative KP, among which 94 and 24 strains were only trh and both tdh and trh-positive ones, respectively.

Am J Trop Med Hyg, 1997 May, 56(5), 533 - 7
Volunteer studies investigating the safety and efficacy of live oral El Tor Vibrio cholerae O1 vaccine strain CVD 111; Tacket CO et al.; A live oral cholera vaccine should ideally protect against both classical and El Tor biotypes of Vibrio cholerae O1 . An El Tor biotype vaccine strain, therefore, would complement classical cholera vaccine strain CVD 103-HgR, a strain already in use in some countries . In this study, 25 healthy adult volunteers received a single dose of 10s colony-forming units of El Tor vaccine strain CVD 111, a derivative of El Tor Ogawa strain N16117 deleted in the virulence cassette . Three (12%) volunteers developed mild diarrhea (mean stool volume = 813 ml) but no systemic symptoms; 23 (92%) of the 25 volunteers developed serum vibriocidal antibodies (geometric mean titer = 1:2,291) . Five weeks after vaccination, 18 vaccines and eight uninimunized control volunteers underwent wild-type challenge with El Tor Ogawa strain 3008 . Three (16.7%) of 18 vaccinees and seven (87.5%) of eight controls developed diarrhea (P = 0.001) (vaccine efficacy = 80.9%) . Further studies are underway to determine a dosage of CVD 111 that will be more clinically acceptable but equally immunogenic and protective.

Eur J Cell Biol, 1997 May, 73(1), 1 - 9
Effects of cell surface ganglioside sialidase inhibition on growth control and differentiation of human neuroblastoma cells; Kopitz J et al.; Gangliosides on the external side of the plasma membrane are important modulators of cellular functions . In previous work we had found that in cultured human SK-N-MC neuroblastoma cells a cell surface sialidase activity specifically cleaved terminal sialic acids from gangliosides, leading to a shift from higher sialylated species to GM1 and a decrease of GM3 . To further elucidate the function of the enzyme, we have now examined the consequences of ganglioside sialidase inhibition . When present in the culture medium, the ganglioside sialidase inhibitors 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), heparin, and heparan sulfate caused dramatic changes in cell behavior . Thus, the inhibitors uniformly led to a complete release from contact inhibition of growth, and to the loss of the differentiation markers neuron-specific enolase and neurofilaments, and a decrease of cyclic AMP . In presence of NeuAc2en, cells that normally were spread out evenly and were firmly attached, appeared smaller, rounded, and only loosely adherent to the culture vessel . Exogenous addition of vibrio cholerae sialidase mimicked the action of the plasma membrane ganglioside sialidase by retarding cell proliferation and increasing intracellular acetylcholinesterase . That the ganglioside sialidase inhibitors in the culture medium indeed affected solely the cell surface enzyme and not also a lysosomal sialidase, was demonstrated in an experiment where the desialylation of exogenously added radioactive gangliosides was determined in absence and presence of NeuAc2en and NH4Cl, an inhibitor of lysosomal function . Taken together, our results suggest that the ganglioside sialidase on the surface of SK-N-MC cells is responsible for growth control and differentiation in this neuronal cell line.

South Med J, 1997 May, 90(5), 544 - 5
Vibrio fluvialis: an underrecognized enteric pathogen in infants?
Kolb EA, Eppes SC, Klein JD.
We report a case of Vibrio fluvialis gastroenteritis in an infants 3 1/2 weeks old . The case was unusual because no likely epidemiologic risk factors were involved . Since several other such cases in young infants have been reported, V fluvialis should be considered in the differential diagnosis of infantile gastroenteritis.

J Med Microbiol, 1997 May, 46(5), 398 - 402
Detection of Vibrio cholerae and V . mimicus heat-stable toxin gene sequence by PCR; Vicente AC et al.; Previously the heat-stable enterotoxin in Vibrio cholerae and V . mimicus has been detected by suckling mouse assay, a non-specific approach, and by DNA probes, a time-consuming method . This report describes a polymerase chain reaction (PCR) procedure for the detection of the stn (NAG-ST) and sto (O1-ST) gene sequences that is rapid and specific, allowing toxin gene molecular characterisation . A total of 34 V . cholerae and V . mimicus isolates was examined for ST and CT genes . The NAG-ST gene sequence was amplified in 13 of 22 non-O1/non-O139 V . cholerae and three of five V . mimicus strains . A new enterotoxin gene sequence pattern was found with MseI and TaqI restriction endonuclease PCR fragment digestion of two V . cholerae isolates, in addition to the pattern anticipated from the Genbank sequence, and found with the other ST+ . These results show that ST-PCR detection is useful for the characterisation of V . cholerae and V . mimicus.

J Bacteriol, 1997 May, 179(10), 3116 - 21
The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity; Brumlik MJ et al.; Vibrio and Aeromonas spp . secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase . The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge . Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding . However, when either of the cysteines is replaced, the enzyme is more readily denatured by urea and more sensitive to degradation by trypsin than is the wild-type enzyme, evidence that the bridge has an important role in stabilizing the protein's structure . The two mutant proteins with serine-for-cysteine replacements were secreted by Aeromonas salmonicida containing the cloned genes, although the levels of both in the culture supernatants were lower than the level of the wild-type enzyme . When the general secretory pathway was blocked with carbonyl cyanide chlorophenylhydrazone, the cell-associated pools of the mutant enzymes appeared to be degraded, whereas the wild-type pool remained stable . We conclude that reduced extracellular levels of the mutant proteins are the result of their increased sensitivities to proteases encountered inside the cell during export.

Appl Environ Microbiol, 1997 May, 63(5), 1674 - 8
Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus; Hoi L et al.; A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer . The RAPD-PCR results were scanned, and the images were analyzed with a computer program . Ribotype membranes were evaluated visually . Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous . Ribotyping enabled us to differentiate U.S . and Danish strains and V . vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V . vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes . Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V . vulnificus biotype 2 is an opportunistic pathogen for humans . One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains . This is, to our knowledge, the first time the isolation of V . vulnificus biotype 2 from coastal waters has been described.

J Bacteriol, 1997 May, 179(9), 3004 - 12
Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone; Milton DL et al.; Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers . This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators . Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout . In this study, we sought to determine whether V . anguillarum employs AHLs to regulate virulence gene expression . Spent V . anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum . This finding suggested that V . anguillarum may produce multiple AHL signal molecules . Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V . anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis . The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases . Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators . Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed . However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E . coli and C . violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.

J Infect Dis, 1997 May, 175(5), 1134 - 41
Molecular characterization of Vibrio cholerae O1 biotype El Tor strains isolated between 1992 and 1995 in Calcutta, India: evidence for the emergence of a new clone of the El Tor biotype; Sharma C et al.; Sixty-one clinical strains of Vibrio cholerae O1 El Tor isolated in Calcutta before, during, and after the V . cholerae O139 Bengal outbreak were examined to see if the O1 strains of the post-O139 period were different from those in existence before . Comparison of the restriction fragment length polymorphism of the rRNA genes (ribotyping) and the CTX genetic element revealed that all "before" strains except 1 belonged to a single known ribotype, whereas all "after" strains except 2 belonged to a hitherto undescribed ribotype . Also, 23 of 25 "before" strains harbored two or more copies of CTX in tandem and also a "free" RS1 element away from CTX, whereas 19 of 21 "after" strains had a single copy of CTX and no free RS1 element . CTX occupied different chromosomal locations in "before" and "after" strains . These studies clearly showed that El Tor O1 strains, which displaced V . cholerae O139 in Calcutta, belonged to a new clone and suggested that there is a continuous genetic reassortment among El Tor strains of V . cholerae O1.

Infect Immun, 1997 May, 65(5), 1830 - 5
Purification and characterization of a hemolysin produced by Vibrio mimicus; Miyoshi S et al.; Vibrio mimicus is a causative agent of human gastroenteritis . This pathogen secretes a pore-forming toxin, V . mimicus hemolysin (VMH), which causes hemolysis by three sequential steps: binding to an erythrocyte membrane, formation of a transmembrane pore, and disruption of the cell membrane . VMH with a molecular mass of 63 kDa was purified by ammonium sulfate precipitation and column chromatography with phenyl Sepharose HP and Superose 6 HR . The hemolytic reaction induced by VMH continued up to disruption of all erythrocytes in the assay system . Moreover, VMH that bound preliminarily to erythrocyte ghosts showed a sufficient ability to attack intact erythrocytes . These results suggest reversible binding of the toxin molecule to the membrane . The final cell-disrupting stage was effectively inhibited by various divalent cations . Additionally, some cations, such as Zn2+ and Cu2+, blocked the pore-forming stage at high concentrations . Although VMH could disrupt all kinds of mammalian erythrocytes tested, those from horses were most sensitive to the hemolysin . Horse erythrocytes were found to have the most toxin-binding sites and to be hemolyzed by the least amount of membrane-bound toxin molecules, suggesting that toxin binding to and pore formation on erythrocytes are more effective in horses than in other mammals . Purified VMH induced fluid accumulation in a ligated rabbit ileal loop in a dose-dependent manner . In addition, the antibody against the hemolysin obviously reduced enteropathogenicity of living V . mimicus cells . These findings clearly demonstrate that VMH is probably involved in the virulence of this human pathogen.

J Clin Microbiol, 1997 May, 35(5), 1260 - 2
Food-borne disease outbreaks due to bacteria in Taiwan, 1986 to 1995; Pan TM et al.; Between 1986 and 1995, 852 outbreaks of food-borne disease involving 26,173 cases and 20 deaths were reported in Taiwan . About 80% of the outbreaks occurred in the warmer months, i.e., between April and October . Of the 852 reported outbreaks, 555 (65%) were caused by bacterial pathogens . The three most common bacteria involved were Vibrio parahaemolyticus (35%, 197 of 555 outbreaks), Staphylococcus aureus (30%, 169 of 555 outbreaks), and Bacillus cereus (18%, 104 of 555 outbreaks).

J Clin Microbiol, 1997 May, 35(5), 1151 - 6
Molecular evolution of Vibrio cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995; Dalsgaard A et al.; Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized . Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V . cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically . While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments . All strains were susceptible to nine antibacterial agents studied . In 1991, more than 95% of the clinical V . cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995 . The small differences in genotypes of V . cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source . However, the relative high rate of genetic changes within V . cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V . cholerae O1 associated with cholera in Latin America are evaluated.

Curr Microbiol, 1997 May, 34(5), 314 - 7
Urea hydrolysis and suppressed production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus associated with presence of TDH-related hemolysin genes; Okitsu T et al.; A total of 18 strains of V . parahaemolyticus isolated from patients of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were assayed for presence of the thermostable direct hemolysin (TDH) gene and the TDH-related hemolysin (TRH) genes (trh 1 and trh 2) with specific reference to their ability to hydrolyze urea and TDH production . A polymerase chain reaction assay revealed that all urea-hydrolyzing strains (9 strains) carried either trh 1 gene or trh 2 gene . The strains carrying the trh genes as well as the tdh gene produced TDH less by a factor of 4 to 16 than those carrying only the tdh gene, suggesting the expression of the tdh gene was suppressed by the presence of trh gene through a mechanism yet to be defined.

J Mol Biol, 1997 Apr 25, 268(1), 137 - 46
Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae; Hu SH et al.; The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme . In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors . Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pili and they no longer secrete cholera toxin . TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera . The crystal structure of oxidized TcpG (refined at a resolution of 2.1 A) serves as a starting point for the rational design of such inhibitors . As expected, TcpG has the same fold as E . coli DsbA, with which it shares approximately 40% sequence identity . In addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role . While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences . For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect) . Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion . Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E . coli DsbA . The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors.

Gene, 1997 Apr 21, 189(2), 203 - 7
The rpoH gene encoding sigma 32 homolog of Vibrio cholerae; Sahu GK et al.; The Vibrio cholerae rpoH gene coding for the heat-shock sigma factor, sigma 32, has been cloned and shown to functionally complement Escherichia coli rpoH mutants . The nt sequence of the gene has been determined and the deduced aa sequence is more than 80% homologous to the E . coli rpoH gene product . Downstream of the V . cholerae rpoH gene, an unidentified dehydrogenase gene (udhA) is present on the opposite strand facing rpoH . The predicted secondary structure of the 5'-proximal region of V . cholerae rpoH mRNA is apparently different from the conserved secondary structures of the rpoH mRNA reported for several bacterial species . The 'RpoH box', a stretch of 9 aa (QRKLFFNLR) unique to sigma 32 factors, and the 'downstream box' sequence complementary to a part of the 16S rRNA, have been detected.

Gene, 1997 Apr 21, 189(2), 163 - 8
Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus; Chuang YC et al.; A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced . When the empV gene was expressed in minicells, a unique peptide of approx . 46 kDa was identified . Protease activity staining experiments also indicated a similar M(r) for the protease . The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it . The crude enzyme prepared from the periplasmic fraction of recombinant E . coli was inhibited by a metalloprotease inhibitor and Zn2+ is essential for its protease activity . Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long 'pro' sequence consisting of 172 aa . The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V . vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference . The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V . vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV . The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining . The deduced aa sequence of EmpV showed high homology to V . anguillarum metalloprotease (EmpA), V . cholerae HA/protease (HprC), and V proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues.

Biochim Biophys Acta, 1997 Apr 12, 1360(2), 102 - 4
Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio mimicus; Kim GT et al.; The structural gene (vmhA) of hemolysin from Vibrio mimicus (ATCC33653) was cloned and sequenced . The vmhA gene contains an open reading frame consisting of 2232 nucleotides which can code for a protein of 744 amino acids with a predicted molecular mass of 83,059 . The similarity of amino acid sequence shows 81.6% identity with Vibrio cholerae El Tor hemolysin.

Lancet, 1997 Apr 5, 349(9057), 981 - 5
Epidemic cholera in Burundi: patterns of transmission in the Great Rift Valley Lake region; Birmingham ME et al.; BACKGROUND: After a 14-year hiatus, epidemic cholera swept through Burundi between January and May, 1992 . The pattern of transmission was similar to that in 1978, when the seventh pandemic first reached this region . Communities affected were limited to those near Lake Tanganyika and the Rusizi River . The river connects Lake Tanganyika with Lake Kivu to the north in Zaire and Rwanda . METHODS: To identify sources of infection and risk factors for illness, an epidemiological study was carried out in Rumonge, a lake-shore town where 318 people were admitted to hospital with cholera between April 9 and May 31, 1992 . The investigation included a case-control study of 56 case-patients and 112 matched controls . FINDINGS: Attack rates according to street increased with the street's proximity to Lake Tanganyika (chi 2 test for linear trend, p < 0.01) which suggests that exposure to the lake was a risk factor for illness . Comparison of the 56 case-patients with matched controls showed that bathing in the lake (odds ratio 1.6, attributable risk percentage 37%) and drinking its water (2.78, 14%) were independently and significantly (p < 0.05) linked with illness . No food-borne risk factors were identified . Vibrio cholera 01 was isolated from Lake Tanganyika during, but not after, the outbreak in Rumonge . Isolates from the lake and from patients with acute watery diarrhoea had the same serotype, biotype, and antimicrobial susceptibility profiles . The number of cases rapidly declined when access to the lake was blocked . INTERPRETATION: This study identifies bathing in contaminated surface water as a major risk factor for cholera in sub-Saharan Africa, and suggests that improving the quality of drinking water alone will have only limited impact on the transmission of the disease in the Great Rift Valley Lake region . The similarity in the patterns of transmission during the 1978 and 1992 epidemics suggests that extensive use of the Great Lakes and connecting rivers for transportation and domestic purposes may be the reason for the explosive cholera outbreaks that occur sporadically in this region.

Rev Environ Health, 1997 Apr-Jun, 12(2), 63 - 80
Pathogenic vibrios in the natural aquatic environment; Chakraborty S et al.; In recent years, members belonging to the genus Vibrio of the family Vibrionaceae have acquired increasing importance because of the association of several of its members with human disease . The most feared of the Vibrio species is Vibrio cholerae, the causative agent of cholera, a devastating disease of global significance . Other important vibrios of medical importance are V . parahemolyticus, V . vulnificus, V . mimicus, and to a lesser extent V . fluvialis, V . furnissii, V . hollisae, and V . damsela . Recent studies have also implicated V . alginolyticus and V . metschnikovii in human disease, although their complete significance has not yet been established . The virulence of all medically important vibrios is aided by a variety of traits that help breach human defenses . In this review, we provide an overview of the environmental distribution of the pathogenic vibrios and the important virulence traits that enable them to cause disease.

Genes Genet Syst, 1997 Apr, 72(2), 115 - 8
Molecular cloning and functional analysis of an rpoS homologue gene from Vibrio cholerae N86; Hiratsu K et al.; A homologue of the rpoS gene of Escherichia coli was cloned from Vibrio cholerae N86 by complementation of the phenotypes of the E . coli rpoS mutant strain . We determined the DNA sequence of this gene . Sequence alignments have indicated that the rpoS gene of V . cholerae N86 encoding a 39-kDa protein is very similar to that of E . coli . In addition, the nlpD-like gene was found in the upstream region of the rpoS gene in the same order as in E . coli . These results suggest that the organization of these genes is highly conserved between E . coli and V . cholerae.

J Clin Microbiol, 1997 Apr, 35(4), 951 - 3
Rapid screening method for identification of cholera toxin-producing Vibrio cholerae O1 and O139; Osawa R et al.; A novel method of identifying cholera enterotoxin (CT)-producing Vibrio cholerae serogroups O1 and O139 was developed . The method uses degradation of NAD as a specific biochemical marker for the CT-producing strains . The substrate NAD at a concentration of 100 mumol/liter was markedly degraded when it was incubated at 37 degrees C for 2 h with the CT-producing stains at a final cell density equivalent to that of a twofold dilution of a McFarland no . 1 standard . NAD degradation was monitored by an enzyme-amplified color development assay . Subsequent tests conducted with a total of 119 strains of V . cholerae, including both clinical and environmental isolates, confirmed a significant correlation between NAD degradation and CT production for all V . cholerae strains belonging to serogroups O1 and O139 . Since 2 of 11 non-O1, non-O139 V . cholerae strains not carrying the CT gene degraded NAD, serotyping of the strains prior to the test is recommended.

J Vet Med Sci, 1997 Apr, 59(4), 277 - 9
Selective survival of a thermostable direct hemolysin-producing Vibrio parahaemolyticus in the alimentary tract of a juvenile estuarine gastropod (Clithon retropictus); Kumazawa NH et al.; Juvenile estuarine gastropods (Clithon retropictus), maintained in ultraviolet ray-irradiated recirculating artificial seawater with a salinity of 20/1000 at 28 degrees C, preserved thermostable direct hemolysin (TDH)-producing strain D-3 of Vibrio parahaemolyticus at a level of 10(4)-10(5) colony forming units per gram (cfu/g) and TDH-non-producing strains N-18 and R-13 at a level of 10(1)-10(2) cfu/g in the alimentary tract for at least 21 days after ingestion . In adults, the numbers of the three strains decreased to a level of 10(0) cfu/g within 21 days under the same conditions . This evidence supports our recent observations that TDH-producing strains increased to a high level in the summer months in the presence of high levels of TDH-non-producing strains in the alimentary tract of juvenile C . retropictus at estuaries in Japan.

Indian J Med Res, 1997 Apr, 105, 153 - 4
Resistance of Vibrio cholerae 01 to nalidixic acid; Jesudason MV et al.; Until 1987 all isolates of V . cholerae 01 at a tertiary care hospital in south India were susceptible to drugs commonly used to treat gastroenteritis including cholera . Since July 1987 strains resistant to co-trimoxazole have been encountered and since October 1995 strains resistant to nalidixic acid are being isolated . In this study the latter strains were examined by determining minimum inhibitory concentration levels of nalidixic acid as well as norfloxacin, the fluoroquinolone extensively used to treat diarrhoea . No cross resistance to norfloxacin was found in any of the nalidixic acid resistant V . cholerae 01 strains.

Hybridoma, 1997 Apr, 16(2), 195 - 9
Detection of two isotypically different antibodies produced by a murine hybridoma; Chen D et al.; A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized . This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells . The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains . Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality . Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted . PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa . A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products . These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa.

Zentralbl Bakteriol, 1997 Apr, 285(4), 486 - 90
Arsenate resistance as a possible marker in the differentiation of environmental and clinical isolates of Vibrio parahaemolyticus; Chauhan BS et al.; Strains of Vibrio parahaemolyticus were isolated from clinical, marine and freshwater fish of Calcutta, West Bengal, India . Drug and metal resistance characteristics were compared for differentiation of clinical and environmental strains . Eighteen out of the twenty environmental isolates were resistant to arsenate, unlike the clinical isolates which were all susceptible . All the thirty-five isolates of V . parahaemolyticus were resistant to ampicillin and streptomycin.

Trends Microbiol, 1997 Apr, 5(4), 161 - 5
The evolution of epidemic Vibrio cholerae strains; Mooi FR et al.; The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms . Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.

Microb Pathog, 1997 Apr, 22(4), 199 - 208
A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae; Ghosh C et al.; Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element) . Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin . Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis . The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay . The presence of RS element was demonstrable in ctxA+ strains only . Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element . Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable . These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions . Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same . Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci . These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15 . The significance of these results in terms of the pathogenic and epidemic potential of V . cholerae strains is discussed.

Toxicon, 1997 Apr, 35(4), 515 - 27
Biochemical and physiological characteristics of HlyA, a pore-forming cytolysin of Vibrio cholerae serogroup O1; Sathyamoorthy V et al.; Among the various toxins produced by the bacterial species Vibrio cholerae is HlyA, a cytolytic protein commonly called the E1 Tor hemolysin . HlyA is synthesized and processed in a complex manner involving various processed or degraded forms, that may co-purify and complicate the interpretation of biochemical and physiological experiments . In this study a single form of HlyA was purified by gel filtration and chromatofocusing using fast protein liquid chromatography in the presence of protease inhibitors . A 45-fold purification was obtained, with a final recovery of 17% of pure 60,000 mol . wt HlyA . A significant improvement in specific activity to 8.5 x 10(6) Chinese hamster ovary tissue culture units per mg protein was obtained . Physiological activity studies indicated that cytolysis of erythrocytes (hemolysis) was inhibited by oxygen: storage of HlyA under oil, and experimentation in N2-flushed buffers maintained activity . HlyA-mediated lysis of human erythrocytes was characterized by a significant lag phase, followed by a rapid induction of hemolysis . Hemolysis was inhibited by sucrose, an osmotic protectant, suggesting that the initial action of HlyA on erythrocytes is to raise the basal cation permeability of the cell membrane . The most likely cytolytic mechanism is thus the formation of transmembrane lesions such as homopolymer pores in target cells, as has been found for toxins from numerous other bacterial pathogens.

Infect Immun, 1997 Apr, 65(4), 1561 - 5
Promoter activities in Vibrio cholerae ctx phi prophage; Fando R et al.; Comparison of cholera toxin (CT) production directed by different gene constructs and S1 nuclease mapping revealed the presence of a ctxB-specific promoter within the ctxA coding sequence . Initiation of transcription in this region occurred in wild-type El Tor and classical biotype choleragenic vibrios . We propose that transcription from the ctxB-specific promoter and a stronger ribosomal binding site on the ctxB mRNA synergistically contribute to achieve the correct (5B:1A) subunit stoichiometry . Plasmid pB, a CT promoterless vector expressing only CTB, was used to detect promoter activity by restoration of A-subunit synthesis . Promoter activity expressed in vitro and in vivo was detected upstream of the zonula occludens toxin gene, suggesting that this factor could be produced in vivo to contribute to fluid accumulation . No promoter activity was detected in vitro and in vivo upstream from the accessory cholera enterotoxin gene.

Infect Immun, 1997 Apr, 65(4), 1387 - 94
Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women; Kozlowski PA et al.; To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit . Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks . The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents . Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions . Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina . In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women . Vaginal immunization did not generate antibodies in the rectum, however . Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.

Infect Immun, 1997 Apr, 65(4), 1293 - 8
Potent membrane-permeabilizing and cytocidal action of Vibrio cholerae cytolysin on human intestinal cells; Zitzer A et al.; Many strains of Vibrio cholerae non-O1 and O1 El Tor that cause diarrhea do not harbor genes for a known secretogenic toxin . However, these strains usually elaborate a pore-forming toxin, hitherto characterized as a hemolysin and here designated V . cholerae cytolysin, whose action on intestinal cells has not yet been described . We report that V . cholerae cytolysin binds as a monomer to Intestine 407 cells and then assembles into detergent-stable oligomers that probably represent tetra- or pentamers . Oligomer formation is accompanied by generation of small transmembrane pores that allow rapid flux of K+ but not influx of Ca2+ or propidium iodide . Pore formation is followed by irreversible ATP depletion and cell death . Binding of fewer than 10(4) toxin molecules per cell in vitro is lethal . The possibility is raised that production of this toxin by bacteria that are in close contact with intestinal cells is rapidly cytocidal in vivo, and death of intestinal cells may be a cause of diarrhea.

Clin Pharmacokinet, 1997 Apr, 32(4), 313 - 23
Oral delivery of antibodies . Future pharmacokinetic trends; Reilly RM et al.; Antibodies have been investigated as specific targeting agents for cancer diagnosis and therapy, to inactivate toxic substances including drugs and also as passive immunotherapy for neoplastic or infectious diseases . In most cases the antibodies were administered systemically by the intravenous route . More recently, however, there has been increasing interest in the oral administration of antibodies for localised treatment of infections or other conditions in the gastrointestinal tract . The normal physiological handling of ingested proteins is degradation by proteases in the stomach and intestine into small peptides or amino acids which are subsequently absorbed . Proteolytic enzymes involved in the degradation of orally administered immunoglobulins include pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase . These enzymes initially degrade the antibodies to F(ab')2 . Fab and Fc fragments . The F(ab')2 and Fab fragments, however, retain some of their neutralising activity locally in the gastrointestinal tract . Various approaches are possible to increase the stability of orally administered antibodies against proteolysis, including formulation in liposomes, coating with polymers and genetic engineering of resistant forms . The clinical application of orally administered antibodies includes the treatment and prevention of gastrointestinal infections caused by enteric pathogens such as rotavirus, Escherichia coli or Vibrio cholerae in susceptible individuals including those with immunodeficiency diseases and patients with bone marrow transplants . There is also a suggestion that such agents may be useful in preventing chemotherapy-induced gastrointestinal mucositis . Future opportunities for research include the design of oral dosage forms of antibodies which resist proteolysis and can deliver a greater fraction of immunoreactive antibody locally in the gastrointestinal tract for the treatment of infections or perhaps even to allow the absorption of antibodies for the treatment or prevention of systemic conditions.

J Biomed Mater Res, 1997 Apr, 35(1), 101 - 5
Evaluation of toxicity of medical devices using Spirotox and Microtox tests: I . Toxicity of selected toxicants in various diluents; Nalecz-Jawecki G et al.; Significant effort has been directed toward developing in vitro alternatives, which can be the first step of toxicity analysis . Tissue culture assays are currently the most popular in vitro tests for evaluating acute toxicity . The possibility of applying two bioassays using microorganisms in assessing the toxicity of extracts of medical devices was investigated . The Microtox test system--a luminescent bacteria toxicity test--assesses changes in light output from a luminescent bacteria, Vibrio fischeri . Spirotox used a large ciliate protozoan: Spirostomum ambiguum . The most widely used extraction solvent, 0.9% NaCl, must be concentrated up to 2% for Microtox and diluted nine times for the Spirotox test . The organic solvents ethanol, DMSO, and polyethylene glycol 400 were not toxic in either test in concentrations of 1-2% . The toxicity of reference compounds Hg, Cd, Zn, Pb, and SDS was examined in various diluents . The sequence of toxicity of the tested compounds in Spirotox and Microtox was: Hg > Cd > Zn > Pb > SDS, and Hg > Pb = Zn > SDS > Cd, respectively . Addition of organic solvents changed the toxicity of compounds tested in 60% of Spirotox tests and only in 25% of Microtox tests . Changes were low, not exceeding 100% in almost all cases . No correlation was observed between diluent and toxicant in either bioassay.

Appl Environ Microbiol, 1997 Apr, 63(4), 1460 - 6
Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar; Biosca EG et al.; The classification of Vibrio vulnificus strains into two biotypes has been maintained on the basis of phenotypic properties and eel virulence . Biotype 2 is virulent for eels, negative for the indole reaction, and serologically homogeneous (serogroup E), whereas strains of biotype 1 are avirulent, indole positive, and serologically heterogeneous . In the present study, we phenotypically and genotypically characterized 21 V . vulnificus isolates, recovered mainly from northern Europe, by comparing them with reference strains of both biotypes to look for new isolates of biotype 2 . The results of this work revealed that the majority of isolates virulent for eels presented phenotypic traits previously considered characteristics of biotype 2 and specific ribotypes with HindIII . However, among the new isolates we found (i) a serogroup E strain virulent for eels but indole positive and (ii) one isolate not belonging to serogroup E but pathogenic for eels . Since no biochemical test for specific serogroup can with certainty be associated with eel virulence, we propose to classify V . vulnificus strains into serovars instead of biotypes . Thus, we suggest serovar E as the denomination of those strains previously classified as biotype 2 . Finally, the occurrence of serogroup E in eels cultured in Norway and Sweden, as well as from human infections and shrimp, has been demonstrated.

Appl Environ Microbiol, 1997 Apr, 63(4), 1449 - 52
Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling; Castro D et al.; A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis . A total of 90.9% of V . tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands . In contrast, the V . tapetis strains showed a low degree of similarity with six reference Vibrio species tested . All V . tapetis strains harbored a large plasmid of 74.5 kb . This plasmid was not detected in any of the other Vibrio species . In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V . tapetis possessed the same cleavage pattern . The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb . The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V . tapetis strains belonged to the same ribotype when the enzyme PvuII was used.

Curr Microbiol, 1997 Apr, 34(4), 258 - 63
Molecular cloning and nucleotide sequence of the porphobilinogen deaminase gene, hemC, from Chlorobium vibrioforme; Majumdar D et al.; We previously reported the DNA sequence and expression of the Chlorobium vibrioforme glutamyl-tRNA reductase (hemA) gene (Majumdar et al., Arch Microbiol 156:281, 1991) . The sequence downstream of the hemA gene indicated homology to Escherichia coli and Bacillus subtilis porphobilinogen deaminase (hemC) gene . The Chlorobium gene was confirmed to be the porphobilinogen deaminase gene, and complete sequence of the structural gene was obtained . A 2.8-kb DNA fragment containing the 1.3-kb hemA gene of Chlorobium was cloned into a hemC auxotroph (Sz16) of Bacillus subtilis, and complementation of the auxotroph to prototrophy was achieved . DNA sequence data showed a single open reading frame of 840 bp coding a protein of 279 amino acid residues . The deduced amino acid sequence of the Chlorobium porphobilinogen deaminase revealed 39% to 46% homology with the corresponding prokaryotic and eukaryotic sequences.

J Commun Dis, 1997 Mar, 29(1), 15 - 22
Changing patterns of Vibrio cholerae isolation over three consecutive cholera seasons (1992-1994) in east Delhi; Rudra S et al.; The emergence of new strains of Vibrio Cholerae has added a new dimension to the variability in pathogenicity and potential virulence of the organisms precipitating diarrhoeal diseases . Considering the shifting patterns of V . cholerae 01 there is a continuous need to monitor the strain characteristics . In this study total 541 stool specimens of acute secretory diarrhoea were investigated between May 1992 and November 1994 for strains of Vibrio Cholerae and anti-microbial susceptibility testing of all the confirmed V . Cholerae strains . In 1992, 50 of the 125 strains (40%) were positive for V . cholerae 01 predominantly biotype El Tor serotype ogawa, and 10 (80%) of non 01 type, with most strains susceptible to tetracycline (100%), chloramphenicol (98%) and Cotrimoxazole (98%), but all resistant to polymyxin B and furazolidine . In 1993, 44 (43.6%) of the 010 strains were positive for V . cholerae 0139 and the rest V . cholerae 01 . In 1994, another sero group of V . cholerae 010 emerged, with 42 (13.3%) being positive . Isolates did not agglutinate with any of these antisera and have been labelled as 'other than non-01 vibrio cholerae'.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Mar-Apr, (2), 43 - 6
{The characteristics of the protectiveness of a preparation of Vibrio cholerae outer membrane based on the data from a study using the ligated intestinal loop}; Urbanovich LIa et al.; The study aimed at finding out the antiadhesive capacity of antigenic preparation, earlier obtained from V . cholerae outer membrane and highly effective with respect to cholera infection, was undertaken . The study was made on previously immunized adult rabbits who had been subjected to laparotomy under anesthesia and the ligation of intestinal loops, subsequently inoculated with the broth culture of V . cholerae eltor (P-3122, serovar Ogawa) . The intestinal loops were studied histologically and bacteriologically with the calculation of the number of vibrios, the deduction of the adhesion index and the coefficient of the efficacy of immunization . The data thus obtained indicated that the specific immunization of rabbits with their subsequent inoculation with V . cholerae virulent strain suppressed the adhesive activity of the infective agent which was more pronounced in rabbits immunized with the preparation of V . cholerae outer membrane.

J Med Microbiol, 1997 Mar, 46(3), 233 - 8
Isolation of a contact-dependent haemolysin from Mycobacterium tuberculosis; Deshpande RG et al.; Contact-dependent haemolytic activity was observed with cells of Mycobacterium tuberculosis H(37)Rv and M . tuberculosis H(37)Ra, but not with those of M . bovis, M . bovis BCG and M . africanum . Culture filtrates of all these strains did not exhibit any haemolytic activity . M . tuberculosis H(37)Rv was subsequently used for the isolation of haemolysin . Haemolytic activity was retained in the cell debris even after sonication of the cells and treatment with Tween 80 and lysozyme . Solubilisation of haemolysin was possible only after the cell debris was washed with ethanol 70% and then treated with Tween 80 0.1% . The haemolysin thus obtained showed a micellar M(r) of >200000 by gel-filtration on Sephadex G-200 and a subunit M(r) of 66000 by SDS-PAGE . It was sensitive to trypsin but stable when heated at 60 degrees C for 10 min . Polyclonal serum raised in rabbits against the haemolysin neutralised the haemolytic activity . The N-terminal amino-acid sequence of the 66-kDa subunit of haemolysin showed identity with TB66, the 66-kDa secretory protein of M . tuberculosis, and 30% homology with the haemolysin A precursor of Vibrio cholerae . Phosphatidylglycerol inhibited lysis of sheep erythrocytes by the haemolysin and is probably the receptor for the haemolysin . Haemolysin not only lysed erythrocytes, but was also cytotoxic to human lung cells . It appears that, among the members of the M . tuberculosis complex, the cell-bound contact-dependent haemolysin/cytolysin is restricted to M . tuberculosis and it may be associated with the pathogenesis of M . tuberculosis.

Ecotoxicol Environ Saf, 1997 Mar, 36(2), 189 - 95
Mixture toxicity of nitrobenzene and trinitrobenzene using the marine bacterium Vibrio harveyi as the test organism; Thomulka KW et al.; Vibrio harveyi, a bioluminescent marine bacterium, was used to evaluate combined or mixture toxicity of two organic compounds, nitrobenzene and trinitrobenzene . An estimated median effective concentration (EC50) and confidence interval were determined for each chemical . These chemicals at their EC50 were evaluated in combination and an additive index method was used to determine a numerical toxicology value . Combinations at 20% intervals of the EC50 were performed using isopleths . The isopleths employed were the isobole plot and the isobologram . Bioluminescent change was also determined and graphed for evaluation of toxicity . Statistical evaluation of isopleths and the additive index method were employed by incorporating confidence intervals . Bioluminescent change and isopleths suggest that mixtures of nitrobenzene and trinitrobenzene are additive, while the additive index method is suggestive of synergism . Statistical evaluation between mixtures and single values, using the z test, was in some cases different at the 5% level . These data suggest that interaction of combinations should be evaluated and described by multiple methodologies . Evaluation of these data suggests, in part, that one mixture is statistically different for antagonism . This study supports the use of bioluminescent microbial toxicity tests with various evaluative methodologies for the determination of mixture interactions.

Ecotoxicol Environ Saf, 1997 Mar, 36(2), 180 - 2
Effect of river and wetland sediments on toxicity of metolachlor; Karuppiah M et al.; Metolachlor is a preplant, preemergent herbicide applied to corn and soybean fields . Agricultural runoff after application can cause the herbicide to enter into natural waters . The objective of this study was to determine the effect of river and wetland sediments on the toxicity of metolachlor . Sediments from a river and a wetland (separately) were mixed with Ottawa sand (0, 25, 50, 75, and 100%) . Metolachlor (in water) and sediment were mixed in an orbital shaker for 8 hr; the mixture was centrifuged, and the supernatant liquid was tested for toxicity (EC50%) using the marine luminescent bacteria Vibrio fischeri (Microtox) . The toxicity (EC50%) of metolachlor with the river sediment was 64.61 . Metolachlor after interaction with the wetland sediment demonstrated no toxicity possibly due to increased adsorption on the higher amount of organic matter (10 times) and clay (3.5 times) present in the wetland sediment than the river sediment.

Eur J Biochem, 1997 Mar 1, 244(2), 454 - 61
Redox titration of two {4Fe-4S} clusters in the photosynthetic reaction center from the anaerobic green sulfur bacterium Chlorobium vibrioforme; Scott MP et al.; Anaerobic green sulfur bacteria contain photosynthetic reaction centers analogous to photosystem I (PS I) of plants and cyanobacteria . These reaction centers, termed type I, are characterized by the presence of bound iron-sulfur clusters as the terminal electron acceptors . In this work, the iron-sulfur clusters in Chlorobium vibrioforme were studied using selective light-induced reduction protocols, spin quantifications, and chemical redox titrations coupled with EPR detection . Illumination of a dark-frozen sample at 12 K results in the appearance of a spectrum termed signal I . Chemical reduction in darkness at solution potentials between -414 mV and -492 mV results in the appearance of a different spectrum termed signal II . Illumination of these chemically poised samples at 12 K results in the appearance of signal I such that the sum of the intensity of signal I + signal II is nearly constant for every ratio of signal I/signal II . As the solution potential is lowered to -545 mV, the spectrum shifts to yet a third set of resonances, termed signal III . Concomitant with this shift is a loss of low temperature light-induced reduction of signal I . Photoaccumulation of a sample poised at a solution potential of -50 mV results also in the appearance of signal III at nearly the same spin concentration as the chemically reduced sample . Spin quantifications imply that signals I and II are both derived from the reduction of one iron-sulfur cluster, termed center I; signal III is derived from simultaneous reduction of two iron-sulfur clusters, centers I and II . By measuring the EPR signal intensities over a range of solution potentials, centers I and II were shown to have Em (pH 10.0) values of -446 mV and -501 mV, respectively . The observations are consistent with a structural and functional analogy of centers I and II with F(A) and F(B) of PS I.

J Med Entomol, 1997 Mar, 34(2), 226 - 33
Parasites of the Asian tiger mosquito and other container-inhabiting mosquitoes (Diptera:Culicidae) in northcentral Florida; Fukuda T et al.; Seven microorganisms including 4 protozoans, 2 fungi, and a bacterium infected Aedes albopictus (Skuse) larvae collected from 12 counties in northecentral Florida . Ae albopictus and 14 other species of mosquitoes were collected from tires, flower-holding vases in cemeteries, other types of artificial containers, and treeholes . Ascogregarina taiwanensis (Lien & Levine) was the most common parasite of Ae . albopictus throughout the year . The microsporidium Vavraia culicis (Weiser) infected Aedes aegypti (L.), Ae . albopictus, Aedes triseriatus (Say), and Orthopodomyia sinifera (Coquillett) . A vibrio bacterium and 2 fungi (Leptolegnia sp . and Smittium culisetae Lichtwardt), infected Ae . albopictus larvae but were observed infrequently . A . taiwanensis, S . culisetae, and the vibrio bacterium previously have been reported from Ae . albopictus . This is the 1st report of the other 4 microorganisms parasitizing Ae . albopictus larvae.

J Bacteriol, 1997 Mar, 179(6), 2038 - 46
Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B; Iredell JR et al.; The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways . Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V . cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export . Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains . While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA . Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP . In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation . These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber . Possible candidates for the outer membrane defect are discussed.

FEMS Microbiol Lett, 1997 Mar 1, 148(1), 101 - 6
Characterization of a mutant of Vibrio vulnificus for heme utilization; 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 isolated and characterized a mutant for protoheme utilization . One mutant isolated by treatment with a chemical mutagen was shown to be unable to use either protoheme or heme proteins, but multiplied in a medium supplemented with an iron siderophore, such as iron-vulnibactin . Like a wild-type strain, the mutant sensed iron depletion, so that the 74- and 79-kDa outer membrane proteins were expressed under iron-regulated conditions . Both the parent and mutant strains secreted hemolysin independent of the iron concentration of the medium . Whole cells of either of the strains were equally capable of binding of hematin . Taken together, the data suggest that the mutant may have a mutation in a gene encoding an inner membrane or a periplasmic protein which transports protoheme or iron dissociated from protoporphyrin IX into the cell.

J Invertebr Pathol, 1997 Mar, 69(2), 177 - 82
In Vitro Activity of the Limulus Antimicrobial Peptide Tachyplesin I on Marine Bivalve Pathogens
Morvan A, Iwanaga S, Comps M, Bachere E.
Tachyplesin 1 is an antimicrobial peptide extracted from hemocytes of the Japanese horseshoe crab Tachypleus tridentatus . We studied the in vitro activity of tachyplesin I against bivalve pathogens: the oyster parasites Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis and Perkinsus marinus, the histozoic parasite of the Eastern oyster Crassostrea virginica, and the bacterium Vibrio P1, pathogenic for the clam Tapes philippinarum . Viability of the protozoans was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide . Following exposure to tachyplesin I, B . ostreae and P . marinus viabilities were reduced in a dose-dependent manner, up to, respectively, 94 and 62% within a 500 mug/ml peptide concentration . The fine structure of P . marinus was highly altered by the peptide . Tachyplesin I also displayed a potent activity against marine vibrios, with a MIC of 0.4-0.8 mug/ml against Vibrio P1 . We examined the morphology of oyster hemocytes treated by tachyplesin I, together with the cell functional capabilities to produce chemiluminescence . No effect of the peptide was found on bivalve host cells . As transgenic technology is currently being applied to marine invertebrates, these results indicate that tachyplesin I may provide effective gene sequences to be manipulated in order to produce disease-resistant bivalves.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 47 - 54
Purification and characterization of isobutylamine N-hydroxylase from the valanimycin producer Streptomyces viridifaciens MG456-hF10; Parry RJ et al.; Streptomyces viridifaciens MG456-hF10 produces the antitumor agent valanimycin, which is a member of a family of antibiotics containing the azoxy group . An enzyme involved in the biosynthesis of valanimycin has been purified 360-fold from S . viridifaciens . This enzyme, isobutylamine N-hydroxylase, catalyzes the oxidation of isobutylamine to isobutylhydroxylamine in the presence of oxygen and a reduced flavin cofactor . Unlike other known N-hydroxylases, isobutylamine N-hydroxylase cannot carry out the reduction of the flavin cofactor . Rather, the reduced flavin is supplied by a separate flavin reductase that is present in extracts of S . viridifaciens . The reduced flavin cofactor could also be supplied by the flavin mononucleotide reductase of Vibrio fischeri . The requirement for molecular oxygen and a reduced flavin indicates that the N-hydroxylase is a flavin monooxygenase and that the mechanism for the hydroxylation is likely to proceed via the formation of a flavin 4a-hydroperoxide . Isobutylamine N-hydroxylase exhibited a subunit molecular mass of 40 kDa and existed in dimeric or trimeric form depending upon buffer conditions . The pI of the protein was found to be ca . 5.1 and the enzyme exhibited a sensitivity to thiol-directed reagents.

J Bacteriol, 1997 Mar, 179(5), 1805 - 8
Primary structure and properties of the Na+/glucose symporter (Sg1S) of Vibrio parahaemolyticus; Sarker RI et al.; Previously, we cloned and sequenced a DNA fragment from Vibrio parahaemolyticus and found four open reading frames (ORFs) . Here, we clearly demonstrate that one of the ORFs, ORF1, is the gene (sglS) encoding a Na+/glucose symporter (SglS) . We characterize the Na+/glucose symporter produced in Escherichia coli mutant (JM1100) cells which lack original glucose transport activity and galactose transport activity . We also show that phlorizin, a potent inhibitor of the SGLT1 Na+/glucose symporter of animal cells, inhibited glucose transport, but not galactose transport, via the SglS system.

J Bacteriol, 1997 Mar, 179(5), 1591 - 7
The light organ symbiont Vibrio fischeri possesses two distinct secreted ADP-ribosyltransferases; Reich KA et al.; We have previously described the purification, cloning, and initial characterization of a secreted ADP-ribosyltransferase, halovibrin (gene designation hvn), from the luminescent light organ symbiont Vibrio fischeri . This report describes a strategy for overexpression of halovibrin, the production and refinement of antihalo-vibrin antisera, and the molecular biological construction of a V . fischeri halovibrin null strain . Biochemical analysis of this mutant revealed that V . fischeri hvn null still possessed ADP-ribosyltransferase activity and that this activity is immunologically, genetically, and structurally distinct from the previously described enzyme . This unusual finding, of two ADP-ribosyltransferase enzymes produced by a microorganism, is complemented by the details of the purification to apparent homogeneity and in vitro regulation of this new protein, halovibrin-beta.

J Clin Microbiol, 1997 Mar, 35(3), 624 - 30
Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V . cholerae O139 Bengal in Bangladesh; Faruque SM et al.; The emergence of Vibrio cholerae O139 Bengal in 1993, its rapid spread in an epidemic form, in which it replaced existing strains of V . cholerae O1 during 1992 and 1993, and the subsequent reemergence of V . cholerae O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged El Tor vibrios . We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and Africa before the emergence of V . cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V . cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V . cholerae O139 to determine whether the reemerged El Tor vibrios were genetically different from the El Tor vibrios which existed before the emergence of V . cholerae O139 . Analysis of restriction fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin (zot) or in DNA sequences flanking the genes showed that the El Tor strains isolated before the emergence of V . cholerae O139 belonged to four different ribotypes and four different ctx genotypes . Of 32 El Tor strains isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to a single new ribotype and a distinctly different ctx genotype . These results provide evidence that the reemerged El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor vibrios which were replaced by the O139 vibrios . Further analysis showed that all the strains carried the structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR) . All strains of the new clone produced cholera toxin (CT) in vitro, as assayed by the GM1-dependent enzyme-linked immunosorbent assay, and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios . Further studies are required to assess the epidemic potential of the newly emerged clone of V . cholerae O1 and to understand the mechanism of emergence of new clones of toxigenic V . cholerae.

Gastroenterology, 1997 Mar, 112(3), 839 - 46
The enterotoxic effect of zonula occludens toxin on rabbit small intestine involves the paracellular pathway; Fasano A et al.; BACKGROUND & AIMS: Zonula occludens toxin is a novel toxin elaborated by Vibrio cholerae that modulates intestinal tight junctions . The aim of this study was to establish whether the permeabilizing effect of the toxin leads to intestinal secretion . METHODS: Rabbit intestine was mounted in Ussing chambers and exposed to increasing concentrations of purified toxin . The tissues were also fixed, exposed to zonula occludens toxin, and processed for fluorescence microscopy to determine the distribution of the toxin receptor within the intestine . Then purified toxin was simultaneously perfused in three distinct rabbit intestinal segments in vivo, and water and electrolyte absorption were measured . RESULTS: Zonula occludens toxin induced a time- and dose-dependent decrease of tissue resistance starting at a toxin concentration of 1.1 x 10(-13) mol/L . When tested in vivo, the toxin induced a secretion of water and chloride and the passage of polyethylene glycol 4000 in the bloodstream . Both the in vitro and in vivo effects of the toxin were observed only in the small intestine but not in the colon and paralleled the distribution of the toxin receptor within the intestine . CONCLUSIONS: The intestinal secretion induced by zonula occludens toxin follows the opening of tight junctions caused by the toxin, possibly representing a novel mechanism of intestinal secretion.

Infect Immun, 1997 Mar, 65(3), 1131 - 4
Bile affects production of virulence factors and motility of Vibrio cholerae; Gupta S et al.; The effect of bile on the expression of cholera toxin (CT) and the major subunit of the toxin-coregulated pilus (TcpA) and on motility was examined in the Vibrio cholerae O1 classical-biotype strains 0395 and 569B . Although the motility of the cells increased significantly in the presence of bile, transcription of the ctxAB genes, encoding CT, and of the tcpA gene was drastically reduced . In toxR mutant strains, motility is higher than in the wild-type strain and was further increased, by about 150%, in the presence of bile . Bile represses CT production in strain 569B-55, a toxR mutant of strain 569B, which normally produces more than 80% of the amount of CT synthesized in the wild-type cells . These results suggest that bile may target some factor other than ToxR that is involved in the regulation of CT production and motility . Bile has no effect on the relative amounts of the two outer membrane porins, OmpU and OmpT, which are under ToxR control.

Biochemistry (Mosc), 1997 Feb, 62(2), 225 - 30
The role of the sodium cycle of energy coupling in the emergence and persistence of natural foci of modern cholera; Brown II et al.; A hypothesis on the appearance and persistence of natural foci of cholera based on ecological and bioenergetic features of the process has been developed . The main causes of persistence and propagation of modern cholera are: 1) inability of various bacteria, including the genus Vibrio and many cyanobacterial species, to perform energy coupling, depending on external conditions, by means of two cycles (the proton and sodium cycles); induction of the sodium cycle of energy coupling increases the resistance of bacteria to various environmental factors, such as high concentrations of sodium, alkaline pH, and a high proton conductance of coupling membranes {1}, and probably the virulence of the vibrios; 2) development of cyanobacteria in an aquatic environment enriched with Na+ accelerates alkalization of the medium and stimulates the development of the community of cyanobacteria with Vibrio cholerae, an autochthonous inhabitant of saline water bodies and marine shallow waters; 3) salinization of water bodies accelerates their blooming and enriches them with soluble organic matter, a substrate for vibrios inhabiting the biotope; 4) further propagation of cholera infection is related to eating heat-untreated hydrobionts from blooming water bodies {2}.

Hum Exp Toxicol, 1997 Feb, 16(2), 101 - 5
Membrane attack induced by HlyA, a pore-forming toxin of Vibrio cholerae; Huntley JS et al.; Determining the activity of purified toxins has generally provided the basis for establishing their role in the host-pathogen relationship . The bacterial genus Vibrio produces a number of exotoxins in addition to cholera toxin, including haemolysin A (HlyA; Vibrio cholerae) and thermostable direct haemolysin (TDH; Vibrio parahaemolyticus), both of which possess membrane-targeting cytolytic activity . The action of HlyA has been analyzed using protocols previously applied to TDH: lysis and flux experiments on human erythrocytes showed that HlyA similarly causes lysis after cell swelling (by colloid osmosis) due to an elevation of cation permeability . However, kinetic measurements of flux, haemolysis and cation selectivity showed that HlyA and TDH form pores with distinct and characteristic features.

FEMS Microbiol Lett, 1997 Feb 1, 147(1), 115 - 20
Purification and characterisation of a hemolysin with phospholipase C activity from Vibrio cholerae O139; Pal S et al.; A hemolysin was purified from a Vibrio cholerae O139 strain which moved as a single protein band of 67 kDa in SDS-PAGE . The hemolysin showed high level of phospholipase C activity . The purified phospholipase C-hemolysin demonstrated enterotoxic activity in rabbit ileal loop, suckling mice and enhanced permeability of rabbit skin . The pI of the purified hemolysin was 6.4 . Erythrocytes from rabbit, chicken, guinea pig, sheep and horse were sensitive to the purified hemolysin in decreasing order of intensity . Erythrocytes from human and cow were unaffected by purified hemolysin.

Appl Environ Microbiol, 1997 Feb, 63(2), 537 - 42
An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies; Biosca EG et al.; Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species . In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen . The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V . anguillarum, V . furnissii, A . hydrophila, and Y . ruckerii . The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state . Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment) . With this methodology, V . vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.

Curr Microbiol, 1997 Feb, 34(2), 110 - 7
Alkaline serine protease is an exotoxin of Vibrio alginolyticus in kuruma prawn, Penaeus japonicus; Lee KK et al.; An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn(Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose Hig hPerformance) chromatography and gel filtration columns . The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),and showed maximal activity at pH 10, having a molecular weight of about 33kDa estimated by SDS-PAGE and gel filtration chromatography . In addition, the toxin was also completely inhibited by FeCl2 but partially inhibited by CaCl2, CuCl2, CoCl2,MnCl2, and ZnCl2, and not inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecylsulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK) . Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD50 values of 0.30 and 0.27 microg protein/g body weight, respectively . The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium . The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V . alginolyticus strain Swy.

J Neurochem, 1997 Feb, 68(2), 878 - 81
Expression of sulfated gangliosides in the central nervous system; Farrer RG et al.; Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with {35S} sulfate . Separation of the ganglioside fraction by two-dimensional TLC showed that, except for galactosylceramide-sulfate, none of the sulfate-labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components . At least eight sulfate-labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate . Six of the sulfate-labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A . ureafaciens neuraminidase . In vivo labeling of lipids from 14-day-old rat brain with {35S}-sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue . These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.

Gene, 1997 Jan 31, 185(1), 43 - 7
The rfaD locus: a region of rearrangement in Vibrio cholerae O139; Vimont S et al.; We analyzed the rfaD locus of the novel epidemic Vibrio cholerae strain O139, a putative region of rearrangement . This region includes 4 orfs in the same orientation . Two orfs, rfaD(O139) and orf2(O139) were almost identical to those described in V . cholerae O1 . In contrast, the two other orfs upstream from rfaD(O139), designated orfA(O139) and orfB(O139), were absent from V . cholerae O1, but present in environmental strains of V . cholerae O22, O141 and O155 . These results suggest that a chromosomal rearrangement might have occurred in the vicinity of rfaD in V . cholerae O1, resulting in the emergence of V . cholerae O139 . The putative source of exogenous DNA might have been V . cholerae O22, O141 and O155.

Biochemistry, 1997 Jan 28, 36(4), 665 - 72
Structure of bacterial luciferase beta 2 homodimer: implications for flavin binding; Tanner JJ et al.; The crystal structure of the beta 2 homodimer of Vibrio harveyi luciferase has been determined to 2.5 A resolution by molecular replacement . Crystals were grown serendipitously using the alpha beta form of the enzyme . The subunits of the homodimer share considerable structural homology to the beta subunit of the alpha beta luciferase heterodimer . The four C-terminal residues that are disordered in the alpha beta structure are fully resolved in our structure . Four peptide bonds have been flipped relative to their orientations in the beta subunit of the alpha beta structure . The dimer interface of the homodimer is smaller than the interface of the heterodimer in terms of buried surface area and number of hydrogen bonds and salt links . Inspection of the subunits of our structure suggests that FMNH2 cannot bind to the beta 2 enzyme at the site that has been proposed for the alpha beta enzyme . However, we do uncover a potential FMNH2 binding pocket in the dimer interface, and we model FMN into this site . This proposed flavin binding motif is consistent with several lines of biochemical and structural evidence and leads to several conclusions . First, only one FMNH2 binds per homodimer . Second, we predict that reduced FAD and riboflavin should be poor substrates for beta 2 . Third, the reduced activity of beta 2 compared to alpha beta is due to solvent exposure of the isoalloxazine ring in the beta 2 active site . Finally, we raise the question of whether our proposed flavin binding site could also be the binding site for flavin in the alpha beta enzyme.

Lancet, 1997 Jan 25, 349(9047), 231 - 5
Field trial of a locally produced, killed, oral cholera vaccine in Vietnam; Trach DD et al.; BACKGROUND: Several studies have shown that orally administered killed cholera vaccines are safe and protective in populations at risk of cholera in developing countries . However, these vaccines have not been adopted for use in developing countries because of their expense and limited efficacy in young children . We have tested an inexpensive, killed whole-cell cholera vaccine developed and produced in Vietnam . METHODS: The efficacy of the vaccine was assessed in a large-scale, open field trial in people at least 1 year old residing in 22,653 households in the central coastal city of Hue . Alternate households were assigned vaccine (67,395 people; two doses per person) or no vaccine (67,058 people) . Surveillance for cholera was conducted in all Ministry of Health facilities serving this population . Analysis was by intention to treat . FINDINGS: During an outbreak of El Tor cholera 8-10 months after vaccination, 37 cases of cholera requiring inpatient care occurred among age-eligible people allocated to the vaccine group, and 92 cases among age-eligible people allocated to the no-vaccine group (protective impact 60% {95% CI 40-73}) . Among the 51,975 people who received the complete two-dose vaccine regimen, the protective efficacy was 66% (46-79): in this subset, the protective efficacy was similar for children aged 1-5 years (68%) and for older people (66%) . INTERPRETATION: These findings suggest that oral killed whole-cell vaccines can confer substantial protection against El Tor cholera in young children, who are at highest risk of cholera in endemic settings . An inexpensive, locally produced, and effective oral cholera vaccine may be within reach of the limited health-care budgets of poor countries with endemic cholera, if our findings can be replicated in a randomised double-blind trialPIP: Vibrio cholera 01, El Tor biotype, entered Vietnam in 1964 and during 1990-94 an average of 3240 cases were reported annually with a case-fatality rate of about 1% . The efficacy of an inexpensive, killed whole-cell cholera vaccine developed in Vietnam was assessed in a large-scale, open field trial in the city of Hue . The vaccine contained V . cholera 01 constituents: heat-killed V . cholera Inaba, heat-killed V . cholera Ogawa, and formalin-killed V . cholera Inaba . All 134,453 residents, aged 1 year or older, of 22,653 households in 19 communes were eligible to take part in the trial . Alternate households were assigned vaccine (67,395 people; 77% received 2 doses per person) or no vaccine (67,058 people serving as controls) during December 1992 and January 1993 by 80 vaccination teams . Following the vaccination no cases of cholera were detected until late August 1993 . Between August 20 and October 4, 1993, there were 129 cases of cholera requiring inpatient care among age-eligible participants . The isolates were 01 serogroup and Ogawa serotype . There were 37 cases of cholera in the vaccine group and 92 cases in the control group . The risk of cholera was 0.5/1000 and 1.4/1000, respectively . The protective impact was 60% (95% confidence interval {CI} 40-73; p 0.001) . Among 51,975 recipients of 2 vaccine doses the protective efficacy was 66% (CI 46-79; p 0.001) . The protective efficacy was similar for children 1-5 years old (68%) and for older people (66%) . The protective efficacy was somewhat higher among the vaccinated living in homes with unclean water sources (74% vs . 62%) . The protective efficacy was also higher against severe than against non-severe cholera (76% vs . 58%) . Oral killed whole-cell vaccines can protect against El Tor type cholera in children who are highest risk . The government has lately added a killed V . cholera 0139 strain to the existing formulation . Phase 2 tests of safety are under way, and a large-scale, randomized, double-blind field trial will start in 1997 .

Lancet, 1997 Jan 25, 349(9047), 220 - 1
Oral vaccines against cholera: lessons from Vietnam and elsewhere; Levine MM; PIP: Cholera epidemics, often involving new etiologic agents, are a major public health problem in developing countries . Although two new types of oral cholera vaccines--inactivated whole Vibrio cholerae O1 and attenuated strains of V cholerae O1--have been demonstrated to be safe, immunogenic, and effective, there has been a lag in their systematic use in areas with endemic and epidemic cholera . An open field trial conducted by Trach et al of a locally manufactured killed oral cholera vaccine resulted in a 60% reduction in the incidence of cholera in vaccinated versus nonvaccinated households in Hue, Viet Nam . Levels of protection in children 1-5 years of age were similar to those achieved in adults . Although the requirement for two spaced doses of this vaccine is a drawback, the potential for local manufacture overcomes the obstacle of high costs associated with imported vaccines . Attenuated V cholerae O1 vaccine strain CVD 103-HgR represents the most advanced application of the live oral vaccine approach . Small clinical trials in Asia, Latin America, Europe, and North America have indicated that a single dose confers complete protection against severe or moderate cholera caused by El Tor or classical biotype V cholerae O1 within eight days of administration .

J Mol Biol, 1997 Jan 24, 265(3), 310 - 8
Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor; Kojima S et al.; The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor . To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil . The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil . The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain . However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain . The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor . We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil . The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors . The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.

J Biol Chem, 1997 Jan 10, 272(2), 1338 - 43
Intramolecular chaperone activity of the pro-region of Vibrio cholerae El Tor cytolysin; Nagamune K et al.; Vibrio cholerae synthesizes a toxin named El Tor cytolysin/hemolysin, which lyses erythrocytes and other mammalian cells . This toxin is encoded by the hlyA gene and is synthesized as a precursor form, prepro-HlyA . Prepro-HlyA consists of, from the amino terminus of this protein, a signal peptide, a pro-region, and a mature region . The pro-region is cleaved off extracellularly resulting in activation . To analyze the role of the pro-region, we substituted the native hlyA gene with the pro-region-deleted hlyA gene (hlyA delta pro) . The hemolytic activity of the mutant organism was markedly decreased; the product of the hlyA delta pro gene, secreted in the periplasm, was degraded . To compare their abilities to form tertiary structure, the purified mature- and pro-HlyA were denatured and then renatured by reducing the concentration of denaturant; the denatured pro-HlyA recovered almost all activity while the mature-HlyA was not renatured . The sequences of the pro-region and a molecular chaperone, Hsp90, were similar . The pro-region expressed in Escherichia coli containing the hlyA delta pro gene increased the cytolytic activity . The purified pro-region peptide also facilitated renaturation of the denatured mature HlyA . These results suggest that the pro-region possibly guides the folding of the cytolysin similar to a molecular chaperone; the pro-region and molecular chaperones share common function and structure.

Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 265 - 70
Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilus in Vibrio cholerae; Skorupski K et al.; Insertion mutations in two Vibrio cholerae genes, cya and crp, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP), respectively, derepressed the expression of a chromosomal cholera toxin (CT) promoter-lacZ fusion at the nonpermissive temperature of 37 degrees C . In the classical biotype strain O395, the crp mutation increased the production of both CT and toxin-coregulated pilus (TCP) in vitro under a variety of growth conditions not normally permissive for their expression . The most dramatic increase in CT and TCP was observed with the crp mutant in Luria-Bertani (LB) medium pH 8.5, at 30 degrees C . El Tor biotype strains differ from classical strains in that they do not produce CT or TCP when grown in LB media . Incorporation of the crp mutation into El Tor strain C6706 permitted production of these proteins in LB medium pH 6.5, at 30 degrees C . In the infant mouse cholera model, the crp mutation decreased colonization in both biotypes at least 100-fold relative to the wild-type strains . The data presented here suggest a model whereby cAMP-CRP negatively regulates the expression of CT and TCP in both classical and El Tor biotypes under certain environmental conditions and also influences pathogenesis by regulating other processes necessary for optimal growth in vivo.

J Biol Chem, 1997 Jan 3, 272(1), 162 - 7
Carbohydrate-mediated regulation of interaction of Vibrio cholerae hemolysin with erythrocyte and phospholipid vesicle; Saha N et al.; Vibrio cholerae hemolysin is an extracellular pore-forming monomeric protein with a native molecular weight of about 60,000 . In this study, we showed that the hemolysin interacted with immobilized phospholipids and cholesterol and formed oligomers in vesicles constituted from phospholipids alone with a stoichiometry identical to those produced in rabbit erythrocyte membrane . However, the hemolysin bound to glycoproteins with terminal beta1-galactosyl residues and an association constant of 9.4 x 10(7) M(-1) was estimated for the hemolysin-asialofetuin complex by solid phase binding assay . Oligomerization of the hemolysin in lipid bilayer converted the sugar-binding monomer to a lectin with strong carbohydrate-dependent hemagglutinating activity accompanied by inactivation of hemolytic activity and loss in ability to interact with phospholipids . There was no evidence for erythrocyte surface carbohydrates playing an essential role in interaction of the hemolysin with the cell . However, specific glycoproteins inhibited hemolysis of rabbit erythrocytes as well as interaction of the hemolysin with phospholipid . The results suggest (i) V . cholerae hemolysin is a monomer with distinct domains associated with specific binding to carbohydrates and interaction with lipids, (ii) the pore-forming property depends solely on the protein-lipid interaction with no evidence for involvement of sugars, and (iii) specific sugars can down-regulate the ability of the hemolysin to form pores in lipid bilayers.

World Health Stat Q, 1997, 50(1-2), 111 - 8
The role of food in the epidemiology of cholera; Albert MJ et al.; Cholera is an acute dehydrating diarrhoeal disease, traditionally caused by vibrio cholerae O1, and also more recently by V . cholerae O139 (Bengal) . Traditionally, water was recognized as the primary vehicle for transmission of cholera, but in the past 30 years, outbreaks of cholera associated with eating contaminated food have demonstrated that food also plays an important role, although in many instances water is the source of contamination of foods . Most commonly associated with cholera is seafood, both molluscan shellfish and crustaceans . Seafood may be contaminated in its natural environment or during preparation . Other food items associated with outbreaks are fruit and vegetables, meat, cooked grains, etc . Vegetables are usually contaminated by contact with sewage in soil and fruits when injected with contaminated water to increase weight and turgor . Food items initially free from V . cholerae organism may become contaminated when mixed with water, or other contaminated food, or through handling by infected persons who have not observed proper hygiene . Refrigeration, freezing, alkaline pH, high concentration of carbohydrate, humidity and absence of competing flora enhance the survival of V . cholerae in food . Survival of V . cholerae is shorter in food with acidic pH . Foodborne cholera can be averted by the hygienic preparation of food and its consumption . However, since the vehicles of transmission vary markedly from place to place, being affected by local customs and practices, selected control and preventive measures that are most important locally must be implemented . To this end, application of the Hazard Analysis and Critical Control Point system to food preparation is essential in order to identify the practices which may present a risk . Restrictions on importation of foods which do not present a risk of being contaminated from areas where cholera is endemic is not warranted.

Dev Biol Stand, 1997, 90, 413 - 21
Glucan-induced disease resistance in tiger shrimp (Penaeus monodon); Song YL et al.; Non-specific disease resistance induced by yeast cell wall extract, beta-1,3-1,6-glucan, was demonstrated in the tiger shrimp . In this study beta-1,3-1,6-glucan was administered to shrimps by immersion before culturing and orally during the culturing period . Challenge of the treated shrimps with the virulent pathogens, Vibrio vulnificus and viral agents extracted from the white spot syndrome victims, yielded promising results . The tolerance of glucan-treated shrimps was slightly enhanced to stresses including catching, transport and ammonia . The growth and survival rates of treated and untreated shrimps were not significantly different . Therefore, we suggest that beta-1,3-1,6-glucan can be used as an immuno-stimulant of cultured shrimps and may benefit shrimp farmers.

Dev Biol Stand, 1997, 90, 401 - 8
Vaccination strategies in seawater cage culture of salmonids; Lillehaug A; Successful vaccination depends both on the development of protective vaccines and their correct use . In addition to deciding which diseases to vaccinate against, the choice of the method, timing, and use of revaccination must be considered . In seawater culture of salmonids, vibriosis and furunculosis are the most important diseases against which to vaccinate in many parts of the world, while cold-water vibriosis is of great significance in Atlantic salmon in some areas with low water temperatures . A vaccine against infectious pancreatic necrosis (IPN) has also been introduced recently . For optimal protection of salmonids in sea-water, vaccination should be carried out some time before sea transfer, in order to give immunity sufficient time to develop, and to avoid handling stress during smoltification . On the other hand however, vaccination should not be carried out too early, as the degree of immunity declines with time . Water temperature is an important factor when deciding when to vaccinate . Recent research has demonstrated that Atlantic salmon may be vaccinated successfully at low water temperatures . In general, vaccination by the injection method gives superior protection . Vaccines against the Vibrio-infections can also be administered successfully by immersion . However, due to lower levels of immunity, the need for a booster vaccination is greater when such a method is used . As regards vaccines against furunculosis, adjuvants are needed in order to achieve good protection, and, consequently, administration by injection is the only option.

Dev Biol Stand, 1997, 90, 257 - 65
Adjuvants and immunostimulants for enhancing vaccine potency in fish; Anderson DP; Adjuvants combined with immunogens are effective enhancers of the immune response in fish . As in other animals, substances such as Freund's complete and incomplete adjuvants, light oils and bacterial lipopolysaccharides have been shown to induce elevated antibody production when added to bacterins and administered to fish . Light oil adjuvants are now used successfully with injectable bacterins of Aeromonas salmonicida, and in multivalent fish vaccines where A . salmonicida is combined with Yersinia sp., and/or Vibrio spp . antigen preparations . The oils are thought to act as depots or reservoirs, holding the antigens in globules at the site of injection, thus allowing prolonged dosage . Additions to the oils such as muramyl dipeptide, bacterial lipopolysaccharides and other bacterial and animal-extracted products may activate specific immune cell populations such as T-cells, neutrophils and other phagocytic cells important in the cellular mediated response . Conjugation of antigens with alum is another traditional approach which has been used for A . salmonicida and Y . ruckeri bacterins with varying results . The process enables antigen to be held in a reservoir and also may detoxify harmful substances . Recent research on substances such as beta-1,3 glucans, chitosan, levamisole, and other inflammatory agents shows enhancing effects on the specific immune response when added to immunogens and administered by injection, bath or by feeding . These substances may also act to elevate non-specific defence mechanisms against disease agents as most of them are active even when given alone.

Dev Biol Stand, 1997, 90, 93 - 105
Immunization with bacterial antigens: Vibrio infections; Toranzo AE et al.; Within the genus Vibrio, the species causing the most economically important diseases in marine culture are Vibrio anguillarum, V . ordalii, V . salmonicida and V . vulnificus biotype 2 . For these bacterial fish pathogens host range, clinical importance, virulence mechanisms, the antigenic variants relevant to vaccination, the existence of genetic intraspecific diversity and the available vaccines including commercial or domestically produced will be described . Among the 10 serotypes described in V . anguillarum, only serotypes O1, O2 and O3 have been associated with mortality in a great variety of farmed and feral fish worldwide . Whereas serotype O1 is a very homogeneous group from the biochemical, serological and genetic stand-point, within serotype O2 and O3 two antigenic entities have been detected . Moreover these two serotypes present a remarkable genetic heterogeneity . However, many of the available commercial vibriosis vaccines include in their formulations only V . anguillarum serotype O1 in combination with V . ordalii (formerly V . anguillarum biotype 2) . In addition no commercial vaccine provides information about the subgroup(s) used as representative of V . anguillarum O2 . Recently, Vibrio species taxonomically related to V . anguillarum (VAR) have been isolated from diseased fishes . An extensive characterization of these VAR organisms allowed us to distribute them into at least seven O-serogroups . The inclusion of representative VAR strains in the vibriosis vaccines need to be discussed . V . ordalii, V . salmonicida and V . vulnificus are homogeneous species with respect to biochemical reactions, serology and degree of virulence, possess a narrow host range and seem to be restricted to some geographic areas . Although iron acquisition systems can be involved in the virulence mechanisms of these pathogens, only in V . anguillarum has it been clearly demonstrated that the ability to scavenge iron from the host is a crucial virulence determinant . The role of exotoxins and cell surface associated properties in the Vibrio infections remains to be elucidated.

Bol Asoc Med P R, 1997 Jan-Mar, 89(1-3), 31 - 2
Septicemia due to a non-0:1, non-0:139 Vibrio cholerae; Christenson B et al.; We describe a patient with a non-0:1, non-0:139 Vibrio cholerae septicemia associated with ecythema gangrenosa-like skin lesions . The patient acquired the infection in Puerto Rico . Given the high fatality rate, it is important for the medical community to consider the diagnosis in high risk patients with exposures in Puerto Rico tropical waters.

Rev Panam Salud Publica, 1997 Jan, 1(1), 3 - 8
{Ecology of Vibrio cholerae serogroup 01 in aquatic environments}; Borroto RJ; The endemic and seasonal nature of cholera depends upon the survival of Vibrio cholerae 01 in a viable but not necessarily culturable state in ecologic niches in aquatic environments during interepidemic periods . To understand the ecology of V . cholerae it is necessary to know which aquatic ecosystems can harbor it and thus contribute to the endemic presence of cholera in Latin America . This article presents a summary of the ecology of V . cholerae 01, organized according to the abiotic and biotic factors that are relevant to the microbe's survival in aquatic environments . This pathogen finds favorable conditions in waters characterized by moderate salinity, high nutrient content, warm temperature, neutral or slightly alkaline pH, and the presence of aquatic macrophages, phytoplankton, zooplankton, fish, mollusks, and crustaceans . These ecologic conditions are typical of estuaries and coastal swamps, and toxigenic V . cholerae 01 is now considered an autochthonous member of the microbial flora of these environments . The microorganism has also shown the ability to colonize freshwater ecosystems in its viable but not necessarily culturable form, if organic or inorganic substrates that favor its survival are available.

J Infect, 1997 Jan, 34(1), 83 - 4
Vibrio cholerae non-O1 septicaemia in a patient with liver cirrhosis and Billroth-II-gastrectomy; Halabi M et al.; This report deals with the first diagnosed case of Vibrio cholerae non-O1 septicaemia in Austria . After a vacation in Tunisia, a 51-year-old male patient with liver cirrhosis and Billroth-II-gastrectomy was admitted to hospital because of abdominal pain, growing ascites, and jaundice . Four days later, the patient developed a single peak of high fever (39.6 degrees C) . A blood culture was drawn and treatment with amoxycillin/clavulanic acid commenced . The blood culture yielded Gram-negative comma-like rods which were identified as V . cholerae non-O1.

Microbiol Immunol, 1997, 41(1), 1 - 6
Cryptic appearance of a new clone of Vibrio cholerae serogroup 01 biotype El Tor in Calcutta, India; Yamasaki S et al.; In this study, pulsed-field gel electrophoresis (PFGE) was applied to determine if the Vibrio cholerae 01 strains which reappeared after being temporarily displaced in Calcutta by the 0139 serogroup were different from those isolated before the advent of the 0139 serogroup . NotI digestion generated a total of 11 different patterns among the 24 strains of V . cholerae randomly selected to represent different time frames . Among the V . cholerae 01 strains isolated after July 1993, 4 PFGE banding designated as H through K were observed with pattern H dominating . Pattern H was distinctly different from all other patterns encountered in this study including patterns A, B and C of V . cholerae 01 E1 Tor, which dominated before November 1992, and pattern F, which was the dominant V . cholerae 0139 pattern . Further, pattern H was also different from the NotI banding patterns of the representative strains of the 4 toxigenic clonal groups of V . cholerae 01 E1 Tor currently prevailing in different parts of the world . NotI fragments of the new clone of V . cholerae 01 did not hybridize with an 0139 specific DNA probe, indicating that there was no 0139 genetic material in the new clone of V . cholerae 01 . Hybridization data with an 01-specific DNA probe again differentiated between the clones of V . cholerae 01 existing before the genesis of the 0139 serogroup and the 01 strains currently prevalent.

Microbiol Immunol, 1997, 41(2), 169 - 73
Analysis of the structural gene encoding a hemolysin in Vibrio mimicus; Rahman MM et al.; An environmental isolate of V . mimicus, strain E-33, has been reported to produce and secrete a hemolysin of 63 kDa . The hemolysin is enterotoxic in test animals . The nucleotide sequence of the structural gene of the hemolysin was determined . We found a 2,232 bp open reading frame, which codes a peptide of 744 amino acids, with a calculated molecular weight of 83,903 Da . The sequence for the structural gene was closely related to the V . cholerae el tor hlyA gene, coding an exocellular hemolysin . The amino terminal amino-acid sequence of the 63 kDa hemolysin, purified from V . mimicus, was determined by the Edman degradation method and found to be NH2-S-V-S-A-N-N-V-T-N-N-N-E-T . This sequence is identified from S-152 to T-164 predicted from the nucleotide sequence . So, it seems that the mature hemolysin in V . mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da . Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal . Our results suggest that, like V . cholerae hemolysin, two-step processing also exists in V . Mimicus hemolysin.

Natl Med J India, 1997 Jan-Feb, 10(1), 17 - 8
Oral vaccines for cholera control; Chaturvedi S et al.; Two oral cholera vaccines-inactivated WC/rBS and live CVD 103 HgR-have recently been marketed in Europe . Though the efficacy of the live vaccine is yet to be supported by field trials, the inactivated oral vaccine has shown encouraging results in field trails on different population groups . Since the role of cholera vaccines-including oral vaccines-as a public health tool in epidemic situations is debatable and cholera immunization for travellers will result in a high cost-benefit ratio, endemic cholera remains the main area of their application . The questions raised in the Bangladesh trial about the protective efficacy of WC/rBS vaccine in people infected with the EI Tor biotype, in 'O' blood group people and in those having no previous immunity to cholera have been reconsidered and explored during the recent field trail in South America, with satisfactory results . However, none of these vaccines provide protection against Vibrio cholerae 0139 Bengal . With their widely demonstrated safety and efficacy, oral cholera vaccines are set to make injectable vaccines obsolete.

Zentralbl Bakteriol, 1997 Jan, 285(2), 245 - 51
Typing of the fish pathogen Listonella (Vibrio) anguillara by pyrolysis mass spectrometry; Manfio GP et al.; Twenty-eight representatives of Listonella (Vibrio) anguillara serovars O1, O2 and O3 were compared by Curie-point pyrolysis mass spectrometry (PyMS) . The representatives of serovars O1 and O3 formed discrete, homogeneous groups in ordination plots of the PyMS data . Strains from serovar O2 were recovered in two groups, one of which encompassed six strains including the type strain of the species and the reference strain for serovar O2, and the other included two strains which showed cross-reactions between serovars O2 and O5 . The almost complete agreement found between the PyMS and the serological data suggests that pyrolysis mass spectrometry will prove to be an effective method for interstrain comparison within the species Listonella anguillara.

Mol Microbiol, 1997 Jan, 23(2), 323 - 31
A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains; Champion GA et al.; Co-ordinate expression of genes associated with pathogenicity in Vibrio cholerae requires two transcription activators, ToxR and ToxT . Work carried out to date suggests that ToxR activates transcription of the toxT gene and that ToxT directly activates transcription of several genes whose products play a role in colonization or CT production by V . cholerae . Previous work also suggests that ToxR can directly activate transcription of the CT operon (ctxAB) independently of ToxT, thereby implying a degree of complexity in control of the cixAB operon not found with other genes of the ToxR regulon . We tested the regulatory cascade model of virulence gene expression by constructing strains of classical and El Tor V . cholerae deleted for the coding sequence for the putative DNA-binding domain of toxT . Phenotypic analysis of these strains suggests that V . cholerae has ToxT-dependent and ToxT-independent branches of its virulence regulon . The results also raise questions about the precise role for ToxR in activation of ctxAB transcription.

Biochem Mol Biol Int, 1997 Jan, 41(1), 41 - 7
Genetic defect of the sodium pump-defective mutant Nap-1 from the marine Vibrio alginolyticus; Hayashi M et al.; The marine bacterium, Vibrio alginolyticus, has a respiratory chain-linked Na(+)-translocating NADH-quinone reductase (NQR) . Among several mutant cells defective in Na+ pump activity, Nap1 was a very stable mutant and a spontaneous revertant could not be isolated from Nap1 . Using genetic information from the recently sequenced nqr operon, the genetic defects in Nap1 were examined, and the sodium pump-defective mutant Nap1 was found to be caused by the insertion of a 1.2 kbp DNA fragment into the C-terminal region of nqr6 gene.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 96 - 101
Novel extracellular alkaline metalloendopeptidases from Vibrio sp . NUF-BPP1: purification and characterization; Fukuda K et al.; We found two types of novel alkaline metalloendopeptidases (AP1 and AP2) from a marine bacterium, isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) and identified as Vibrio sp . (NUF-BPP1) . We studied the structure-function relationship of these marine bacterial proteases . The electrophoretically homogeneous proteases had a molecular mass of 48 kDa for AP1 and 36 kDa for AP2 on SDS-PAGE, and showed optimum activity at around pH 9.5-10.0 (casein as substrate) . Calcium chloride (5 mM) stabilized the activities over pH 6-11, but o-phenanthroline and EDTA inhibited the activities of both AP1 and AP2 . The EDTA-inactivated proteases were partly restored to activity by addition of either zinc or calcium . Sodium chloride (3.5 M) increased the activities toward Z-Gly-Leu-NH2 . N-Terminal sites of hydrophobic amino acid residues (Leu, Ile, Phe, Tyr, and Trp) of the peptide substrates were cleaved by AP1 and by AP2 . Autolysis of AP1 in the absence of calcium ion probably produced AP2 by releasing a fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1.

Microbiology, 1997 Jan, 143 ( Pt 1), 135 - 45
An iron-regulated outer-membrane protein specific to Bordetella bronchiseptica and homologous to ferric siderophore receptors; Beall B et al.; The bfrA (Bordetella bronchiseptica ferric iron repressed outer-membrane protein) gene was cloned from Bordetella bronchiseptica by screening a library of TnphoA insertion mutants for iron-repressed fusions to phoA . The bfrA gene encoded an 80 kDa outer-membrane protein with a high level of amino acid sequence identity to several bacterial proteins belonging to the family of Ton B-dependent outer-membrane receptors . BfrA was especially homologous to Cir of Escherichia coli, IrgA of Vibrio cholerae and to three previously characterized ferric enterobactin receptors . DNA hybridization results indicated that bfrA was not present in other Bordetella species . Expression of the bfrA gene was induced by low iron availability from a promoter overlapped by a sequence resembling a consensus Fur-binding sequence, and bfrA expression was derepressed in a B . bronchiseptica fur mutant . Utilization of the Bordetella siderophore alcaligin and the exogenous siderophore enterobactin was unaffected in bfrA mutants . Upon attempting to find the specificity of BfrA, 2,3-dihydroxybenzoylserine (DHBS) was shown to be utilized in a bfeA (Bordetella ferric enterobactin receptor gene)-dependent manner by B . bronchiseptica and B . pertussis . In addition, the hydroxamate siderophores ferrichrome and desferrioxamine B, and the iron source haemin were shown to be utilized independently of bfeA and bfrA in B . bronchiseptica and B . pertussis.

Microbiology, 1997 Jan, 143 ( Pt 1), 23 - 34
Colonial opacity variations among the choleragenic vibrios; Finkelstein RA et al.; Cultures of Vibrio cholerae 01, biotype El Tor, from the current epidemic of cholera in the Western Hemisphere, and of the new V . cholerae serogroup O139, from the current outbreak in India and Bangladesh, revealed marked colonial heterogeneity when received by the authors . By comparison with reference colony types, using a stereoscope and transmitted oblique illumination, colonies of approximately 10 different degrees of opacity could be distinguished . In contrast, strains freshly isolated from patients and rapidly and carefully preserved were more homogeneous although still differentiable by this technique . These (and older) observations prompted the questions: (1) why is a V . cholerae colony opaque or translucent? and (2) what benefit is it to the vibrios to vary their colonial appearance? The observed changes in colonial opacity, which are reversible, are sometimes (rarely) accompanied by changes in virulence for infant rabbits and, more frequently, by other phenotypic variations including the ability to produce poly-beta-hydroxybutyrate inclusion bodies on glycerol-containing medium, the degree of encapsulation in 0139, changes in outer-membrane proteins, alteration in lipopolysaccharide structure, changes in expression of glycolytic pathways, and differences in ability to survive under adverse conditions . Colonial variations in choleragenic vibrios are phenotypically multifactorial . The genetic mechanisms(s) underlying the observed phenotypic changes remain to be defined.

FEMS Immunol Med Microbiol, 1997 Jan, 17(1), 21 - 5
Rapid and differential detection of two analogous enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli by a modified enzyme-linked immunosorbent assay; Koike N et al.; The principle of a novel ELISA (nylon-slip immuno-test, NSIT) was applied to the differential detection of two analogous enterotoxins, cholera toxin (CT) of Vibrio cholerae and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli . The results obtained for CT and LT detection by a single test were sufficiently sensitive (87.9 and 100%) and specific (100 and 94.7%) in the differential detection test, when compared with the result of a colony hybridization test with DNA probes . The results suggest that the novel ELISA is applicable to the diagnosis of bacterial infections, by means of differential immunological detection of toxins in a single test.

Protein Sci, 1997 Jan, 6(1), 13 - 23
Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap; Thoden JB et al.; Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer . When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C . This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO . 1994 . Kinetic partitioning during protein folding yields multiple native states . Nature Struct Biol 1: 320-326) . Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V . harveyi determined and refined at 1.95 A resolution . Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit . Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I . 1995 . Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution . Biochemistry 34: 6581-6586) . The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A . This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms . In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

J Pediatr, 1997 Jan, 130(1), 45 - 51
Hyperglycemia during childhood diarrhea; Ronan A et al.; OBJECTIVE: To determine the cause of hyperglycemia in childhood diarrhea . METHODS: During an 8-month period, patients admitted to a diarrhea treatment center in Bangladesh had their blood glucose concentrations determined . Sixteen patients aged 2 to 10 years with hyperglycemia (blood glucose concentration >10.0 mmol/L) and 20 patients in the same age group with a normal blood glucose concentration (3.3 to 9.0 mmol/L) had blood samples obtained on admission and 4 and 24 hours later for determination of glucoregulatory hormones and gluconeogenic substrates . RESULTS: Prevalence of hyperglycemia among patients aged 2 to 10 years was 9.4% . Compared with the normoglycemic patients, hyperglycemic patients more often had severe dehydration (100% versus 10%, p <0.001), infection with Vibrio cholerae 0 1 or toxigenic Escherichia coli (94% vs 25%, p <0.001), and had similar duration of fasting (16 vs 14 hours, p = 0.677) . Concentrations of epinephrine (7.15 vs 2.00 micromol/L), norepinephrine (10.35 vs 3.50 micromol/L), cortisol (1.38 vs 0.82 micromol/L), glucagon (36 vs 14 pmol/L), and C-peptide (1.22 vs 0.35 nmol/L) were all significantly (p < or = 0.014) higher in patients with hyperglycemia than in normoglycemic patients . CONCLUSIONS: The development of hyperglycemia in diarrhea is caused by a stress response to hypovolemia.

Int J Syst Bacteriol, 1997 Jan, 47(1), 144 - 9
Desulfonatronovibrio hydrogenovorans gen . nov., sp . nov., an alkaliphilic, sulfate-reducing bacterium; Zhilina TN et al.; A new alkaliphilic, sulfate-reducing bacterium, strain Z-7935T (T = type strain), was isolated from a soda-depositing lake, Lake Magadi in Kenya . This organism is a motile vibrio which utilizes only hydrogen and formate as electron donors and sulfate, sulfite, and thiosulfate, but not sulfur, as electron acceptors . Thiosulfate is dismutated . Strain Z-7935T is an obligately sodium-dependent alkaliphile which grows in sodium carbonate medium and does not grow at pH 7; the maximum pH for growth is more than pH 10, and the optimum pH is 9.5 to 9.7 . The optimum NaCl concentration for growth is 3% (wt/vol) . The optimum temperature for growth is 37 degrees C . The G + C content of the DNA is 48.6 mol% . 16S ribosomal DNA sequence analysis revealed that strain Z-7935T represents a new lineage with genus status in the delta subclass of the Proteobacteria . The name Desulfonatronovibrio hydrogenovorans gen . nov., sp . nov . is proposed for this organism; the type strain of D . hydrogenovorans is strain Z-7935 (= DSM 9292).

Int J Syst Bacteriol, 1997 Jan, 47(1), 58 - 61
Vibrio scophthalmi sp . nov., a new species from turbot (Scophthalmus maximus); Cerda-Cuellar M et al.; Six strains isolated from the intestines of juvenile turbot in a fish hatchery in the north of Spain were found to be phenotypically members of the genus Vibrio . However, the phenotypic traits of these organisms did not place them in any of the currently known Vibrio species . These isolates formed an homogeneous group in which the DNA-DNA similarity values (the differences between the thermal denaturation midpoints of the homologous and heterologous duplexes) with reference strain A089T (T = type strain) ranged from 0 to 1.7 degrees C . The results of a 16S rRNA sequence analysis of A089T placed this strain in the genus Vibrio in the gamma subclass of the Proteobacteria . The closest relative is Vibrio aestuarianus, with a sequence similarity of 97.8% . This group of strains can be easily differentiated from the other Vibrio species by their clear phenotype . We propose the name Vibrio scophthalmi sp . nov . for these strains; the type strain is strain A089 (= CECT 4638).

J Bacteriol, 1997 Jan, 179(2), 557 - 62
Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes; Stevens AM et al.; LuxR is required for cell density-dependent activation of the Vibrio fischeri luminescence (lux) genes . It has not been possible to study full-length LuxR in vitro, but a polypeptide containing the C-terminal transcriptional-activator domain of LuxR (LuxRdeltaN) has been purified, and its binding to lux regulatory DNA has been investigated . By itself, LuxRdeltaN interacts with a region of lux regulatory DNA that is upstream of the lux box, which is a 20-bp element that is required for LuxR activation of the luminescence operon . Individually, neither the purified LuxRdeltaN nor RNA polymerase binds to the lux box region, but together the two proteins bind in synergy to the lux box-luxI promoter region . We show that binding of LuxRdeltaN to the upstream region is not a prerequisite for its synergistic binding with RNA polymerase to the lux box and the luxI promoter region . We also show that LuxRdeltaN and RNA polymerase are both required and sufficient for transcriptional activation of the lux operon . This argues against the hypothesis that LuxR functions to alleviate repression of the lux operon by another cellular factor . Rather, our data support the view that LuxR functions as an accessory factor that enables RNA polymerase to bind to and initiate transcription from the promoter of the lux operon.

Arch Biochem Biophys, 1997 Jan 1, 337(1), 89 - 95
Vibrio harveyi NADPH:FMN oxidoreductase: preparation and characterization of the apoenzyme and monomer-dimer equilibrium; Liu M et al.; A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with > or =80% yields . The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 microM at 23 degrees C . The reconstituted holoenzyme was catalytically as active as the native enzyme . FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme . Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme . 2-ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding . The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2-thioFMN as a substrate . The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations . Both the holoenzyme and the apoenzyme was shown at 4 degrees C by equilibrium ultracentrifugation to undergo dimerization with dissociation constants of 1.8 and 3.3 microM, respectively.

J Bacteriol, 1997 Jan, 179(1), 293 - 6
Identification, sequencing, and enzymatic activity of the erythrose-4-phosphate dehydrogenase gene of Vibrio cholerae; Carroll PA et al.; We have identified a gene in Vibrio cholerae (epd) which encodes an erythrose-4-phosphate dehydrogenase activity and is located immediately downstream of an iron-regulated virulence gene, irgA, and immediately upstream of a gene encoding phosphoglycerate kinase (pgk) . Expression of epd in V . cholerae is not regulated by iron, nor is it required for virulence in an infant mouse model.

J Bacteriol, 1997 Jan, 179(1), 243 - 7
Purification of Vibrio cholerae fur and estimation of its intracellular abundance by antibody sandwich enzyme-linked immunosorbent assay; Watnick PI et al.; The Vibrio cholerae fur gene was previously cloned and sequenced . A putative Fur box was identified in the divergent promoters of irgA, a virulence factor of V . cholerae, and irgB, a transcriptional activator of irgA . In this work, V . cholerae Fur was overexpressed in Escherichia coli and purified to approximately 95% homogeneity . The purified protein bound a DNA fragment containing the irgA-irgB promoter in a gel shift assay . The purified protein was used to raise monoclonal and polyclonal antibodies to V . cholerae Fur, and a Fur sandwich enzyme-linked immunosorbent assay was developed to estimate the intracellular abundance of Fur under a variety of growth conditions . The number of Fur molecules per cell during exponential growth was approximately 2,500, which is higher than most measurements for other bacterial repressors but comparable to the intracellular concentration of the leucine-responsive regulatory protein . The number of Fur molecules per cell increased in the late logarithmic and stationary phases . Growth of V . cholerae in low-iron medium did not alter the intracellular abundance of Fur significantly . Growth under microaerophilic conditions resulted in a significant, approximately twofold decrease in the intracellular levels of Fur . The measurements of intracellular Fur abundance indicate that a large amount of this repressor is produced constitutively and that the concentration of Fur in the cell varies by less than a factor of 2 under the conditions studied . We hypothesize that the high constitutive expression of Fur is necessary for its role as an iron-responsive regulator.

J Bacteriol, 1997 Jan, 179(1), 107 - 14
The lonS gene regulates swarmer cell differentiation of Vibrio parahaemolyticus; Stewart BJ et al.; Vibrio parahaemolyticus differentiates from a polarly flagellated, short, rod-shaped cell known as the swimmer to the elongated, hyperflagellated, and multinucleated swarmer cell type when it is grown on a surface . The swarmer is adapted to movement over and colonization of surfaces . To understand the signal transduction mechanism by which the bacterium recognizes surfaces and reprograms gene expression, we isolated a new class of mutants defective in surface sensing . These mutants were constitutive for swarmer cell gene expression, inappropriately expressing high levels of a swarmer cell gene fusion product when grown in liquid . They showed no defect in the swimming motility system, unlike all previously isolated constitutive mutants which have defects in the alternate, polar motility system . The lesions in the majority of the newly isolated mutants were found to be in a gene, lonS, which encodes a polypeptide exhibiting 81% sequence identity to the Escherichia coli Lon protein, an ATP-dependent protease . Upstream sequences preceding the lonS coding region resemble a heat shock promoter, and the homology extends to sequences flanking lonS . The gene order appears to be clpX lonS hupB, like the organization of the E . coli locus . V . parahaemolyticus lonS complemented E . coli lon mutants to restore UV resistance and capsular polysaccharide regulation to that of the wild type . Vibrio lonS mutants were UV sensitive . In addition, when grown in liquid and examined in a light microscope, lonS mutant cells were extremely long and thus resembled swarmer cells harvested from a surface.

Curr Microbiol, 1997 Jan, 34(1), 38 - 42
Immunological reactivity of Vibrio anguillarum sero-subgroups O2a and O2b, and comparison of their lipopolysaccharide profiles; Tiainen T et al.; One hundred and twenty-nine Vibrio anguillarum serogroup O2 strains were compared by slide agglutination and Western blotting for their lipopolysaccharide (LPS) structure . The strains showed six different LPS profiles, four different reaction patterns in Western blotting, and four different kinds of reaction in slide agglutination, when both unabsorbed and absorbed anti-O2a and anti-O2b sera were used . All in all, nine different groups were detected when the combination of these three methods was applied . The two serological methods gave corresponding results for almost all strains (96%) . Most of these strains (84%) belonged to sero-subgroup O2a, while 12% of the strains belonged to sero-subgroup O2b . The remaining six strains had varying reactions in the used serological methods; therefore, their sero-subgroups could not be determined . These results suggest the existence of additional sero-subgroups within serogroup O2.

J Biol Chem, 1996 Dec 27, 271(52), 33468 - 75
Sugar transport by the marine chitinolytic bacterium Vibrio furnissii . Molecular cloning and analysis of the mannose/glucose permease; Bouma CL et al.; We have previously reported that the chitin catabolic cascade in Vibrio furnissii involves multiple signal transducing systems, and that mono- and disaccharide chemoreceptors/transporters are essential components of some of these systems . This and the accompanying papers (Bouma, C . L., and Roseman, S . (1996) J . Biol . Chem 271, 33457-33467; Keyhani, N . O., Wang, L.-X., Lee, Y . C., and Roseman, S . (1996) J . Biol . Chem . 271, 33409-33413) describe some of the sugar transporters . A 13-kilobase pair fragment of V . furnissii DNA was found to impart a Glc+, Man+ phenotype to Escherichia coli ptsG ptsM mutants, and encodes the mannose transporter, ptsM, of the phosphoenolpyruvate:glycose phosphotransferase system . Unlike the E . coli mannose permease, V . furnissii IIMan is inactive with GlcNAc and Fru, and is encoded by four genes rather than three . The gene order is manXYZW, where the product of manY corresponds to IIPMan, manZ to the mannose receptor IIBMan, and manX and manW to the single E . coli gene, manX (which encodes IIIMan, viz . IIAMan) . Thus, in V . furnissii, the E . coli manX equivalent comprises two genes, which are separated in the genome by two other genes of the ptsM complex . Two additional open reading frames were detected in the V . furnissii DNA fragment . One encodes a GlcNAc-6-P deacetylase, and the other is similar to aldolase.

J Biol Chem, 1996 Dec 27, 271(52), 33457 - 67
Sugar transport by the marine chitinolytic bacterium Vibrio furnissii . Molecular cloning and analysis of the glucose and N-acetylglucosamine permeases; Bouma CL et al.; Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose . We report the properties of the respective permeases that complemented E . coli Glc- Man- mutants . Although the V . furnissii Glc-specific permease (55,941 Da) shares 38% identity with E . coli IIGlc (ptsG), it is 67% identical to MalX of the E . coli maltose operon (Reidl, J., and Boos, W . (1991) J . Bacteriol . 173, 4862-4876) . An adjacent open reading frame encodes a protein with 52% identity to E . coli MalY . Glc phosphorylation requires only V . furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins . The V . furnissii equivalent of IIGlc was not found in the 25,000 transformants screened . The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E . coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro . While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V . furnissii and enteric bacteria, the differences may be important for survival of V . furnissii in the marine environment.

J Biol Chem, 1996 Dec 27, 271(52), 33433 - 9
Molecular cloning and characterization of a novel beta-N-acetyl-D-glucosaminidase from Vibrio furnissii; Chitlaru E et al.; The accompanying papers (Keyhani, N . O., and Roseman, S . (1996) J . Biol . Chem . 271, 33414-33424; Keyhani, N . O., and Roseman, S . (1996) J . Biol . Chem . 271, 33425-33432) describe two unique beta-N-acetylglucosaminidases from Vibrio furnissii . A third, ExoII, is reported here . The gene, exoII, was cloned into Escherichia coli, sequenced, and ExoII purified to apparent homogeneity (36 kDa) . The molecular weight and N-terminal 16 amino acids of the protein conform to the predicted sequence . ExoII exhibited unique substrate specificity . It rapidly cleaved p-nitrophenyl and 4-methylumbelliferyl beta-GlcNAc, was slightly active with p-nitrophenyl-beta-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N'-diacetylchitobiose and (GlcNAc)n, n = 3-6 . Unlike GlcNAc (Ki, 210 microM), (GlcNAc)n are poor inhibitors of ExoII . The predicted protein sequence is unique among beta-N-acetylglucosaminidases excepting Cht60, recently cloned from a marine Alteromonas (Tsujibo, H., Fujimoto, K., Tanno, H., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y . (1994) Gene (Amst.) 146, 111-115) . Cht60, a chitobiase, is 26.9% identical to ExoII in a 182-amino acid overlap, but the two enzymes differ in substrate specificity and other properties . ExoII shares similarity with five bacterial and yeast beta-glucosidases, up to 44% identity in the 25-amino acid catalytic domain . By analogy, ExoII may play a role in signal transduction between invertebrate hosts and V . furnissii.

J Biol Chem, 1996 Dec 27, 271(52), 33425 - 32
The chitin catabolic cascade in the marine bacterium Vibrio furnissii . Molecular cloning, isolation, and characterization of a periplasmic beta-N-acetylglucosaminidase; Keyhani NO et al.; We have described some steps in chitin catabolism by Vibrio furnissii, and proposed that chitin oligosaccharides are hydrolyzed in the periplasmic space to GlcNAc and (GlcNAc)2 . Since (GlcNAc)2 is an important inducer in the cascade, it must resist hydrolysis in the periplasm . Known V . furnissii periplasmic hydrolases comprise an endoenzyme (Keyhani, N . O . and Roseman, S . (1996) J . Biol . Chem . 271, 33414-33424), and the beta-N-acetylglucosaminidase, ExoI, reported here . ExoI was isolated from a recombinant strain of Escherichia coli, and hydrolyzes aryl-beta-GlcNAc, aryl-beta-GalNAc, and chitin oligosaccharides . No other beta-GlcNAc glycosides were cleaved . The pH optimum was 7.0 for (GlcNAc)n, n = 3-6, but 5.8 for (GlcNAc)2 . At the pH of sea water (8.0-8.3), the enzymatic activity with (GlcNAc)2 is virtually undetectable . These results explain the stability of (GlcNAc)2 in the periplasmic space . The cloned beta-GlcNAcidase gene, exoI, encodes a 69,377-kDa protein (611 amino acids); the predicted N-terminal 20 amino acid residues matched those of the isolated protein . The protein amino acid sequence displays significant homologies to the alpha- and beta-chains of human hexosaminidase despite their marked differences in substrate specificities and pH optima.

J Biol Chem, 1996 Dec 27, 271(52), 33414 - 24
The chitin catabolic cascade in the marine bacterium Vibrio furnissii . Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase; Keyhani NO et al.; Chitin catabolism in Vibrio furnissii comprises several signal transducing systems and many proteins . Two of these enzymes are periplasmic and convert chitin oligosaccharides to GlcNAc and (GlcNAc)2 . One of these unique enzymes, a chitodextrinase, designated EndoI, is described here . The protein, isolated from a recombinant Escherichia coli clone, exhibited (via SDS-polyacrylamide gel electrophoresis) two enzymatically active, close running bands ( approximately mass of 120 kDa) with identical N-terminal sequences . The chitodextrinase rapidly cleaved chitin oligosaccharides, (GlcNAc)4 to (GlcNAc)2, and (GlcNAc)5,6 to (GlcNAc)2 and (GlcNAc)3 . EndoI was substrate inhibited in the millimolar range and was inactive with chitin, glucosamine oligosaccharides, glycoproteins, and glycopeptides containing (GlcNAc)2 . The sequence of the cloned gene indicates that it encodes a 112,690-kDa protein (1046 amino acids) . Both proteins lacked the predicted N-terminal 31 amino acids, corresponding to a consensus prokaryotic signal peptide . Thus, E . coli recognizes and processes this V . furnissii signal sequence . Although inactive with chitin, the predicted amino acid sequence of EndoI displayed similarities to many chitinases, with 8 amino acids completely conserved in 10 or more of the homologous proteins . There was, however, no "consensus" chitin-binding domain in EndoI.

J Biol Chem, 1996 Dec 27, 271(52), 33409 - 13
The chitin catabolic cascade in the marine bacterium Vibrio furnissii . Characterization of an N,N'-diacetyl-chitobiose transport system; Keyhani NO et al.; The disaccharide N,N'-diacetyl-chitobiose, (GlcNAc)2, is critical in chitin dissimilation by Vibrio furnissii and, as reported here, is taken up by a specific permease . Since (GlcNAc)2 is rapidly catabolized by V . furnissii, a non-hydrolyzable thioglycoside analogue was used: methyl beta-N,N'-{3H}diacetyl-thiochitobioside (Me-{3H}TCB) . Me-TCB and TCB substitute for (GlcNAc)2 as chemoattractants and inducers of beta-N-acetylglucosaminidase activity . The {3H}Me-TCB uptake system was induced only by (GlcNAc)2 and by (GlcNAc)n that can be converted to (GlcNAc)2 . The Km for {3H}Me-TCB uptake was </=1 microM and was not affected by Na+ or K+ . Uptake appeared to be unidirectional, and in 0.4 M sucrose (+/- K+) the cells accumulated {3H}Me-TCB until it was depleted from the medium, giving an internal concentration of 0.1 M and an internal/external ratio > 1,000 . The only effective inhibitors of uptake were: (GlcNAc)n, n = 2-4 > cellobiose > (GlcNAc)5 . In 50% artificial sea water (or sucrose/Na+), {3H}Me-TCB accumulation attained a constant steady state level because of efflux, a Na+-dependent process . The physiological implications of these results are considered.

Science, 1996 Dec 20, 274(5295), 2025 - 31
Global climate and infectious disease: the cholera paradigm; Colwell RR; The origin of cholera has been elusive, even though scientific evidence clearly shows it is a waterborne disease . However, standard bacteriological procedures for isolation of the cholera vibrio from environmental samples, including water, between epidemics generally were unsuccessful . Vibrio cholerae, a marine vibrio, requiring salt for growth, enters into a dormant, viable but nonculturable stage when conditions are unfavorable for growth and reproduction . The association of Vibrio cholerae with plankton, notably copepods, provides further evidence for the environmental origin of cholera, as well as an explanation for the sporadic and erratic occurrence of cholera epidemics . On a global scale, cholera epidemics can now be related to climate and climatic events, such as El Nino, as well as the global distribution of the plankton host . Remote sensing, with the use of satellite imagery, offers the potential for predicting conditions conducive to cholera outbreaks or epidemics.

Biochim Biophys Acta, 1996 Dec 18, 1277(3), 201 - 8
Cloning and sequencing of novel genes from Vibrio alginolyticus that support the growth of K+ uptake-deficient mutant of Escherichia coli; Nakamura T et al.; Novel genes that functionally complement the growth of K+ uptake-deficient mutant strain of Escherichia coli TK420 have been cloned from the marine bacterium Vibrio alginolyticus . The nucleotide sequence revealed three open reading frames . The second gene was homologous to proC gene and allowed the growth of proC-defective mutant strain of E . coli chi 342 in the absence of proline . The first and third genes, but not proC, were required for the growth of TK420 in a synthetic medium containing 10 mM K+ and 100 mM Na+ . Since K+ uptake activity of TK420 was restored by the introduction of these genes, these two genes were considered to be directly related to K+ transport . Homologous genes were found in E . coli, but their functions have not been reported.

Biochemistry, 1996 Dec 17, 35(50), 16069 - 76
A pH-dependent conformational change in the B-subunit pentamer of Escherichia coli heat-labile enterotoxin: structural basis and possible functional role for a conserved feature of the AB5 toxin family; Ruddock LW et al.; The non-covalently associated B-subunit moieties of AB5 toxins, such as cholera toxin and related diarrheagenic enterotoxins, exhibit exceptional pH stability and remain pentameric at pH values as low as 2.0 . Here, we investigate the structural basis of a pH-dependent conformational change which occurs within the B5 structure of Escherichia coli heat-labile enterotoxin (EtxB) at around pH 5.0 . The use of far-UV CD and fluorescence spectroscopy showed that EtxB pentamers undergo a fully reversible pH-dependent conformational change with a pKa of 4.9 +/- 0.1 (R2 = 0.999) or 5.13 +/- 0.01 (R2 = 0.999), respectively . This renders the pentamer susceptible to SDS-mediated disassembly and decreases its thermal stability by 18 degrees C . A comparison of the pH-dependence of the structural change in EtxB5, with that of a mutant containing a Ser substitution at His 57, revealed that the pKa of the conformational change was shifted from ca . 5.1 to 4.4 . This finding suggests that protonation of the imidazole side chain of His 57 might facilitate disruption of a spatially adjacent salt bridge, located between Glu 51 and Lys 91 in each B-subunit, thus triggering the conformational change in the pentameric structure . The pH-dependent conformational change was found to be inhibited when B-subunits bound to monosialoganglioside, GMI; and to have no effect on the stability of interaction between A- and B-subunits within the AB5 complex . This suggests that the conformational change is unlikely to have a direct involvement in toxicity . Conservation of the pH-dependent conformational change in the AB5 toxin family, combined with the potential exposure of the hydrophobic core of beta-barrel in the monomeric units, leads to the proposal that the conformational change may be the common feature that ensures the secretion of these proteins from the Vibrionaceae.

Gene, 1996 Dec 12, 183(1-2), 255 - 7
Cloning and nucleotide sequencing of the protease gene of Vibrio vulnificus; Cheng JC et al.; The gene (vvp) encoding a thermolabile protease of Vibrio vulnificus was cloned and sequenced . The transcription start point was also determined by primer extension . The product of this gene is very likely the secretory neutral metalloprotease that has been purified and characterized previously.

Gene, 1996 Dec 12, 183(1-2), 237 - 42
Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies; Setterquist RA et al.; A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18 . Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins . A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA . This approach should allow rapid characterization of L18 from large numbers of bacteria . Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein . The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro . Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo . This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

Parassitologia, 1996 Dec, 38(3), 547 - 8
A rapid method for restriction analysis of large plasmids from enteric pathogens; Pazzani C et al.; A modified version of the method of Kado and Liu (J Bacteriol 1981, 145: 1365) has been developed for rapid detection and direct cleavage analysis of large plasmids from Vibrio cholerae and other enteric pathogens.

J Diarrhoeal Dis Res, 1996 Dec, 14(4), 248 - 54
Differentiation of Vibrio cholerae O1 isolates with biochemical fingerprinting and comparison with ribotyping; Ansaruzzaman M et al.; The Phene Plate (PhP) system is a commercially available typing system based on the measurements of kinetics of selected biochemical reactions of bacteria grown in liquid medium in 96-well microplates . The system uses numerical analysis to identify biochemical phenotypes among the tested strains . In the present study, a set of 16 discriminatory tests were used to differentiate 117 strains of Vibrio cholerae O1 from MExico and Bangladesh . The stability of PhP types of 16 isolates under different storage temperatures and after repeated subcultures were also evaluated . The PhP system had a reproducibility of 95% . Storage either at +4 degrees C or -70 degrees C, did not affect the reactions of the isolates, whereas 4 strains (25%) stored at room temperature and 5 strains (31%) subjected to 30 consecutive subcultures, exhibited minor changes in their biochemical reactions . Endemic isolates of V . cholerae O1 from Bangladesh were more diverse (diversity index = 0.84 to 0.93) than epidemic isolates from Mexico (diversity index = 0.73) . Using a collection of 33 heterogeneous isolates of classical biotype of vibrios, PhP typing and ribotyping were compared . PhP typing discriminated more types (n = 23) than ribotyping (n = 5), whereas a combination of both yielded 27 types . The PhP system appears to be a simple, reliable and highly discriminating method for typing of V . cholerae, and may prove especially useful as a first screening method in epidemiological studies of V . cholerae.

J Diarrhoeal Dis Res, 1996 Dec, 14(4), 243 - 7
Erythromycin and trimethoprim-sulphamethoxazole in the treatment of cholera in children; Kabir I et al.; To evaluate the efficacy of erythromycin and trimethoprim-sulphamethoxazole (TMP-SMX) in the treatment of cholera in children aged 1-8 years, a randomised clinical trial was conducted at a diarrhoea treatment centre in Bangladesh from December 1991 to June 1992 . Fifteen children received erythromycin, 50 mg/kg per day, in four equally divided doses, 18 children received 10 mg/kg per day of trimethoprim and 50 mg/kg per day of sulphamethoxazole in two equally divided doses (12 hourly) for five days, and 15 children received no antibiotic; children in all three groups received intravenous cholera saline for severe dehydration and for mild to moderate dehydration, a rice-based oral rehydration solution . The mean stool volumes in mL/kg body weight in the two treatment groups were less than that of the control group, and there were no significant differences in stool volume among the two treatment groups . However, 67% of the children in the erythromycin group and 82% in the TMP-SMX group recovered within 72 hours compared to 33% in the control group (p < 0.01) . Similarly, the bacteriological cures were 80% in the erythromycin group and 83% in the TMP-SMX group compared to only 27% in the control group (p < 0.001) . These results confirm that both erythromycin and trimethoprim-sulphamethoxazole are effective antimicrobials in the treatment of cholera . These drugs are of value specially in younger children in whom tetracycline is contraindicated or when the infecting Vibrio cholerae are resistant to tetracycline.

Int J Biochem Cell Biol, 1996 Dec, 28(12), 1365 - 9
Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus; Yoh M et al.; Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus . Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain . Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane . Phosphorylation of proteins was studies using {gamma-32P} ATP and SDS-PAGE . A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events . TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH . The estimated molecular weight of these proteins was 25 and 22.5 kDa . Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes . Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes . Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25kDa protein . In addition to phosphorylation, these protein kinase C inhibitors suppressed hemolysis by TDH . These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.

Zentralbl Veterinarmed B, 1996 Dec, 43(10), 579 - 84
Detection of Vibrio anguillarum by a sandwich enzyme-linked immunosorbent assay performed with monoclonal antibodies; Adone R et al.; Monoclonal antibodies (Mabs) against V . anguillarum were produced and characterized by Western blotting analysis, competitive binding assays and cross-reactivity tests . Their ability to detect V . anguillarum in a liquid culture was tested in a sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of these Mabs used as capture or tracer antibodies . One combination was selected as the most suitable for diagnostic applications, showing the highest sensitivity and specificity.

Kansenshogaku Zasshi, 1996 Dec, 70(12), 1234 - 41
{Isolation and incidence of Vibrio cholerae from river water}; Yamai S et al.; The prevalence of Vibrio cholerae contamination in river water derived from 20 sites of 18 rivers in Kanagawa, Japan, was investigated during a period from July to September, 1987, and from one of the 20 sites in August, 1988 and in February, 1989, V . cholerae non-O1 was found in all samples at concentrations of 0.9-->1,400 MPN/100 ml . Higher amounts of the organism were observed in the samples from estuaries . V . cholerae O1 was detected in samples collected in August, 1988 and in February, 1989 at concentrations of 150 MPN/100 ml and 1.5 MPN/100 ml, respectively . From 1989 to 1995, water samples were collected monthly from 10 sites of 10 rivers to detect V . cholerae . V . cholerae O1 and non-O1 were detected in 3.6% (30 of 840) and in 61.1% (513 of 840) in the water samples examined, respectively . Overall, V . cholerae was found in 62.9% (528 of 840) . Both types, O1 and non-O1, of organisms were detected in 15 samples . These results indicated that river water was contaminated frequently with V . cholerae non-O1 and sporadically with V . cholerae O1 throughout the year . Only one strain of V . cholerae O1 out of 543 V . cholerae strains was found to be a producer of cholera toxin . During these studies, the selectivity of 3 media for V . cholerae O1 was evaluated, and PMT agar was found to be the best.

Trop Med Int Health, 1996 Dec, 1(6), 854 - 8
Isolation of Vibrio cholerae O139 from the drinking water supply during an epidemic of cholera; Ramakrishna BS et al.; In mid-1994, the public water supply was investigated in a medium-sized town in south India during an epidemic of cholera due to Vibrio cholerae O139 . Vibrio cholerae O139 was isolated from the public water supply including one of the wells supplying the town, the central overhead tank, and domestic taps connected to the public supply . Following chlorination, the organism was no longer isolated from the water supply and the epidemic subsided . This demonstration of V . cholerae O139 in the drinking water supply of a town underlines the need for adequate treatment of the water supply.

Epidemiol Infect, 1996 Dec, 117(3), 471 - 8
Plasmid profiles, restriction fragment length polymorphisms and O-serotypes among Vibrio anguillarum isolates; Pedersen K et al.; A total of 279 Vibrio anguillarum strains were serotyped and examined for plasmid content . Plasmids were subjected to digestion with restriction enzymes . Most strains belonged to serogroup O1 (39%) and O2 (16%) . In total 164 strains (53%) carried plasmids . Of the O1 and O2 isolates, 92% and 30%, respectively, carried one or more plasmids . Restriction fragment length polymorphism (RFLP) analysis of plasmid DNA indicated that plasmids belonged to several groups . Each group seemed to be restricted to a single O-serovar . The largest group was the pJM1-like plasmids among most serovar O1 strains . Most of these plasmids were about 67 kb like the pJM1 plasmid, but various derivatives ranged from 26-77 kb . RFLP studies of the 67 kb plasmids revealed 17 different restriction patterns . Some patterns were dominant among European strains whereas others were dominant among North American strains . The results confirmed the applicability of O-serotyping together with plasmid profile and restriction analysis of plasmids for typing of V . anguillarum . They also indicated that plasmids among strains which belonged to the traditional fish pathogenic serogroups, O1 and O2, showed more homology than did strains from most other serogroups, that were usually non-pathogenic, environmental bacteria.

J Med Microbiol, 1996 Dec, 45(6), 494 - 500
Clinical manifestations and molecular epidemiology of five cases of diarrhoea in children associated with Vibrio metschnikovii in Arequipa, Peru; Dalsgaard A et al.; In April 1994, Vibrio metschnikovii was isolated from five infants with watery diarrhoea in Arequipa, Peru, as part of a passive cholera surveillance system . The children ranged in age from 11 to 20 months and had acute diarrhoea, with two cases showing moderate dehydration . Two children also had traces of blood in liquid stool . The children were seen at two different hospitals, and no evidence of a common source of infection was found . No additional V . metschnikovii isolates were identified in the remaining surveillance period that covered the rest of 1994 and 1995 . However, stool samples were not screened for enteric pathogens other than vibrios . V . metschnikovii strains isolated from stool samples produced opaque and translucent colonies on agar plates, suggesting capsular material . All isolates were resistant to ampicillin, erythromycin and streptomycin . Plasmid analysis revealed a common 200-kb plasmid in isolates from all cases and an additional 2.7-kb plasmid in three of the isolates . Ribotyping of each isolate after restriction with BglI and HindIII endonucleases demonstrated identical ribotyping patterns . The cases reported suggest that V . metschnikovii may be associated with diarrhoea in man by mechanisms so far unknown.

Infect Immun, 1996 Dec, 64(12), 5406 - 9
Cloning and characterization of the gene encoding the OmpU outer membrane protein of Vibrio cholerae; Sperandio V et al.; The OmpU outer membrane protein is a member of the ToxR regulon of Vibrio cholerae and has recently been shown to be a potential adherence factor for this species . Using PCR and degenerate oligonucleotide primers based on internal peptide sequences of purified OmpU, we have cloned and sequenced the gene encoding OmpU . The ompU gene is predicted to encode a 36,646-molecular-weight protein which is present in both cholera toxin-positive and -negative V . cholerae O1 and O139 strains.

Infect Immun, 1996 Dec, 64(12), 5233 - 8
Purification and characterization of a pilus of a Vibrio cholerae strain: a possible colonization factor; Yamashiro T et al.; A new flexible type of pilus was purified from Vibrio cholerae non-O1, non-0139 strain NAGV14 and characterized . The molecular mass of the pilin was estimated to be 20 kDa, and the antigenicity differed from that of known pili such as toxin-coregulated pili, mannose-sensitive hemagglutinating pili, V10 pili, and Al-1841 pili . The NAGV14 pilus was regarded as a colonization factor because the purified pili adhered to rabbit intestine and adhesion was inhibited by treating the organisms with the Fab fraction of an antipilus antibody . An intestinal receptor blockade using purified pili failed to inhibit adhesion of the organisms . The NAGV14 pili adhered to the surface of live V . cholerae . An antigen cross-reacting with the NAGV14 pili was widely and specifically distributed among V . cholerae strains irrespective of serotype and biotype . The amino acid sequence of the pilin was homologous with that of MshA . The NAGV14 pili did not agglutinate human and rabbit erythrocytes.

J Clin Microbiol, 1996 Dec, 34(12), 2968 - 72
Active surveillance for Vibrio cholerae O1 and vibriophages in sewage water as a potential tool to predict cholera outbreaks; Madico G et al.; The 1991 Peruvian cholera epidemic has thus far been responsible for 600,000 cholera cases in Peru . In an attempt to design a cholera surveillance program in the capital city of Lima, weekly sewage samples were collected between August 1993 and May 1996 and examined for the presence of Vibrio cholerae O1 bacteria and V . cholerae O1 bacteriophages (i.e., vibriophages) . During the 144 weeks of surveillance, 6,323 cases of clinically defined cholera were recorded in Lima . We arbitrarily defined an outbreak as five or more reported cases of cholera in a week . The odds of having an outbreak were 7.6 times greater when V . cholerae O1 was present in sewage water during the four previous weeks compared with when it was not (P < 0.001) . Furthermore, the odds of having an outbreak increased as the number of V . cholerae O1 isolations during the previous 4 weeks increased (P < 0.001) . The odds of having an outbreak were 2.4 times greater when vibriophages were present in sewage water during the four previous weeks compared with when they were not, but this increase was not statistically significant (P = 0.15) . The odds of having an outbreak increased as the number of vibriophage isolations during the previous 4 weeks increased (P < 0.05) . The signaling of a potential cholera outbreak 1 month in advance may be a valuable tool for implementation of preventive measures . In Peru, active surveillance for V . cholerae O1 and possibly vibriophages in sewage water appears to be a feasible and effective means of predicting and outbreak of cholera.

J Clin Microbiol, 1996 Dec, 34(12), 2904 - 8
Cloning and sequence of a region of Vibrio cholerae O139 Bengal and its use in PCR-based detection; Falklind S et al.; We isolated and characterized a Vibrio cholerae O139 Bengal-specific DNA region by arbitrary PCR . The fragment contains open reading frames encoding two potential glycosyltransferases possibly involved in capsular polysaccharide or lipopolysaccharide biosynthesis . In order to evaluate the possibility that this region could be used for the specific detection of V . cholerae O139 Bengal, a PCR system was established . The specificity and sensitivity of the PCR were investigated by analyzing 240 strains within the family Vibrionaceae and 178 stains of other gram-negative bacteria . All V . cholerae O139 Bengal strains tested were positive, and none of the 384 control strains were amplified . The sensitivity of the assay was 10(2) CFU/ml.

J Infect Dis, 1996 Dec, 174(6), 1364 - 8
Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is virulent for humans; Morris JG Jr et al.; Vibrio cholerae can shift to a "rugose" colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation . Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells . Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine; strains also showed resistance to killing by complement-mediated serum bactericidal activity . Six volunteers fed 10(6) cfu of a rugose variant of V . cholerae O1 El Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3) . Isolates recovered from the stool of infected volunteers retained the rugose phenotype . The data suggest that rugose strains cause human disease . The role of these strains in the epidemiology of cholera remains to be determined.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13683 - 8
A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the Euprymna-Vibrio mutualism; Weis VM et al.; Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts . It has been assumed that these specialized organs develop through a program of symbiosis-specific or -enhanced gene expression in one or both partners, but a clear example of this process has been lacking . In this study, we provide evidence for the enhanced production of an enzyme in the symbiotic organ of the squid Euprymna scolopes, which harbors a culture of the luminous bacterium Vibrio fischeri . Our data show that this enzyme has a striking biochemical similarity to mammalian myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine peroxidase that occurs in neutrophils . MPO and the squid peroxidase catalyze the same reaction, have similar apparent subunit molecular masses, and a polyclonal antibody to native human MPO specifically localized a peroxidase-like protein to the bacteria-containing regions of the symbiotic organ . We also provide evidence that a previously described squid cDNA encodes the protein (LO4) that is responsible for the observed dianisidine peroxidase activity . An antibody made against a fragment of LO4 immunoprecipiated dianisidine peroxidase activity from extracts of the symbiotic organ, and reacted against these extracts and human MPO in Western blot analysis . These data suggest that related biochemical mechanisms for the control of bacterial number and growth operate in associations that are as functionally diverse as pathogenesis and mutualism, and as phylogenetically distant as molluscs and mammals.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 17 - 22
Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA; Kar S et al.; An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04 . Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state . The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated . A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI . By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.

Gene, 1996 Nov 14, 179(2), 199 - 204
Cloning and characterization of the Actinobacillus actinomycetemcomitans gene encoding a heat-shock protein 90 homologue; Winston JL et al.; In a search for clones from a lambda gt11 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family . The gene appears to encode a homologue of HtpG, as the nucleotide sequence has approximately 70% identity with the Escherichia coli (Ec) and Vibrio fischeri htpG . Growth of an Aa htpG insertion mutant at 42 degrees C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant . These data suggest that Aa HtpG performs a function similar to Ec HtpG.

Mutat Res, 1996 Nov 11, 372(1), 115 - 8
Characterization of the adaptive response to ionizing radiation induced by low doses of X-rays to Vibrio cholerae cells; Basak J; Pretreatment with sublethal doses of X-rays induced an adaptive response in Vibrio cholerae cells as indicated by their greater resistance to the subsequent challenging doses of X-irradiation . The adaptive response was maximum following a pre-exposure dose of 1.7 Gy X-rays and an optimum incubation period of 40 min at 37 degrees C . Pre-exposure to a sublethal dose of 1.7 Gy X-rays made the Vibrio cholerae cells 3.38-fold more resistant to the subsequent challenge by X-rays . Pretreatment with a sublethal dose of hydrogen peroxide offered a similar degree of protection to the bacterial cells against subsequent treatment with challenging doses of X-ray radiation . However, exposure of Vibrio cholerae cells to mild heat (42 degrees C for 10 min) before X-ray irradiation decreased their survival following X-irradiation.

J Mol Biol, 1996 Nov 8, 263(4), 525 - 30
Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator; Langosch D et al.; Specific interactions between membrane spanning polypeptide segments are important for folding and oligomerisation of integral membrane proteins . Previously the dimerisation of glycophorin A has been shown to depend on interactions between its transmembrane segment by studying chimeric proteins in detergent solution . Here, we examined dimerisation of the glycophorin A transmembrane segment in a natural membrane employing the ToxR transcription activator from Vibrio cholerae . The ToxR protein is integral to the bacterial inner membrane and its activity requires a dimeric state . Therefore, the ToxR protein is suited to monitor quantitative homophilic interactions . We replaced the ToxR transmembrane segment with parts of the glycophorin A transmembrane segment containing the amino acid motif LIxxGVxxGVxxT previously shown to be sufficient for dimerisation in detergent solution . Expression of these chimeric proteins in an indicator strain resulted in strong transcription activation . This is indicative of efficient dimerisation mediated by the glycophorin transmembrane segment inserted into the inner membrane . Analysis of individual point mutants revealed that at least four residues out of this motif are critical for dimer formation in membranes . However, dimerisation of the glycophorin A transmembrane segment appears to be less sensitive to mutations when localised within a natural lipid bilayer compared to measurements in detergent solution . This may be related to a slightly altered structure of the dimer and/or to a higher local concentration and preorientation of the interacting molecules in a membrane . This makes the ToxR system well suited for probing low-affinity interactions between the transmembrane segments of other proteins.

Arkh Patol, 1996 Nov-Dec, 58(6), 51 - 5
{The effect of adrenoblockers on the ultrastructural changes in the epithelium and neural elements of the small intestine of suckling rabbits with experimental cholera}; Lomov IuM et al.; Infant rabbits (10-12-day old) were infected with El Tor 5879 culture via stomach . During the period of cholera vibrio adhesion (within 4 hr) and twice on the following day the animals were injected intraperitoneally with alpha- and beta-adrenoblockers pyrroxan and obsidan . It was established that mono- or combined therapy with adrenergic blocking agents prevents the vibrio adhesion, cholera vibrios were detected mainly in the small intestine lumen . In addition, adrenoblockers arrest adenylate cyclase activation realized through cAMP formation and subsequent diarrhea development.

Bioorg Med Chem, 1996 Nov, 4(11), 1919 - 28
Unexpected carbohydrate cross-binding by Escherichia coli heat-labile enterotoxin . Recognition of human and rabbit target cell glycoconjugates in comparison with cholera toxin; Karlsson KA et al.; The bacterial protein enterotoxins, cholera toxin (CT) of Vibrio cholerae and heat-labile toxin (LT) of Escherichia coli, induce diarrhea by enhancing the secretory activity of the small intestine of man and rabbit (animal model) . This physiological effect is mediated by toxin binding to a glycolipid receptor, the ganglioside GM1, Gal beta 3GalNAc beta 4(NeuAc alpha 3)GAl beta 4Glc beta 1Cer . However, LT, but not CT, was recently shown by us to bind also to paragloboside, Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, identified in the target cells . By molecular modeling of this tetrasaccharide in the known binding site of LT, the saccharide-peptide interaction was shown to be limited to the terminal disaccharide (N-acetyllactosamine) . This sequence is expressed in many glycoconjugates, and we have therefore assayed glycolipids and glycoproteins prepared from the target tissues . In addition to paragloboside, receptor activity for LT was detected in glycoproteins of human origin and in polyglycosylceramides of rabbit . However, CT bound only to GM1 . Two variants of LT with slightly different sequences, human (hLT) and porcine (pLT), were identical in their binding to target glycoproteins and polyglycosylceramides, but different regarding paragloboside, which was positive for pLT but negative for hLT . This difference is discussed on basis of modeling, taking in view the difference at position 13, with Arg in pLT and His in hLT . Although N-acetyllactosamine is differently recognized in form of paragloboside by the two toxin variants, we speculate that this sequence in human glycoproteins and rabbit polyglycosylceramides is the basis for the common binding . Much work remains, however, to clear up up this unexpected sophistication in target recognition.

Eur J Clin Microbiol Infect Dis, 1996 Nov, 15(11), 864 - 6
First European case of gastroenteritis and bacteremia due to Vibrio hollisae; Gras-Rouzet S et al.; Vibrio hollisae is a pathogenic Vibrio species known to cause gastroenteritis in humans after the consumption of shellfish . All cases of infection reported previously were restricted to the Atlantic and Pacific coasts of the United States . A case of gastroenteritis and bacteremia in a previously healthy 76-year-old man who ate cockles from the Quiberon Bay in Brittany, France, is described . This is the first report of Vibrio hollisae infection in Europe.

Lett Appl Microbiol, 1996 Nov, 23(5), 295 - 8
Detection of Hsp60 (Gro-EL)-like proteins in Vibrio parahaemolyticus and Vibrio species by western immunoblotting analysis; Koga T et al.; Detection of heat shock proteins in Vibrio parahaemolyticus was investigated by SDS-PAGE and Western immunoblotting procedure using an anti-Hsp 60 antibody . Results indicate that V . parahaemolyticus elicited at least one Hsp 60 (GroEL)-like protein with apparent molecular weight of about 58,000 (58 kDa) when submitted to a heat shift from 30 to 42 degrees C . Kanagawa phenomenon-positive and -negative strains of V . parahaemolyticus responded the same way . Six other Vibrio species also showed an increased synthesis of GroEL-like (58 kDa) protein after heat shock, while synthesis of 58 kDa protein of V . alginolyticus was at a similar level before and after heat shock . Vibrio nereis showed an increased synthesis of a 60 kDa GroEL-like protein.

Protein Expr Purif, 1996 Nov, 8(3), 381 - 9
Use of Vibrio spp . for expression of Escherichia coli enterotoxin B subunit fusion proteins: purification and characterization of a chimera containing a C-terminal fragment of DNA polymerase from herpes simplex virus type 1; Loregian A et al.; The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells . To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required . High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T . R., 1994, Protein Expression Purif . 5, 198-204) . However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae . In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1) . Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60 . Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57% . Purified EtxB-pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol . This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins.

J Gen Virol, 1996 Nov, 77 ( Pt 11), 2881 - 4
Characterization of Vibrio cholerae EIT or typing phage D10; Chakrabarti BK et al.; The Vibrio cholerae EITor typing phage D10 was characterized . The adsorption kinetics of the phage on V . cholerae MAK757 strain were biphasic in nature . Intracellular growth was characterized by an eclipse period, latent period and burst size which were 20 min, 25 min and 80 particles per cell respectively . The phage yield was dependent on the concentration and time of addition of DNA synthesis inhibitors such as nalidixic acid and novobiocin, and RNA synthesis inhibitors such as rifampicin . The 32+/-0.2 kb linear double-stranded DNA molecule has unique termini . A restriction map of the phage DNA was constructed with the enzymes BamHI, HindIII and PstI.

Eur J Immunol, 1996 Nov, 26(11), 2587 - 94
Identification of an immunodominant T cell epitope on cholera toxin; Cong Y et al.; Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal immunogen as well as a strong mucosal adjuvant to related and unrelated antigens . The mucosal immune response to CT is T cell dependent and MHC class II restricted . The epitopes on CT recognized by T cells have not been identified . The purpose of this study was to determine the fine specificity of T cell recognition of both the CT A subunit (CT-A) and the CT B subunit (CT-B) by using a range of synthetic peptides . After immunization with CT-B or CT-A in CFA subcutaneously, the peripheral lymph node T cells were stimulated with different synthetic peptides in vitro . The peptide specificity of T cell recognition was identified by assaying T cell proliferation and interleukin-3 production . T cells from C57BL/6 (H-2b) high responder mice recognized one immunodominant epitope (peptide 89-100) and one weak epitope (peptide 31-50) on CT-B and two epitopes (peptide 21-39 and 180-194) on CT-A . The immunization of C57BL/6 mice with synthetic immunodominant CT-B peptide 89-100 induced T cell immunity to the pentameric CT-B . Induction of tolerance to CTB peptide 89-100 by i.v . injection in high responder C57BL/6 mice induced unresponsiveness to mucosal immunization with CT, compatible with an immunodominant role for this T cell epitope.

Infect Immun, 1996 Nov, 64(11), 4795 - 801
Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus; Bilge SS et al.; Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood . We performed TnphoA mutagenesis on E . coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression . Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U . H . Stroeher, L . E . Karageorgos, R . Morona, and P . A . Manning, Proc . Natl . Acad . Sci . USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase . This open reading frame was designated rfbE(EcO157:H7) . The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E . coli . rfbE(EcO157:H7) is conserved in nontoxigenic E . coli O157 strains expressing a variety of other flagellar antigens but is not found in E . coli O55:H7 strains, which are more closely related to E . coli O157:H7 . Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E . coli O157:H7 parent, but they did not differ in other phenotypes . Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12 . We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E . coli rfb genes occurred independently in E . coli O157:H7 and unrelated O157 strains, and that the O side chain of E . coli O157:H7 lipopolysaccharide interferes with the adherence of E . coli O157:H7 to epithelial cells.

Infect Immun, 1996 Nov, 64(11), 4655 - 8
In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V . cholerae, trypsin, and other proteases; Nagamune K et al.; Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene . This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA) . The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158 . We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA . We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V . cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein . Along with this, the protease-processed HlyA showed drastically increased hemolytic activity . The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149 . Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity . These data suggest that the active El Tor cytolysin of V . cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.

Infect Immun, 1996 Nov, 64(11), 4495 - 500
Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene; Toma C et al.; The structural gene for the Vibrio cholerae leucine aminopeptidase (lap) was cloned and sequenced . The cloned DNA fragment contained a 1,503-bp open reading frame potentially encoding a 501-amino-acid polypeptide with a calculated molecular mass of 54,442 Da . The deduced amino acid sequence of the entire protein showed high homology with the sequence of Vibrio proteolyticus leucine aminopeptidase . The residues potentially involved in binding the zinc ions were completely conserved in the V . cholerae aminopeptidase as well as in the V . proteolyticus aminopeptidase . The recombinant protein was partially purified and characterized . The molecular mass was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 34 kDa, suggesting a processing of the protein to acquire the mature form . The protease showed maximum activity at pH 9.0 and was thermostable at 70 degrees C . The substrate leucyl-p-nitroanilide was cleaved by the protease, and its activity was inhibited by EDTA and bestatin . These results suggested that the protein was a leucine aminopeptidase . The PCR analysis of lap gene distribution showed that it was widely distributed among the V . cholerae strains . It was not present in the other species examined.

Curr Microbiol, 1996 Nov, 33(5), 287 - 91
The naturally transformable marine bacterium WJT-1C formally identified as "Vibrio" is a pseudomonad; Frischer ME et al.; A marine bacterial isolate, previously identified as Vibrio WJT-1C (ATCC 55351) and used as a model for investigating the process of natural transformation in the marine environment, has been further examined to determine its taxonomic identity . API 20E test strips, phenotypic testing, and flagellar staining had previously assigned the strain to the genus Vibrio, most closely related to V . campbelli . 16S rRNA analysis indicated that WJT-1C was in the Pseudomonas subgroup of the gamma proteobacteria . Bacteriophage typing and natural transformation with chromosomal DNA indicated that it was distinct from previously described marine transforming pseudomonads including Pseudomonas stutzeri strain JM300 . The importance and abundance of the Pseudomonas subgroup of the gamma proteobacteria in the environment suggest that these marine strains are well suited as model organisms for describing the process and importance of natural transformation in nature.

Carbohydr Res, 1996 Oct 31, 293(2), 173 - 94
Synthesis of eight glycosides of hexasaccharide fragments representing the terminus of the O-polysaccharide of Vibrio cholerae O:1, serotype Inaba and Ogawa, bearing aglycons suitable for linking to proteins; Ogawa Y et al.; The title substances were prepared from intermediate, fully acetylated alpha-trimethylsilylethyl (SE) glycosides . The latter were assembled in a blockwise manner, using as the glycosyl donor the alpha-glycosyl chloride of a disaccharide bearing two 4-azido-4-deoxy functions . Next, the azido groups in the assembled hexasaccharides were converted to the corresponding amines, and these were acylated with 4-O-benzyl-3-deoxy-L-glycero-tetronic acid in the presence of a water-soluble carbodiimide . The SE glycosides were then transformed to glycosyl imidates, and these were coupled with methyl 6-hydroxyhexanoate or methyl 2-(2-hydroxyethylthio) propionate . The aglycons in the glycosides thus obtained were then converted to the corresponding carboxylic acids or acyl hydrazides . Such compounds are suitable for linking to proteins to obtain neoglycoproteins.

Biochemistry, 1996 Oct 22, 35(42), 13531 - 9
Flavin reductase P: structure of a dimeric enzyme that reduces flavin; Tanner JJ et al.; We report the structure of an NADPH:FMN oxidoreductase (flavin reductase P) that is involved in bioluminescence by providing reduced FMN to luciferase . The 1.8 A crystal structure of flavin reductase P from Vibrio harveyi was solved by multiple isomorphous replacement and reveals that the enzyme is a unique dimer of interlocking subunits, with 9352 A2 of surface area buried in the dimer interface . Each subunit comprises two domains . The first domain consists of a four-stranded antiparallel beta-sheet flanked by helices on either side . The second domain reaches out from one subunit and embraces the other subunit and is responsible for interlocking the two subunits . Our structure explains why flavin reductase P is specific for FMN as cofactor . FMN is recognized and tightly bound by a network of 16 hydrogen bonds, while steric considerations prevent the binding of FAD . A flexible loop containing a Lys and an Arg could account for the NADPH specificity . The structure reveals information about several aspects of the catalytic mechanism . For example, we show that the first step in catalysis, which is hydride transfer from C4 of NADPH to cofactor FMN, involves addition to the re face of the FMN, probably at the N5 position . The limited accessibility of the FMN binding pocket and the extensive FMN-protein hydrogen bond network are consistent with the observed ping-pong bisubstrate--biproduct reaction kinetics . Finally, we propose a model for how flavin reductase P might shuttle electrons between NADPH and luciferase.

Mol Gen Genet, 1996 Oct 16, 252(5), 622 - 5
Autoinducer-independent mutants of the LuxR transcriptional activator exhibit differential effects on the two lux promoters of Vibrio fischeri; Sitnikov DM et al.; The LuxR protein is a transcriptional activator which, together with a diffusible small molecule termed the autoinducer {N-(3-oxohexanoyl)-L-homo-serine lactone}, represents the primary level of regulation of the bioluminescence genes in Vibrio fischeri . LuxR, in the presence of autoinducer, activates transcription of the luxICDABEG gene cluster and both positively and negatively autoregulates transcription of the divergently oriented luxR gene, activating transcription at low levels of autoinducer, and repressing synthesis at high autoinducer concentration . Seven LuxR point mutants which activate V . fischeri lux transcription in the absence of autoinducer (LuxR*) have been characterized . The LuxR* proteins activated transcription of the bioluminescence genes to levels 1.5-40 times that achieved by wild-type LuxR without autoinducer . All of the LuxR* mutants retained responsiveness to autoinducer . However, in each case the degree of stimulation in response to autoinducer was lower than that observed for wild-type LuxR . The LuxR* proteins retained the requirement for autoinducer for autoregulation of the luxR gene . We propose that the LuxR protein exists in two conformations, an inactive form, and an active form which predominates in the presence of autoinducer . The LuxR* mutations appear to shift the equilibrium distribution of these two forms so as to increase the amount of the active form in the absence of autoinducer, while autoinducer can still convert inactive to active species . The differential effects of the LuxR* proteins at the two lux promoters suggest that LuxR stimulates PluxR transcription by a different mechanism to that used at the PluxI promoter, implying that binding of LuxR to its binding site, known to be necessary for transcriptional activation, may not be sufficient.

Eur J Biochem, 1996 Oct 15, 241(2), 368 - 73
Kinetic characterization of the neutral protease vimelysin from Vibrio sp . T1800; Kunugi S et al.; The kinetics of the hydrolysis of dipeptide and tripeptide substrates by the recently discovered neutral protease from Vibrio species T1800 (vimelysin) were studied . In the pH dependence of the apparent second-order rate constant, the pKa2 value of vimelysin (approximately 6.5) was significantly lower than thermolysin (8.3), although the pKa1 (approximately 5.1) values were comparable (5.0) . The Kcat/Km(lim) parameter for hydrolysis of Fua-Gly-PheNH2 (Fua = furylacryloyl) was more than sevenfold greater than for Fua-Gly-LeuNH2 . This higher specificity for Fua-Gly-PheNH2 was deduced for both Kcat and Km parameters . Fua-Phe-PheNH2 showed the highest Kcat/Km(app) value of the six substrates studied . The discrimination between Phe/Leu at the P1' site was most evident when the P1 site was not sufficiently filled . Reflecting the characteristically high proteolytic activity of vimelysin at lower temperatures {Oda, K., Okayama, K., Okutomi, K., Shimada, M., Sato, R . & Takahashi, S . (1996) Biosci . Biotech . Biochem . 60, 463-467}, the Arrhenius plot of the apparent second-order rate constant for the hydrolysis of Fua-Gly-LeuNH2 showed an inverse temperature dependence; higher reaction rates were observed at lower temperatures . This was not merely due to the pKa shift nor to thermal denaturation of the enzyme coupling, but rather to the Kcat(app) parameter, which alone showed an inverse temperature dependence . A model containing two temperature-dependent forms of the active enzyme was postulated to explain this unique temperature dependence.

Gene, 1996 Oct 10, 175(1-2), 281 - 3
Cloning and characterization of dnaE, encoding the catalytic subunit of replicative DNA polymerase III, from Vibrio cholerae strain C6706; Franco AA et al.; We report that Vibrio cholerae (Vc) contains a gene homologous to Escherichia coli dnaE, the structural gene for the alpha (catalytic) subunit of replicative DNA polymerase III (PolIII) . Despite 24% amino acid (aa) differences in the encoded proteins, the Vc gene strongly complements an E . coli dnaE temperature sensitive (ts) mutant, indicating that all functional features essential for replication are conserved.

Gene, 1996 Oct 10, 175(1-2), 89 - 94
Construction and symbiotic competence of a luxA-deletion mutant of Vibrio fischeri; Visick KG et al.; Bioluminescence by the squid Euprymna scolopes requires colonization of its light organ by the symbiotic luminous bacterium Vibrio fischeri . Investigation of the genetic determinants underlying bacterial symbiotic competence in this system has necessitated the continuing establishment and application of molecular genetic techniques in V . fischeri . We developed a procedure for the introduction of plasmid DNA into V . fischeri by electroporation, and isolated a mutant strain that overcame the apparent restriction barrier between V . fischeri and Escherichia coli . Using the technique of electroporation in combination with that of gene replacement, we constructed a non-luminous strain of V . fischeri (delta luxA::erm) . In addition, we used the transducing phage rp-1 for the first time to transfer a chromosomal antibiotic resistance marker to another strain of V . fischeri . The luxA mutant was able to colonize E . scolopes as quickly and to the same extent as wild type . This result suggested that, at least during the initial stages of colonization, luminescence per se is not an essential factor for the symbiotic infection.

Vaccine, 1996 Oct, 14(15), 1459 - 65
Intestinal and systemic immune responses in humans after oral immunization with a bivalent B subunit-O1/O139 whole cell cholera vaccine; Jertborn M et al.; There is a need for an effective vaccine that can protect against cholera caused by either Vibrio cholerae O1 or by the new pandemic serotype O139 Bengal . An oral bivalent B subunit-O1/O139 whole cell (B-O1/O139 WC) cholera vaccine has been prepared by adding formalin-killed O139 vibrios to the recently licensed oral recombinant B-O1 WC vaccine . When tested in Swedish volunteers, this B-O1/O139 WC vaccine was found to be safe and immunogenic . Two vaccine doses given 2 weeks apart induced statistically significant, P < 0.05, mucosal IgA antibody responses in intestinal lavage fluid against cholera toxin in all of nine vaccinees and against both O1 and O139 vibrios in seven of nine cases . The intestinal responses were associated with similar high frequencies of intestine-derived antibody-secreting cell responses in peripheral blood to the different antigens . A third dose of vaccine given after 5-6 weeks did not result in any further increased response . All of 12 vaccinees responded with significant IgA and IgG antitoxin responses in serum associated with significant vibriocidal antibody titre rises against O1 vibrios in 10 cases (83%) and against O139 vibrios in eight vaccinees (67%) . The frequencies and magnitudes of the serological responses to the B subunit and O1 WC components were similar to those induced by the B-O1 WC vaccine . Thus, the O139 component of the vaccine induced intestinal and systemic antibacterial immune responses in the majority of the vaccinees, and its addition to the vaccine did not interfere with the immunogenicity of the B subunit or O1 WC components.

Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1651 - 4
Substrate specificity of a novel alcohol resistant metalloproteinase, vimelysin, from Vibrio sp . T1800; Takahashi S et al.; Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp . T1800 . The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates . Vimelysin cleaved mainly Pro7-Phe8 bond and slightly Tyr4-Ile5 bond in human angiotensin I . Vimelysin also cleaved mainly Phe24-Phe25 and Tyr16-Leu17 bonds, and slightly His5-Leu6, His10-Leu11, Ala14-Leu15, and Gly23-Phe24 bonds in oxidized insulin B-chain . The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied . The ratio of kcat/Km for Fua-Gly-Phe-NH2/Fua-Gly-Leu-NH2, Fua-Phe-Leu-NH2/Fua-Gly-Leu-NH2, and Fua-Phe-Phe-NH2/Fua-Gly-Leu-NH2 were 15.9, 27.8, and 59.0, respectively . These results indicate that vimelysin easily recognizes phenylalanine in P1' positions, which is different from thermolysin.

J Trop Pediatr, 1996 Oct, 42(5), 305 - 7
Outbreaks of cholera in Kathmandu Valley in Nepal; Ise T et al.; An analysis of the seasonal outbreak of diarrhoea in children in Kathmandu, Nepal, is reported . Vibrio cholera, 01 biotype El Tor Ogawa was the major cause of this epidemic . The pattern of spread suggested a waterborne infection related to contaminated river water and this was confirmed by a field survey . Although the mortality rate was low, younger children were more susceptible . Enteropathogenic E . coli seems to be a major cause for diarrhoea after cholera amongst children in this study.

J Okla State Med Assoc, 1996 Oct, 89(10), 349 - 52
Overwhelming sepsis with Vibrio vulnificus: a coastal pathogen in Oklahoma; Rotz LD et al.; Vibrio vulnificus has been associated with three main clinical syndromes; primary septicemia; wound infection, and gastroenteritis . This organism has increased virulence for persons with underlying medical conditions that predispose to iron overload or an impaired immune system . Since the organism proliferates more readily in warm, coastal waters, such infections are more commonly found in those regions . Infection can result from the ingestion of contaminated, undercooked seafood; contact of a wound with seawater; or a puncture wound sustained from a contaminated surface . Vibrio infections rarely occur in inland areas, but when they do occur, they are usually a result of the contact of wounds with contaminated, brackish water or the ingestion of raw shellfish . Because infections with this organism occur less frequently in non-coastal regions, the diagnosis may not be suspected initially in susceptible individuals and a delay of treatment may result . We present a case of V . vulnificus sepsis occurring in a man with underlying liver disease and a history of row oyster consumption in Oklahoma and discuss the clinical manifestations of primary sepsis with this organism as well as prevention strategies.

Infect Immun, 1996 Oct, 64(10), 4373 - 7
Development of a germfree mouse model of Vibrio cholerae infection; Butterton JR et al.; A mouse model of Vibrio cholerae infection was successfully developed with germfree mice . Three- to four-week-old germfree mice were orally inoculated with strains of V . cholerae to be tested and then moved to normal housing after inoculation . Stool culture, measurement of serum vibriocidal antibody titers, and determination of immune responses to the cholera toxin B subunit demonstrated that germfree mice are readily colonized by V cholerae and develop systemic and mucosal immune responses to antigens expressed by these organisms . Immune responses to the B subunit of Shiga toxin 1, which was expressed from a V . cholerae vaccine vector, were less pronounced . This model should be valuable for studying immune responses to V . cholerae infection and immunization, including responses to heterologous antigens expressed by cholera vector strains.

Infect Immun, 1996 Oct, 64(10), 4035 - 41
Purification and characterization of novel hemagglutinins from Vibrio mimicus: a 39-kilodalton major outer membrane protein and lipopolysaccharide; Alam M et al.; Two hemagglutinins (HAs) mediating the agglutinability to rabbit erythrocytes were isolated from 32-h culture supernatant of enterotoxigenic strain E-33 of Vibrio mimicus by ultrafiltration followed by gel filtration and anion-exchange column chromatography . The HAs were designated R-HA and C-HA on the basis of specific hemagglutinating activity towards rabbit erythrocytes only (R-HA) and towards chicken and rabbit erythrocytes (C-HA) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent staining with Coomassie brilliant blue revealed no detectable protein band and a single band of Mr 39,000 in the case of R-HA and C-HA, respectively . However, silver staining of the gel containing R-HA revealed the appearance of low-molecular-weight material . These two HAs differed from each other and from previously reported HA/protease in receptor specificity, molecular composition, and biochemical and immunochemical properties . No simple sugar other than glycoproteins, including mucin, inhibited hemagglutinating activities of both C-HA and R-HA . Rabbit antibody against R-HA or C-HA could agglutinate E-33 whole cells, implying a possible cell surface origin of the two HAs . The isolated E-33 lipopolysaccharide (LPS) or its polysaccharide moiety conferred biochemical and immunochemical properties identical to those of R-HA, confirming that the R-HA represents polysaccharide of LPS . The LPS preparations from heterologous strains of Vibrio mimicus and Vibrio cholerae non-O1 confirmed that the hemagglutinating ability is a common function of LPS . On the other hand, the antibody against C-HA specifically recognized a major outer membrane protein (OMP) with an Mr of around 39,000 in both homologous and heterologous strains of V . mimicus, suggesting an OMP origin of C-HA . Furthermore, the antibody recognized a major OMP with an Mr of around 37,000 in V . cholerae . Although the immunogenicity of LPS and OMP is well documented for important intestinal pathogens, the hemagglutinating properties of such attractive cell surface components are hitherto unrecognized and will definitely contribute towards understanding their role in bacterial adherence.

Ann Trop Med Parasitol, 1996 Oct, 90(5), 507 - 13
Efficacy of octreotide in diarrhoea due to Vibrio cholerae: a randomized, controlled trial; Abbas Z et al.; Although octreotide, a long-acting analogue of somatostatin, is currently used in the treatment of chronic secretory diarrhoea due to various causes, its role in the management of acute secretory diarrhoea is not well established . In the present study, therefore, the therapeutic value of octreotide in the management of cholera, a classical example of acute secretory diarrhoea, was investigated . During an outbreak of cholera, patients admitted with acute secretory diarrhoea of < or = 24 h duration and a purging rate > 100 ml/h were enrolled on the study and randomly assigned to octreotide (N = 17) and control (N = 16) groups . All 33 patients received intravenous fluid replacement and antibiotic treatment (200 mg ofloxacin twice daily for 3 days, by mouth) . Each patient in the octreotide group was also given a subcutaneous injection containing 100 micrograms octreotide every 8 h for a maximum of six doses . The stool output of each patient was recorded every hour until there had been none for an hour, which was taken as the endpoint . Mean (S.D.) total stool output was lower {6.56 (3.7) v . 9.7 (6.5) litres} and the mean (S.D.) duration of diarrhoea after admission was shorter {32.9 (15.6) v . 47.8 (22.3); P < 0.05} in the octreotide group than in the control group . However, as both groups generally had similar purging rates, the higher volume of stools from the control group was simply the result of the longer period of diarrhoea in this group . Octreotide therefore only decreased the duration of diarrhoea in the cholera patients.

New Microbiol, 1996 Oct, 19(4), 321 - 6
Morphological, physico-chemical and biological variations in Vibrio anguillarum cultured at low osmolarity; Piccininno G et al.; The Authors studied the morphological, biochemical, physico-chemical and biological characteristics of Vibrio anguillarum cultured on different growth conditions, characterized by low osmolarity and high temperature (37 degrees) . One culture was subcultured for several days in tryptone soya agar with 0.5% Nacl at 37 degrees C incubation until the cell morphology was stabilized . The low osmolarity, through an osmotic shock, induced remarkable morphological modifications in the strain, evidenced by optic and electron microscopic studied; in addition SDS-PAGE analysis of saline extracts from the culture at 37 degrees C showed a specific new protein band of about 66KDa . This band was correlated with remarkable differences in outer membrane protein composition (OMPs) evidenced by Ag/Ah cross-reactions with rabbit hyperimmune sera against the modified and the reference V . anguillarum strains . Finally, the modified strain proved to be non pathogenic for trout and sea bass.

Eur J Epidemiol, 1996 Oct, 12(5), 461 - 9
Cholera: foodborne transmission and its prevention; Estrada-Garcia T et al.; The last several years have witnessed a tremendous increase in reported cholera cases across the globe . The explosive arrival of the seventh cholera pandemic in Latin American in 1991, dramatic epidemics of cholera on the Indian subcontinent and in Southeast Asia due to the newly recognized Vibrio cholerae O139 strain, and the often deadly presence of cholera among populations affected by political and social upheaval in Africa and Eastern Europe are evidence that many countries have failed to adopt effective measures for cholera prevention and control . Foodborne transmission of cholera has been well documented by epidemiologic investigations in nearly every continent, and its interruption is a critical component to any integrated programme for cholera prevention and control . We emphasize clear and effective guidelines for the prevention of foodborne cholera transmission that are drawn from a comprehensive review of relevant epidemiologic and laboratory data.

Mol Microbiol, 1996 Oct, 22(1), 127 - 34
The AngR protein and the siderophore anguibactin positively regulate the expression of iron-transport genes in Vibrio anguillarum; Chen Q et al.; Vibrio anguillarum virulence is associated with the presence of a plasmid-mediated iron-uptake system expressed under iron-limiting conditions, which consists of the siderophore anguibactin and specific iron-transport proteins . This system is maximally expressed under iron-limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE-like enzymatic function that may play a role in anguibactin biosynthesis . In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron-transport genes fatA and fatB . We also show that transcription of angR is repressed by Fur under iron-rich conditions . In addition, we present evidence that anguibactin itself enhanced transcription of the iron-transport genes fatA and fatB, independently of AngR and the trans-acting factor (TAF) product(s) . The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron-transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.

Protein Sci, 1996 Oct, 5(10), 2130 - 2
Crystallization and preliminary X-ray analysis of aldehyde dehydrogenase from Vibrio harveyi; Croteau N et al.; Aldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long-chain aliphatic aldehydes to acids . The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+ . The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three-dimensional structure that may be correlated to cofactor specificity . Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique . In order to improve crystal size and quality, iterative seeding techniques were employed . The crystals belong to space group P2(1), with unit cell dimensions a = 79.4 A, b = 131.1 A, c = 92.2 A, and beta = 92.4 degrees . Freezing the crystal to 100 K has enabled a complete set of data to be collected using a rotating anode source (lambda = 1.5418 A) . The crystals diffract to a minimum d-spacing of 2.6 A resolution . Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit . Self-rotation functions show the presence of 3 noncrystallographic twofold symmetry axes.

Microbiology, 1996 Oct, 142 ( Pt 10), 2879 - 85
The O polysaccharide chain of the lipopolysaccharide from Vibrio cholerae O76 is a homopolymer of N-{(S)-(+)-2-hydroxypropionyl}-alpha-L-perosamine; Kondo S et al.; Chemical and serological studies of LPS from Vibrio cholerae O76 (O76) were performed . The LPS of O76 contained D-glucose, D-galactose, L-glycero-D-manno-heptose, D-fructose, D-glucosamine, D-quinovosamine (2-amino-2,6-dideoxy-D-glucose) and L-perosamine (4-amino-4,6-dideoxy-L-mannopyranose) . The sugar composition of the LPS from O76 was quite similar to that of LPS from V . cholerae O1 with the exception of the presence of a small amount of D-galactose in the LPS of O76 . However, perosamine, a major sugar component of the LPS from O76, was in the L configuration in contrast to the D configuration of the perosamine in the LPS of V . cholerae O1 . The L-perosamine was N-acylated with an (S)-(+)-2-hydroxypropionyl group in the LPS from O76 . Structural analysis by NMR spectroscopy, as well as GC/MS, revealed that the O polysaccharide chain of the LPS from O76 was an alpha(1-->2)-linked homopolymer of N-{(S)-(+)-2-hydroxypropionyl}-L-perosamine . The serological cross-reactivity between the LPS of O76 and the LPS from other strains, such as V . cholerae O1 (Ogawa and Inaba O forms), Vibrio bio-serogroup 1875 (Original and Variant strains), V . cholerae O140 (Hakata) and Yersinia enterocolitica O9, was examined in passive haemolysis tests with sheep red blood cells that had been sensitized with LPS and antisera raised against whole cells of these bacteria . The latter six strains have in common the O antigen that includes Inaba antigen factor C, in addition to their own O-antigenic factors . Thus, they crossreact serologically . The O polysaccharide chains of the LPS of these six trains are known to consist exclusively of alpha(1-->2)-linked D-perosamine homopolymers and differences are found only among the N-acyl substituents . In passive haemolysis tests, the LPS of O76 did not cross-react serologically with any of the other LPS examined . Thus, the results obtained in this study support the hypothesis that Inaba antigen factor C, associated with the O antigens of these six strains, which include V . cholerae O1, is related substantially and exclusively to their alpha(1-->2)-linked homopolymers of N-acylated D-perosamine, and not to such homopolymers of N-acylated L-perosamine.

Microbiology, 1996 Oct, 142 ( Pt 10), 2777 - 83
Chemotactic responses to an attractant and a repellent by the polar and lateral flagellar systems of Vibrio alginolyticus; Homma M et al.; Chemotactic responses in Vibrio alginolyticus, which has lateral and polar flagellar systems in one cell, were investigated . A lateral-flagella-defective (Pof+ Laf-) mutant, which has only a polar flagellum, usually swam forward by the pushing action of its flagellum and occasionally changed direction by backward swimming . When the repellent phenol was added, Pof+ Laf- cells moved frequently forward and backward (tumbling state) . The tumbling was derived from the frequent changing between counter-clockwise and clockwise (CW) rotation of the flagellar motor, as was confirmed by the tethered-cell method . Furthermore, we found that the tumbling cells did not adapt to the phenol stimulus . When the attractant serine was added, the phenol-treated cells ceased tumbling and swam smoothly, adapting to the attractant stimulus after several minutes . We isolated chemotaxis-defective (Che-) mutants from the Pof+ Laf- mutant; the tumbling mutants were not isolated . One interesting mutant swam backwards continuously, with its flagellum leading the cell and its flagellar motor rotating CW continuously . A polar-flagella-defective mutant (Pof- Laf+) stopped swimming after phenol addition and then recovered swimming ability within 10 min, indicating that lateral flagella can adapt to the repellent stimulus . This may represent a functional difference between the two flagellar systems in Vibrio cells, and between the chemotaxis systems affecting the two types of flagella.

Microbiology, 1996 Oct, 142 ( Pt 10), 2767 - 76
Motility mutants of Vibrio cholerae O1 have reduced adherence in vitro to human small intestinal epithelial cells as demonstrated by ELISA; Postnova T et al.; Vibrio cholerae must colonize the human small intestine to cause diarrhoeal disease . V.cholerae strains N16961 (EI Tor, Inaba) and 395 (classical, Ogawa) adhered to the epithelial cell surface and the mucus layer of isolated human small intestinal epithelial cells . They adhered specifically to the mucosa and apical membrane in thin sections of small intestine . No binding to the basolateral membrane of dissected epithelial tissue or to intracellular components of the epithelial cells was observed by either light or indirect immunofluorescence microscopy . Based on these results, a modified ELISA was developed to quantitatively study adherence of V . cholerae to human small intestinal epithelial cells . The assay used homogenized human small intestinal mucosal tissue as the substrate for binding . Treatment of the epithelial cell homogenate with 2-mercaptoethanol to disrupt protein and glycoprotein secondary structure inhibited the binding of V . cholerae strains, suggesting that binding was to specific receptors . Several V . cholerae strains and mutants from both biotypes were tested for adherence in the modified ELISA . Wild-type strains of both biotypes and non-enterotoxigenic strains, which were known to colonize humans, adhered . V . cholerae mutants defective in motility, flagellar structure of chemotaxis, which were known to exhibit reduced colonization in animal models, exhibited decreased adherence . The specificity of the assay and its ability to quantify binding should facilitate identification and the study of adherence factors involved in the colonization of human small intestinal epithelial cells by V . cholerae.

J Clin Microbiol, 1996 Oct, 34(10), 2537 - 43
Temporal shifts in traits of Vibrio cholerae strains isolated from hospitalized patients in Calcutta: a 3-year (1993 to 1995) analysis; Mukhopadhyay AK et al.; This study presents results of a surveillance on cholera conducted with hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, from January 1993 to December 1995 . The O139 serogroup of Vibrio cholerae dominated in 1993 but was replaced by O1 as the dominant serogroup in 1994 and 1995 . The isolation rate of V . cholerae non-O1 non-O139 did not exceed 4.9% throughout the study period, while the isolation rate of the O139 serogroup in 1994 and 1995 was below 9% . No temporal clustering of any non-O1 non-O139 serogroup was observed . With the exception of 1 strain, none of the 64 strains belonging to the non-O1 non-O139 serogroup hybridized with ctx, zot, and ace gene probes, while 97.3 and 97.7% of the O139 and O1 strains, respectively, hybridized with all the three probes . Multiplex PCR studies revealed that all the O1 strains belonged to the EIT or biotype . There was a progressive increase in the cytotoxic response on CHO and HeLa cells evoked by culture supernatants of strains of V . cholerae non-O1 non-O139 isolated during 1994 and 1995 compared with the response evoked by those isolated in 1993 . Dramatic shifts in patterns of resistance to antibiotics between strains of V . cholerae belonging to different serogroups and within strains of a serogroup isolated during different time periods were observed . There was a discernible increase in the incidence of multidrug-resistant strains of V . cholerae O1 isolated in 1994 and 1995 compared with that in 1993 . On the basis of the results of this study, we predict the possibility of newer variants of V . cholerae emerging in the future.

Epidemiol Infect, 1996 Oct, 117(2), 385 - 91
Restriction fragment length polymorphism of the pMJ101-like plasmid and ribotyping in the fish pathogen Vibrio ordalii; Pedersen K et al.; A total of 32 Vibrio ordalii strains were studied for their plasmid content and shown to carry a plasmid of approximately 32 kb . This plasmid was subsequently subjected to restriction fragment length polymorphism (RFLP) studies . Using Hind III, three different restriction patterns were identified while BamH I cleaved the plasmid into a single linear fragment . The results suggest that the 32 kb plasmid is highly conserved but that some variation in restriction pattern occurs . The same set of strains was subjected to ribotyping . Using Mlu I, six different restriction patterns were demonstrated . Strains from the USA and Canada shared profiles with strains from Australia and Japan . Strains from Australia generated a single pattern whereas strains from North America were subdivided into three patterns, and the Japanese strains fell into five patterns . The results suggest that ribotyping in combination with RFLP studies of the pMJ101-like plasmid may be useful in epidemiological studies of V . ordalii.

Am J Gastroenterol, 1996 Oct, 91(10), 2241 - 2
Vibrio cholerae 01 isolated in the gallbladder of a patient presenting with cholecystitis; Asnis DS et al.; Cholera is a topical infection of the intestinal tract and rarely causes extraintestinal disease . The gallbladder has been proposed to be the reservoir of this organism . We present a patient from Bolivia who developed symptoms of acute cholecystitis and whose bile culture grew Vibrio cholerae 01 El tor.

J Am Coll Surg, 1996 Oct, 183(4), 329 - 34
Primary skin infections secondary to Vibrio vulnificus: the role of operative intervention; Halow KD et al.; BACKGROUND: Vibrio vulnificus can cause rapidly spreading skin and soft tissue infections with significant associated morbidity and mortality . Patients with underlying chronic illness, such as cirrhosis, diabetes mellitus, or immunosuppression, have been noted to be at high risk for rapid progression of this infection . The importance of early antibiotic therapy has been reported but the role of operative intervention in these patients is less clear . STUDY DESIGN: We report seven patients who were operatively treated from April 28, 1991 to September 22, 1995 for primary skin and soft tissue infections secondary to Vibrio vulnificus . We have also reviewed the recent literature . The impact of several variables including shock, increased white blood cell count, fever, and the timing of operative intervention on the length of hospital stay and intensive care unit stay was analyzed using the Spearman rank correlation . The impact of early compared with late operative intervention was analyzed using the Mann-Whitney U test . All patients had a history of underlying chronic illness and presented with skin infections in association with recent exposure to saltwater and to shellfish . No patient presented earlier than 24 hours from the time of initial infection . All patients underwent operative exploration within 46 hours of admission with thorough operative debridement of all necrotic tissue . Infection was confined to the skin and subcutaneous tissue . RESULTS: There was no mortality among our patients . The presence of perioperative shock, fever, or elevated white blood cell count did not correlate with an increased intensive care unit stay or an increased hospital stay . Earlier operative exploration and debridement correlated with a decrease in the intensive care unit stay (p < 0.02, correlation coefficient = 0.991) and in the hospital stay (p < 0.02, correlation coefficient = 0.929) . Patients who underwent debridement within 72 hours from the time of the infection had a significantly shorter intensive care unit stay (p = 0.0323) and total hospital stay (p = 0.0339) . CONCLUSIONS: We advocate operative exploration and thorough debridement of all necrotic tissue in high-risk patients with primary Vibrio vulnificus skin and soft tissue infections.

Appl Environ Microbiol, 1996 Oct, 62(10), 3875 - 7
Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica); Motes ML et al.; Oysters naturally contaminated with 10(3) to 10(4) most probable numbers (MPN) of Vibrio vulnificus per g were relayed to offshore waters (salinity, 30 to 34 ppt), where they were suspended in racks at a depth of 7.6 m . V . vulnificus counts in oysters were reduced to < 10 MPN/g within 7 to 17 days in five of the six studies . At the end of the studies (17 to 49 days), V . vulnificus levels were reduced further and ranged from a mean of 0.23 to 2.6 MPN/g . Oyster mortalities during relaying were < 6% . The reduction of V . vulnificus in relayed oysters is associated with exposure to high-salinity environments essentially devoid of V . vulnificus . Offshore suspension relaying may be a method that industry can employ to reduce V . vulnificus levels in raw Gulf Coast oysters.

Appl Environ Microbiol, 1996 Oct, 62(10), 3871 - 4
Expression of virulence-related properties by, and intestinal adhesiveness of, Vibrio mimicus strains isolated from aquatic environments; Alam M et al.; A study of the major pathogenic characteristics of Vibrio mimicus was carried out with 77 strains isolated from aquatic environments in Okayama, Japan . Of the strains tested, 96% demonstrated in vitro adherence to the rabbit intestinal mucosa, of which 36, 20, and 43% belonged to the strongly, moderately, and weakly adhesive groups, respectively . Of the 27 strains which appeared to be enterotoxigenic in the experiments using rabbit ileal loops, 74% belonged to the strongly adhesive group . All strains of V . mimicus at early log phase showed cell-mediated hemagglutination, and 70% of strongly hemagglutinative strains belonged to the strongly adhesive group, implying a possible correlation between cell-mediated hemagglutination and bacterial adherence . However, no significant correlation could be detected in the production of putative exocellular pathogenic factors and bacterial adherence or enterotoxigenicity.

Appl Environ Microbiol, 1996 Oct, 62(10), 3727 - 31
Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange; Singer JT et al.; A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775 . Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V . anguillarum H775-3 . A recombinant cosmid carrying the recA gene of V . anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101 . The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V . anguillarum H775-3 by conjugal mobilization . Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant . V . anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E . coli and V . anguillarum . These results indicate that recA is required for DNA repair in V . anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.

Appl Environ Microbiol, 1996 Oct, 62(10), 3650 - 4
Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilt-head sea bream (Sparus aurata L.); Bordas MA et al.; The kinetics of adhesion of Vibrio strains isolated from diseased fish to skin mucus of gilt-head sea bream was studied . A modified Langmuir adsorption isotherm was calculated, and the results obtained indicate that the strains tested (Vibrio alginolyticus DP1HE4 and Vibrio anguillarum-like DC12R8 and DC12R9) showed a saturation kinetics except for V . alginolyticus (CAN), which showed a proportional adsorption kinetics . The adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important . However, the absence of an inhibitory effect of mucus on the pathogenic microorganisms, and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured, stressed fish.

Appl Environ Microbiol, 1996 Oct, 62(10), 3605 - 13
Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil; Rabus R et al.; A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines . Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m-xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m-isopropyltoluene within approximately 2 months . In anaerobic counting series, the highest numbers of CFU (6 x 10(6) to 8 x 10(6) CFU ml-1) were obtained with toluene and benzoate . Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp . In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil . Desulfovibrio species were detected only at low numbers (3 x 10(5) CFU ml-1) . In agreement with this finding, a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (< 5%) of the cells in the enrichment culture . These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture . All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family) . Most members of this family are complete oxidizers . For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange . Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture . The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than Desulfovibrio species have to be considered in attempts to understand the microbiology of sulfide production in oil wells, tanks, and pipelines when no electron donors other than the indigenous oil constituents are available.

Appl Environ Microbiol, 1996 Oct, 62(10), 3572 - 80
Pulsed-field gel electrophoresis and ribotype profiles of clinical and environmental Vibrio vulnificus isolates; Tamplin ML et al.; Vibrio vulnificus belongs to the autochthonous bacterial flora of warm estuarine waters . It can cause life-threatening extraintestinal disease in persons who have underlying illness and who consume raw shellfish or contact wounds with estuarine water . Currently, very little is known about genetic diversity within this species . In this report, we describe high-level variation in restriction fragment length polymorphism profiles among 53 clinical and 78 environmental isolates, as determined by pulsed-field gel electrophoresis . In contrast, ribotype profiles showed greater similarity . When combined ribotype profiles of clinical and environmental isolates were analyzed, four predominant clusters were observed . Interestingly, a low number (16%) of clinical isolates were found in cluster C, compared with clusters A, B, and D (range, 50 to 83%) . In addition, 83% of all Hawaiian isolates were located in a single cluster, indicating a possible relationship between geography and genotype . We also report that spontaneous translucent colonial morphotypes were distinct by both restriction fragment length polymorphism and biochemical profiles, compared with opaque parent strains.

Arch Microbiol, 1996 Oct, 166(4), 234 - 44
Characterization of csmB genes, encoding a 7.5-kDa protein of the chlorosome envelope, from the green sulfur bacteria Chlorobium vibrioforme 8327D and Chlorobium tepidum; Chung S et al.; The csmB gene, encoding the 7.5-kDa "Gerola-Olson" protein of chlorosomes, has been cloned and sequenced from the green sulfur bacteria Chlorobium vibrioforme strain 8327D and Chlorobium tepidum . Two potential start codons were identified, and the csmB gene may be translated into a preprotein with an amino-terminal extension . Two forms of the mature CsmB protein (74 or 75 amino acids in length) were identified that differ by the presence or absence of a methionine residue at the amino terminus . The csmB gene of Chl . tepidum is transcribed as an abundant monocistronic mRNA of approximately 350 nucleotides; primer extension mapping of the 5' endpoint of the csmB mRNA suggests there is strong similarity between the csmB promoter and the sigma70 promoters of Escherichia coli . The CsmB protein of Chl . tepidum was overproduced as a histidine-tagged fusion protein in E . coli, purified to homogeneity by Ni2+ chelation affinity chromatography, and used to raise polyclonal antibodies in rabbits . Protease susceptibility mapping and agglutination experiments with isolated chlorosomes using anti-CsmB antibodies indicate that the CsmB protein is a component of the chlorosome envelope.

J Biol Chem, 1996 Sep 20, 271(38), 23487 - 94
Luciferase assembly after transport into mammalian microsomes involves molecular chaperones and peptidyl-prolyl cis/trans-isomerases; Brunke M et al.; The assembly of a heterodimeric luciferase was studied after de novo synthesis of corresponding precursor proteins in reticulocyte lysate and concomitant transport into dog pancreas microsomes . This cytosolic luciferase from a prokaryotic organism (Vibrio harveyi) was specifically used as a model protein to investigate (i) whether the eukaryotic cytosol and the microsomal lumen have similar folding capabilities and (ii) whether the requirements of a polypeptide for certain molecular chaperones and folding catalysts are determined by the polypeptide or the intracellular compartment . The two luciferase subunits were fused to the preprolactin signal peptide . Data indicate that efficient assembly of luciferase occurs in the mammalian microsomes . Furthermore, it was observed that luciferase assembly can be separated in time from synthesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal proteins is less efficient as compared with the presence of lumenal proteins . Thus, heterodimeric luciferase depends on functionally related molecular chaperones and folding catalysts during its assembly in either the eukaryotic cytosol or the microsomal lumen.

Eur J Biochem, 1996 Sep 15, 240(3), 646 - 54
HlyA hemolysin of Vibrio cholerae O1 biotype E1 Tor . Identification of the hemolytic complex and evidence for the formation of anion-selective ion-permeable channels; Menzl K et al.; Hemolysin (HlyA) was concentrated from supernatants of different Vibrio cholerae O1 biotype E1 Tor strains by ammonium sulfate precipitation . The concentration of the toxin in the supernatants and in the precipitates was quantified using its hemolytic activity . The toxin formed a high molecular-mass band (about 220 kDa) on SDS/PAGE while the toxin monomer had a molecular mass of 60 kDa when it was heated . The addition of the E1 Tor hemolysin oligomers, but not that of the monomers, to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels, which had long lifetimes at small voltages . The hemolysin channel had a single-channel conductance of 350 pS in 1 M KCl . These results defined hemolysin (HlyA) from V . cholerae as a channel-forming component with properties similar to other cytolytic toxins . The long lifetime of the channel suggested that the channel-forming oligomer did not show a rapid association/dissociation reaction . At voltages larger than 50 mV, the hemolysin channel was voltage dependent in an asymmetric fashion dependent on the side of its addition . The single-channel conductance of the hemolysin (HlyA) from V . cholerae O1 biotype E1 Tor channel was a linear function of the bulk aqueous conductance, which suggested that the toxin forms aqueous channels with an estimated minimum diameter of about 0.7 nm . The hemolysin channel of V . cholerae was found to be moderately anion-selective . The pore-forming properties of hemolysin (HlyA) from V . cholerae O1 biotype E1 Tor were compared with those of aerolysin of Aeromonas sobria and alpha-toxin from Staphylococcus aureus . All these cytolytic toxins must probably oligomerize for activity in biological and artificial membranes and form anion-selective channels.

Ugeskr Laeger, 1996 Sep 9, 158(37), 5169 - 71
{The first case of Vibrio cholerae 0139 in Denmark}; Dalsgaard A et al.; We report the first case of V . cholerae O139 in Denmark . In 1994, a 61-year-old Vietnamese woman was admitted to Aalborg Hospital, Denmark due to severe diarrhoea . The diagnosis was confirmed by biochemical characterization and agglutination in O139 antiserum of a strain isolated from a stool specimen . The woman had stayed in Denmark after family reunion for nine months and had not travelled outside the country . She had not eaten any foods imported from Southeast Asia . The V . cholerae O139 isolate had similar antibiotic susceptibility pattern and contained genes encoding a virulence gene cassette previously identified in O139 isolates from Southeast Asia . Ribotyping showed a banding pattern similar to patterns exhibited by O139 isolates from Bangladesh, India and Thailand . However, the exact mode of transmission of this first Danish case of V . cholerae O139 infection was not determined.

J Biol Chem, 1996 Sep 6, 271(36), 21956 - 68
The 1.5-A resolution crystal structure of bacterial luciferase in low salt conditions; Fisher AJ et al.; Bacterial luciferase is a flavin monooxygenase that catalyzes the oxidation of a long-chain aldehyde and releases energy in the form of visible light . A new crystal form of luciferase cloned from Vibrio harveyi has been grown under low-salt concentrations, which diffract x-rays beyond 1.5-A resolution . The x-ray structure of bacterial luciferase has been refined to a conventional R-factor of 18.2% for all recorded synchrotron data between 30.0 and 1.50-A resolution . Bacterial luciferase is an alpha-beta heterodimer, and the individual subunits fold into a single domain (beta/alpha)8 barrel . The high resolution structure reveals a non-prolyl cis peptide bond that forms between Ala74 and Ala75 in the alpha subunit near the putative active site . This cis peptide bond may have functional significance for creating a cavity at the active site . Bacterial luciferase employs reduced flavin as a substrate rather than a cofactor . The structure presented was determined in the absence of substrates . A comparison of the structural similarities between luciferase and a nonfluorescent flavoprotein, which is expressed in the lux operon of one genus of bioluminescent bacteria, suggests that the two proteins originated from a common ancestor . However, the flavin binding sites of the nonfluorescent protein are likely not representative of the flavin binding site on luciferase . The structure presented here will furnish a detailed molecular model for all bacterial luciferases.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9505 - 9
Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein; Schaefer AL et al.; Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response . Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri . This communication resolves two critical issues concerning the synthesis of the V . fischeri signal . (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway . We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone . In V . fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone . The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM . There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate . This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Sep-Oct, (5), 20 - 2
{The determination of the content of the cholera toxin gene in the composition of the DNA from Vibrio cholerae strains by means of the nested polymerase chain reaction}; Glukhov AI et al.; The possibility of detecting cholera toxin genes in V.cholerae enterotoxigenic strains by the method of "nested" polymerase chain reaction with the use of primers on the DNA area of operon ctx of AB genes . The possibility of the detection of several V.cholerae cells by this method was shown with the use of a series of bacterial lysate dilutions . The newly developed test system for the detection of cholera toxin gene on the basis of the analysis of bacterial lysates of V.cholerae nontoxigenic strains, as well as Escherichia coli toxigenic and nontoxigenic strains, was shown to be highly specific.

J Diarrhoeal Dis Res, 1996 Sep, 14(3), 182 - 6
Epidemiology and transmission of V . cholerae O1 and V . cholerae O139 infections in Delhi in 1993; Singh J et al.; In 1993, rectal swabs from clinically suspected cases of cholera admitted to the Infectious Diseases Hospital (IDH), Delhi were examined for Vibrio cholerae O1 and O139 . Epidemiological data of 396 cholera cases were collected before the patients' discharge from IDH . Of the 1528 laboratory-confirmed cholera cases, 46% and 54% were caused by serotype O1 and O139 respectively . Both serotypes appeared and disappeared simultaneously, and peaked during the same time of the year . However, the two serotypes affected persons of different age groups; about 65% of the O1 cases occurred in children aged less than 10 years, whereas this age group accounted for 40% of the cases due to V . cholerae O139 . Although there were some focal outbreaks due to serotype O139, both serotypes had almost similar geographical distributions . Important risk factors for transmission of cholera were almost equally prevalent in the majority of both types of cholera cases . Since the seasonality, geographical distribution, and risk factors for transmission were similar for both serotypes, the study indicates that the preventive and control measures are also likely to be similar . The study also shows that the emergence of V . cholerae O139 in 1993 did not affect the incidence, seasonality, and epidemiology of endemic V . cholerae O1 E1 Tor strains in Delhi.

Nippon Koshu Eisei Zasshi, 1996 Sep, 43(9), 844 - 53
{An ecological study for prediction of Vibrio parahaemolyticus food poisoning in Shizuoka prefecture}; Kubota T; The mean MPN viable cell counts in 15 samples of sea water in which clams were held at the time of the onset of mass outbreaks of food poisoning, the number of food poisoning outbreaks, and prevalence of Kanagawa phenomenon-positive strains, and effectiveness of measures to control food poisoning were investigated over 6 years from 1990 to 1995 . The results obtained were as follows: 1 . Of 6 materials, including sea water and shellfish, which were examined to determine the best marker material for prediction of Vibrio parahaemolyticus food poisoning, sea water in which clams were held was found to be the most appropriate . 2 . Except for the outbreaks in 1994, all Vibrio parahaemolyticus food poisoning occurred after the mean MPN viable cell count in 15 samples of sea water in which clams are kept reached 10(5) cells/100 ml . 3 . The number of outbreaks of Vibrio parahaemolyticus food poisoning could be predicted based on the time at which the mean MPN viable cell count reached 10(5) cells/100 ml . 4 . In 1995, sea water in which clams were held was cultured and examined for thermostable direct hemolysin gene by PCR method . Thermostable direct hemolysin gene was detected in 3 of 82 samples . Thirty-nine Kanagawa phenomenon-positive strains were isolated from 2 of these 3 samples . 5 . Kanagawa phenomenon-positive strains were detected after the mean MPN viable cell count in 15 samples of sea water in which clams were kept reached 10(5) cells/100 ml . 6 . Four serotypes of Kanagawa phenomenon-positive strains were detected, and they were involved in 5 (45%) of the 11 cases of Vibrio parahaemolyticus food poisoning that occurred in the same year . These serotypes were observed also in 28 (38%) of the 74 strains isolated from food poisoning patients . 7 . No conclusion could be made concerning the effectiveness of measures to control food poisoning.

J Vet Med Sci, 1996 Sep, 58(9), 921 - 3
Selective growth of thermostable direct hemolysin-producing Vibrio parahaemolyticus in the alimentary tract of a gastropod, Clithon retropictus, at a selected estuary in Japan; Kumazawa NH et al.; Thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus increased in cell number in the alimentary tract of a gastropod . Clithon retropictus, from an undetectable level in July to 2.0 x 10(3)/g in September 1991 at the Sada estuary in the southern part of Japan . It was not detected in microalgae, foods of the gastropod, at the estuary or muddy sediments at a neighboring fishing port . The strain was not detected at two estuaries and two fishing ports ecologically separated from the Sada estuary . Serotypes of the isolated strains were different from those registered as clinical isolates . Our results confirm that TDH-producing V . parahaemolyticus grows selectively in the gastropod at some estuaries in Japan.

FEMS Immunol Med Microbiol, 1996 Sep, 15(2-3), 143 - 8
Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139; Pal S et al.; The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V . cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE . A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth . As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V . cholerae O139 OM became the major protein component . On immunoblot analysis with rabbit antiserum against V . cholerae O139 OM, it was shown that, apart from the major protein component of V . cholerae O1 OM of around 45 kDa and that of V . cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups . In immunoblot assays with convalescent sera obtained from V . cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the patient . Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.

Microb Pathog, 1996 Sep, 21(3), 173 - 82
Cloning and detection of the hemolysin gene of Vibrio anguillarum; Hirono I et al.; A 5 kb DNA fragment encoding a hemolysin was cloned from the fish pathogenic bacterium Vibrio anguillarum using the cosmid vector charomid9-36 . An open reading frame of the hemolysin gene (VAH1) was 2253 bp and corresponded to a protein of 751 amino acid residues . The deduced amino acid sequence of the VAH1 gene and the previously reported Vibrio cholerae EI Tor hemolysin, V . vulnificus cytolysin-hemolysin, Aeromonas hydrophila AHH1 hemolysin, and A . salmonicida ASH4 hemolysin showed a significant degree of sequence homology, and the overall amino acid identities were 57.3%, 25.8%, 46.2%, and 43.7%, respectively . DNA hybridization analysis under high-stringent conditions using VAH1 as a probe, demonstrated that VAH1 hybridized with 25 out of 28 strains of V . anguillarum including serotypes A to I, but did not hybridize with other species of Vibrio, A . hydrophila or A . salmonicida . The targeted DNA fragment of VAH1 was successfully amplified from V . anguillarum-infected fish tissues by PCR.

Appl Environ Microbiol, 1996 Sep, 62(9), 3516 - 20
Comparison of a fluorogenic assay with a conventional method for rapid detection of Vibrio parahaemolyticus in seafoods; Venkateswaran K et al.; A conventional method and a fluorogenic assay for the detection of Vibrio parahaemolyticus were compared . Among 29 seafood samples examined for the presence of V . parahaemolyticus, 17 samples harbored V . parahaemolyticus, and trypsinlike activity was noticed in 19 seafoods . The added fluorogenic substrate was cleaved in single samples of shrimp, turbo, and cuttlefish from which V . parahaemolyticus could not be isolated by the conventional method . Vibrio alginolyticus, in addition to V . parahaemolyticus, was found to exhibit intracellular trypsinlike activity . Trypsinlike activity in seafoods was observed after the most probable number for the initial density of V . parahaemolyticus-like organisms was found to have reached > 10(2) per g . A V . parahaemolyticus inoculum at 10(4) CFU/ml in arabinose-glucuronate medium was required to attain growth to 10(6) CFU/ml, which is the level necessary for the release of detectable amounts of fluorescent compound from the added substrate.

Appl Environ Microbiol, 1996 Sep, 62(9), 3344 - 9
Enumeration and phylogenetic analysis of polycyclic aromatic hydrocarbon-degrading marine bacteria from Puget sound sediments; Geiselbrecht AD et al.; Naphthalene- and phenanthrene-degrading bacteria in Puget Sound sediments were enumerated by most-probable-number enumeration procedures . Sediments from a creosote-contaminated Environmental Protection Agency Superfund Site (Eagle Harbor) contained from 10(4) to 10(7) polycyclic aromatic hydrocarbon (PAH)-degrading bacteria g (dry weight) of sediment-1, whereas the concentration at an uncontaminated site ranged from 10(3) to 10(4) g of sediment(-1) . Isolates of PAH-degrading bacteria were obtained from these most-probable-number tubes as well as from sediment samples from noncontaminated sites and from bioreactors enriched with PAHs . The 18 resulting strains were grouped by whole-cell fatty acid analysis into two subgroups . The larger group of strains belonged to the newly described genus Cycloclasticus, whereas the other group contained members of the genus Vibrio . The Cycloclasticus group seems to be widespread in noncontaminated sediments . PAH degradation was confirmed in selected strains on the basis of removal of phenanthrene from growing cultures.

J Bacteriol, 1996 Sep, 178(17), 5291 - 4
Quorum sensing in Vibrio fischeri: evidence that S-adenosylmethionine is the amino acid substrate for autoinducer synthesis; Hanzelka BL et al.; Synthesis of the autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI . The autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis . We have measured autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid . Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for autoinducer synthesis . The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent autoinducer synthesis . Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for autoinducer synthesis . The amount of 15N-labeled methionine incorporated into the autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry . The labeling studies show that even in the presence of homoserine, almost all of the autoinducer produced contains the 15N label from methionine . Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V . fischeri autoinducer.

J Bacteriol, 1996 Sep, 178(17), 5188 - 98
Identification and characterization of additional flagellin genes from Vibrio anguillarum; McGee K et al.; Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D . This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE . The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci . The genetic organization of these two loci is conserved between both organisms . For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made . Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament . However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen . Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype . The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes . These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.

Infect Immun, 1996 Sep, 64(9), 3778 - 85
Immune mechanisms and protective antigens of Vibrio cholerae serogroup O139 as a basis for vaccine development; Jonson G et al.; We have characterized 11 isolates of Vibrio cholerae O139 Bengal with regard to properties deemed to be relevant for development of a vaccine against O139 cholera . For most strains two colony variants, A and B, which are nonhemolytic and hemolytic, respectively, were detected on blood agar . The A and B variants were associated with high- and low-level production of soluble hemagglutinin-protease, respectively . However, on Luria-Bertani agar both types formed opaque colonies, which has been shown to be associated with capsule formation . Interestingly, under the stationary tube-shaken flask culture conditions in yeast extract-peptone water medium which were used to stimulate the production of cholera toxin (CT) and toxin-coregulated pili, B variants constitutively produced CT and TcpA, two ToxR-regulated proteins, at 28 and 37 degrees C, whereas the production of these proteins by A variants was downregulated at the higher temperature . One of the strains, 4260B, having a well-exposed O antigen and capsule and the capacity to produce large amounts of TcpA, CT, and mannose-sensitive hemagglutinin pili but minimal amounts of the proteolytic soluble hemagglutinin, was selected to produce antibacterial antisera and as a challenge strain in protection studies using the rabbit ileal loop model . Rabbit antisera to live, heat-killed, or formalin-killed O139 vibrios or to purified O139 lipopoly-saccharide (LPS) as well as monoclonal antibodies (MAbs) to O139 LPS agglutinated all O139 isolates . However, when A and B variants of strain 4260 were tested for sensitivity to vibriocidal activity of these antibody preparations, only the B variant was killed . All of the antisera against live or killed O139 vibrios conferred passive protection against fluid accumulation induced by the challenge strain . The protective effects of the antisera were correlated to anti-LPS antibody titers rather than to titers against whole bacteria that had been grown for toxin-coregulated pilus expression . This protection was considerably higher than that conferred by antisera to classical, EI Tor, or recombinantly produced (classical) CT or CTB . Furthermore, MAbs to O139 LPS and CTB-CT exhibited a strong synergistic protection against O139 challenge irrespective of the level of sensitivity of challenge strains to O139 LPS MAbs in vibriocidal assays in vitro.

Infect Immun, 1996 Sep, 64(9), 3565 - 70
Construction and characterization of a potential live oral carrier-based vaccine against Vibrio cholerae O139; Favre D et al.; The rfb region from Vibrio cholerae O139 strain MO45 was cloned from cosmid gene banks established in Escherichia coli HB101, using an immunoblot assay for screening of the correct clones . Immunoblot analysis of lipopolysaccharide (LPS) preparations revealed the presence of two types of positive clones: (i) those expressing only a short core-linked O polysaccharide (SOPS) and (ii) those also expressing a highly polymerized capsular polysaccharide (CPS) not bound to the E . coli K-12 LPS core . In addition, the latter clones appear to contain a locus which may encode a putative regulator of SOPS and CPS chain length . Further characterization in E . coli showed that CPS constitutes a barrier against large particles such as the bacteriophage Ffm but not against bacteriophage lambda or P1 . In addition, a portion of the K-12 LPS core may not be substituted with SOPS . Loci associated with the two clonal types were transferred into V . cholerae CH19, an rfbAB deletion mutant of CVD103-HgR deficient in the production of the homologous Inaba O polysaccharide . This resulted in the stable expression of SOPS, alone or together with CPS, that was indistinguishable from that of wild-type V . cholerae O139 . Strains CH25 and CH26, which correspond to CH19 bearing the V . cholerae O139 rfb region integrated into the chromosome, were found to be genetically stable and essentially identical to the parent CVD103-HgR with respect to physiological properties such as cell motility, mercury resistance, toxicity, and production of the cholera toxin B subunit . Rabbits immunized with CH25 elicited high titers of anti-O139 SOPS- and CPS-specific serum antibodies . These strains possess characteristics desirable in candidate live oral vaccines against V . cholerae O139.

Carbohydr Res, 1996 Aug 26, 290(1), 43 - 58
Structural analysis of the lipopolysaccharide from Vibrio cholerae O139; Cox AD et al.; The lipopolysaccharide (LPS) from Vibrio cholerae O139 was deacylated with KOH . The following structure of the oligosaccharide resulting from this treatment was established on the basis of monosaccharide and methylation analyses, 1H, 13C and 31P 1D and 2D NMR experiments and 1D analogues of 3D NOESY-TOCSY and 3D TOCSY-NOESY experiments . {formula: see text} 'C' is a beta-L-threo-hex-4-enuronopyranosyl residue . Hep is L-glycero-D-manno-heptose, QuiN is 2-amino-2,6-dideoxy-D-glucose, GlcN is 2-amino-2-deoxy-D-glucose, Glc is D-glucose, Fru is D-fructose, and Kdo is 3-deoxy-D-manno-2-octulosonic acid . All sugars are pyranoses except fructose which is furanosidic . The fructose residue was localised after deacylation of the LPS with anhydrous hydrazine, methylation, acid methanolysis, and remethylation using deuterated iodomethane . The elucidation of this structure allowed for a direct comparison to the previously determined structure for Vibrio cholerae O1 lipid A-core region . The two structures are almost identical, and, therefore, this study is consistent with the genetic data for the biogenesis of strain O139 from O1 . Furthermore, the identification of a structural analogue to the capsular polysaccharide of O139 in the outer core of the LPS in conjunction with the identification of colitose as a constituent of the LPS, provides additional evidence that the O-antigen and capsular polysaccharide of this strain may share the same repeat unit.

Biochemistry, 1996 Aug 6, 35(31), 9967 - 73
Conversion of serine-114 to cysteine-114 and the role of the active site nucleophile in acyl transfer by myristoyl-ACP thioesterase from Vibrio harveyi; Li J et al.; The lux-specific myristoyl-ACP thioesterase (LuxD) is responsible for diverting myristic acid into the luminescent system and can function as an esterase and transferase as well as cleave myristoyl-CoA and other thioesters . The recently elucidated crystal structure of the enzyme shows that it belongs to the alpha/beta hydrolase family and that it contains a typical catalytic triad composed of Asp211, His241, and Ser114 . What is unusual is that the nucleophilic S114 is not contained within the esterase consensus motif GXSXG although the stereochemistry of the turn involving S114 is almost identical to the nucleophilic elbow found in alpha/beta hydrolases . In contrast to mammalian thioesterases, deacylation of LuxD was the rate-limiting step, with the level of acylated enzyme formed on reaction with myristoyl-CoA and the pre-steady-state burst of p-nitrophenol on cleavage of p-nitrophenyl myristate both being 0.7 mol/mol . Cold chase experiments showed that the deacylation rate of LuxD corresponded closely to the turnover rate of the enzyme with ester or thioester substrates . Replacement of S114 by a cysteine residue generated a mutant (S114C) that was acylated with the same pH dependence as LuxD but had greatly diminished capacity to transfer acyl groups to water or glycerol . The acyl group could be removed from the S114C mutant by neutral hydroxylamine, showing that a cysteine residue had been acylated . Mutation of H241 creating the double mutant, S114C.H241N, decreased acylation of the cysteine residue . These results provide direct kinetic and chemical evidence that S114 is the site of acylation linked to H241 in the charge relay system and have led to the recognition of a more general consensus motif flanking the nucleophilic serine in thioesterases.

Lancet, 1996 Aug 3, 348(9023), 296 - 300
Randomised controlled comparison of single-dose ciprofloxacin and doxycycline for cholera caused by Vibrio cholerae 01 or 0139; Khan WA et al.; BACKGROUND: Effective antimicrobial therapy can reduce the duration and volume of cholera diarrhoea by half . However, such treatment is currently limited by Vibrio cholerae resistance to the drugs commonly prescribed for cholera, and by the difficulties involved in the administration of multi-drug doses under field conditions . Because of its favourable pharmacokinetics we thought it likely that single-dose ciprofloxacin would be effective in the treatment of cholera . METHODS: In this double-blind study treatment was either a single 1 g oral dose of ciprofloxacin plus doxycycline placebo, or a single 300 mg oral dose of doxycycline plus ciprofloxacine placebo . 130 moderately or severely dehydrated men infected with V cholerae 01 and 130 infected with V cholerae 0139 were randomly assigned treatment . Patients stayed in hospital for 5 days . We measured fluid intake and stool volume every 6 h, and a sample of stool for culture was obtained daily . The primary outcome measures were clinical success--the cessation of watery stool within 48 h; and bacteriological success--absence of V cholerae from cultures of stool after study day 2 . FINDINGS: Among patients infected with V cholerae 01, treatment was clinically successful in 62 (94%) of 66 patients who received ciprofloxacin and in 47 (73%) of 64 who receive doxycycline (difference 21% {95% Cl 8-33}); the corresponding proportions with bacteriological success were 63 (95%) and 44 (69%) (27% {14-39}) . Among patients infected with V cholerae 0139, treatment was clinically successful in 54 (92%) of 59 patients who received ciprofloxacin and in 65 (92%) of 71 who received doxycycline (< 1% {-9 to 9}), and bacteriologically successful in 58 (98%) and 56 (79%), respectively (19% {9-30}) . Total volume of watery stool did not differ significantly between ciprofloxacin-group and doxycycline-group patients infected with either V cholerae 01 or 0139 . All but one of the V cholerae 01 and all of the 0139 isolates were susceptible in vitro to doxycycline, whereas 48 (37%) of the V cholerae 01 isolates and none of the 0139 isolates were resistant to tetracycline . Treatment clinically failed in 14 (52%) of 27 doxycycline-treated patients infected with a tetracycline-resistant V cholerae 01 strain, compared with three (8%) of 37 patients infected with a tetracycline-susceptible strain (44% {23-65}) . INTERPRETATION: Single-dose ciprofloxacin is effective in the treatment of cholera caused by V cholerae 01 or 0139 and is better than single-dose doxycycline in the eradication of V cholerae from stool . Single-dose ciprofloxacin may also be the preferred treatment in areas where tetracycline-resistant V cholerae are common . In V cholerae, in-vitro doxycycline susceptibilities are not a useful indicator of the in-vivo efficacy of the drug.

J Biol Chem, 1996 Aug 2, 271(31), 18885 - 91
Antisense RNA, fur, iron, and the regulation of iron transport genes in Vibrio anguillarum; Chen Q et al.; The negative regulation of the expression of iron transport genes fatA and fatB in Vibrio anguillarum is mediated by a chromosome-encoded Fur protein and a plasmid pJM1-derived antisense RNA (RNAalpha), which is preferentially expressed under iron-rich conditions . In this work, we characterized the RNAalpha promoter region, and by using promoter fusion and rifampicin experiments we were able to demonstrate that iron regulates RNAalpha synthesis posttranscriptionally by stabilizing RNAalpha half-life rather than enhancing transcription initiation . The Fur protein is also essential for RNAalpha synthesis at the transcription initiation level, independently of the iron status of the cell . From experiments assessing the relative contribution of Fur and RNAalpha, we were able to show that RNAalpha may indeed play an important role on the negative regulation of the expression of the iron transport genes under physiological conditions.

J Ind Microbiol, 1996 Aug, 17(2), 80 - 3
Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations; Erbe JL et al.; The metal-responsive smt operator/promoter region of Synechococcus PCC7942 was fused to the luxCDABE genes of Vibrio fischeri . Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria . Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl2 concentrations within a range of 0.5-4 microM as demonstrated by induction of luminescence . Trace levels of CuSO24 and CdCl2 were also detected.

Biosci Biotechnol Biochem, 1996 Aug, 60(8), 1324 - 30
Purification of membrane-bound ATPase of a psychrophilic marine bacterium, Vibrio sp . strain ABE-1 and characterization as a F0F1-type enzyme; Harashima A et al.; The ATPase bound to the inner membrane of a psychrophilic marine bacterium, Vibrio sp . strain ABE-1 (Vibrio ABE-1) was extracted with Triton X-100 and purified by fractionation with polyethylene glycol, sucrose density gradient centrifugation, and DEAE-Toyopearl 650M column chromatography . The molecular masses of subunits constituting the purified ATPase were estimated as 54, 49, 33.5, 27, 23.5, 18.5, and 15 kDa by SDS-PAGE . The composition and molecular masses of the subunits of the purified ATPase were similar to those of Escherichia coli F0F1-ATPase (EF0F1) . The 54-, 49-, and 18.5-kDa polypeptides of the Vibrio ABE-1 ATPase strongly cross-reacted with the antibodies against the EF0F1 alpha, beta, and b subunits, respectively . However, the Vibrio ABE-1 ATPase contained no cross-reactive polypeptide with the antibodies against A and B subunits of V-type H(+)-ATPase from mung bean tonoplasts . The ATPase activity showed two pH optimum peaks at pH 5.3 and 8.0 and was strongly inhibited by N,N'-dicyclohexyl carbodiimide (DCCD) and NaN3 . It hydrolyzed ATP, GTP, and ITP at similar rates . These properties confirm that the purified ATPase is a F0F1-type . The optimum temperature for the ATP-hydrolyzing activity of the enzyme was observed at 50 degrees C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30 degrees C, suggesting that the F1-moiety is released from the enzyme complex at high temperatures . This characteristic is compatible with the psychrophilic nature of Vibrio ABE-1.

Biosci Biotechnol Biochem, 1996 Aug, 60(8), 1320 - 3
Purification and characterization of N-acetylglucosamine 6-phosphate deacetylase with activity against N-acetylglucosamine from Vibrio cholerae non-O1; Yamano N et al.; An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures . The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da . The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine . The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu2+ . On the other hand, the activity was activated by Co2+ . The amino-terminal amino acids of the purified enzyme were sequenced . Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase.

Genetika, 1996 Aug, 32(8), 1148 - 52
{Lux-regulon from Vibrio fisheri reduces type 1 restriction in Escherichia coli K12 cells}; Zavil'gel'skii GB et al.; The EcoK restriction of nonmodified phage lambda.0 controlled by Escherichia coli K12 lon- cells is alleviated 10-20-fold in the presence of a hybrid plasmid containing the entire lux regulon of Vibrio fischeri . The La protease participates in degradation of the regulatory LuxR-LuxI proteins; therefore, transcription of the right lux operon begins significantly earlier than in the lon+ bacteria and is characterized by high content of the N-(3-oxohexanoyl) homoserine lactone autoinducer in medium . The effect of lux-induced alleviation of type I restriction in the lon mutants of E.coli K12 is proposed to depend on the reduction at the intracellular level of S-adenosylmethionine, one of the LuxI synthase substrates responsible for autoinducer synthesis.

Vaccine, 1996 Aug, 14(12), 1137 - 42
Serum antibacterial and antitoxin responses in clinical cholera caused by Vibrio cholerae O139 Bengal and evaluation of their importance in protection; Nandy RK et al.; Vibrio cholerae O139 Bengal strain was the causative agent of the recent epidemics of cholera in India and Bangladesh . We studied antibacterial and antitoxin immune responses in acute and convalescent phase paired sera collected from seven of these cholera patients . Significant rise in the levels of both antibacterial and antitoxin antibodies was demonstrable in the sera of convalescent cholera patients . Antibacterial antibodies, directed primarily against O139 lipopolysaccharides (LPS), belonged to IgM class, while antitoxin antibodies were of IgG and IgA class and neutralized cholera toxin . The convalescent sera, however, showed no increase in the reactivity towards V . cholerae O1 whole cells or their LPS preparation . Immunoblotting experiments revealed that the convalescent, but not the acute, phase serum recognized the truncated form of LPS characteristics of O139 strains . Convalescent serum also induced definite protection against O139, but not O1, challenge in experimental animal model . Further studies showed that such protection was probably mediated by antibodies inhibiting intestinal colonization of O139 organisms . These results suggest that critical difference(s) exists between the immunogenic somatic components of V . cholerae O1 and O139 organisms that are of considerable importance in protection against cholera.

Zentralbl Bakteriol, 1996 Aug, 284(4), 496 - 500
Immunocytochemical localization of 60-kDa heat shock protein in Vibrio cholerae; Taguchi H et al.; The immunocytochemical localization of the 60-kDa heat shock protein (HSP) in Vibrio cholerae strain 569B was studied by transmission electron microscopy using a combination with the antigen-specific monoclonal antibody (5C3) and immunogold labelling . The labelling with gold particles in V . cholerae detected 2 types; the gold particles were exclusively detected in the cytoplasm for one type and in the periplasmic space for another type, suggesting that the 60-kDa HSP of V . cholerae corresponding immunologically to Escherichia coli GroEL may translocate from the cytoplasm to the periplasmic space in the V . cholerae cell.

Kansenshogaku Zasshi, 1996 Aug, 70(8), 815 - 20
{Detection of the trh gene in Vibrio parahaemolyticus isolated from overseas travellers' diarrhea and their biochemical characteristics}; Obata H et al.; A total of 478 Vibrio parahaemolyticus strains isolated from overseas travellers' diarrhea during the last 7 years of 1989 to 1995 were examined for the production of Kanagawa hemolysin by reversed passive latex agglutination (RPLA) test . Three hundred-seventy (77.4%) out of 478 strains were positive for Kanagawa hemolysin, whereas 108 strains were weakly positive or negative . For those Kanagawa hemolysin-weakly positive or negative strains, the tdh and trh genes associated with the production of TDH (thermostable direct hemolysin) and TRH (TDH-related hemolysin), respectively, were studied by the polymerase chain reaction method . The trh gene was detected in 98 (90.7%) out of 108 strains . In 35 strains belonging to 13 serotypes such as O3: K6, O1:K33, O3:K59, the trh gene alone was detected . On the other hand, both trh and tdh genes were detected in 63 strains of 17 serotypes including O1:K69, O3:K72, O6:K46 . Among the strains of 4 serotypes including O1:K56, O1:KUT, O3:KUT and O5:KUT, two types of the trh positive alone and the trh and tdh positive were observed . Of interest, all of the 98 trh-positive strains were positive for the urease hydrolysis, whereas all Kanagawa hemolysin-positive strains were not . Furthermore, the strains of serotype O6:K18 (4 strains) were positive for the fermentation of dulcitol, and the strains of serotype O1:K1 (5 strains) were indole negative . These characteristics of the strains were completely different from those of typical V . parahaemolyticus strains.

Int J Food Microbiol, 1996 Aug, 31(1-3), 317 - 23
Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods; Karunasagar I et al.; Detection of Kanagawa positive strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR) was tested . Primer pairs for specific amplification of tdh gene fragment is described . The assay could detect contamination of seafood homogenate when PCR was performed using lysate prepared directly from fish homogenates . The sensitivity of the assay could be improved to detect less than 10 cells of V . parahaemolyticus by performing PCR after 8 h enrichment in alkaline peptone water.

Int J Food Microbiol, 1996 Aug, 31(1-3), 197 - 204
Survival of Vibrio cholerae O1 on plastic materials; Fernandez-Escartin E; Survival of environmental and clinical strains of Vibrio cholerae O1 was studied on glass and on two varieties of plastic materials . V . cholerae survived at least 2 days on glass, but was not recovered from polystyrene spoons after 15-20 min . Escherichia coli survived for at least 2 days on both glass slides and plastic spoons . Extracts, 10 and 50% (w/v) of ground plastic spoons in isotonic saline water, inactivated 10(4) vibrios in less than 2 h . Isotonic saline water rinses from polyethylene bags inactivated (0% survival) 15 out of 23 strains of V . cholerae in 1 h . A strain of V . cholerae (100-200 CFU/ml) directly suspended in 25 ml of isotonic saline per bag and maintained at 20 degrees C was progressively inactivated . The number of viable cells diminished 95% in 4 h in bags taken from non-sterile rolls . In sterile bags the decrease was 44% and 93% after 4 and 24 h, respectively . A variability up to 50% was observed in the antibacterial effect among the different bags, either sterile or taken from non-sterile rolls . These decreases in V . cholerae viability may result in false negative reports if water samples are collected and carried out in plastic bags.

Histochem Cell Biol, 1996 Aug, 106(2), 197 - 207
Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study; Cao Y et al.; A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens, TF (Gal beta 1-3GalNAc alpha 1-R), Tn (TF precursor, GalNAc alpha 1-R), sialosyl-Tn (NeuAc alpha 2-6GalNAc alpha 1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF . These antigens or, more correctly, glycotopes, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry . For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence . Staining with mAbs appeared to be more restricted than with the lectins used . Distribution patterns among normal epithelia were different for all four antigens . These antigens were also detected in some non-epithelial tissues . They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF > > sialosyl-Tn > Tn > TF . Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas . The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase from Vibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF {NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-R and Gal beta 1-3 (NeuAc alpha 2-6)GalNAc alpha 1-R} in normal human tissues . From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.

Toxicon, 1996 Aug, 34(8), 857 - 69
Toxic effects of blooms of marine species of Oscillatoriales on farmed prawns (Penaeus monodon, Penaeus japonicus) and brine shrimp (Artemia salina); Smith PT; Benthic and planktonic blooms of species of Oscillatoriales coincided with mortalities of Penaeus monodon during four episodes at Australian prawn farms . Oscillatoria corakiana was the dominant planktonic species at 65-90,000 cells/ml, but Spirulina sp., Lyngbya sp., Oscillatoria sp . and Nodularia sp . were also identified from the water column, benthic layers or surface mats . The levels and variety of Vibrionaceae in prawn tissue, suggest that mortalities were caused by secondary infections of bacteria . However, experimental results indicate that toxicity of the blooms of Oscillatoriales was the primary cause of disease . Pond water and extracts from a tank culture of benthic Oscillatoriales caused mortalities when injected into P . monodon and P . japonicus . Immersion of artemia in extracts from the tank culture also caused mortalities, with L.D50 values for the supernatant extract of 70 mg/litre for artemia cysts and 50 mg/litre for adult artemia, and LD50 values for the pellet extract of 110 mg/litre for artemia cysts and 200 mg/litre for adult artemia . Experiments with artemia suggested the blooms of Oscillatoriales produced water-soluble, heat-labile toxin/s . Mortalities may have been caused by a neurotoxin because: (a) there was a lack of histopathological evidence of damage to the digestive tracts of prawns during each episode; and (b) artemia cysts immersed in extracts of Oscillatoriales died before they developed digestive tracts . PSP toxin, anatoxin-a, homoanatoxin-a and microcystins were not detected when pond water from a diseased pond was tested . It is proposed that sub-lethal levels of toxin weakened the prawns, causing reduced feeding behaviour and an impaired immune system . As a result, prawns were prone to secondary infection by pathogenic bacteria . Because Oscillatoriales are ubiquitous in prawn farms, the findings have significant implications for the assessment of disease in the prawn farming industry.

FEMS Immunol Med Microbiol, 1996 Aug, 15(1), 9 - 15
Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production; Whayeb SA et al.; The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L2 of Vibrio cholerae, was investigated . Introduction of lypA into the V . cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity . Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L2 activity and the production of CT but not the haemolytic activity . Furthermore, constructed mutants of E1 Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test . These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V . cholerae.

Microb Pathog, 1996 Aug, 21(2), 111 - 23
Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function; Wu Z et al.; In this report, we describe the cytotoxic activity of the cholera hemagglutinin/protease (HA/protease) . A concentrated protein sample from the 37 degrees C overnight culture supernatant of CVD110, a delta ctxA, delta zot, delta Ace and hlyA::(ctxB mer) mutant of El Tor biotype Ogawa serotype strain E7946 caused morphological changes in cultured MDCK-I epithelial cells and altered their arrangement of filamentous actin (F-actin) and Zonula occludens-associated protein ZO-1 . The drastic morphological changes can be inhibited by Zincov, a specific bacterial metalloprotease inhibitor . The cytotoxic fractions of the sample after FPLC gelfiltration fractionation showed two visible protein bands with molecular weights of approximately 34- and 32 kDa . Microsequencing of these two proteins revealed that they were the cholera HA/protease.

J Clin Microbiol, 1996 Aug, 34(8), 1930 - 3
Further studies on biochemical characteristics and serologic properties of the genus Aeromonas; Janda JM et al.; We characterized a collection of 268 Aeromonas isolates from diverse sources (clinical, animal, and environmental sources) for their species and serogroup designations . Overall, 97% of these strains could be identified to the genomospecies level by using an expanded battery of biochemical tests . Members of the Aeromonas hydrophila complex (A . hydrophila, HG2, and A . salmonicida), a group that has previously been difficult to separate biochemically, could easily be distinguished from one another by using a number of recently described phenotypic properties which included utilization of DL-lactate and urocanic acid . Differences in species distributions on the basis of the source of isolation were noted . Serogroup analysis of these 268 isolates plus a number of reference cultures indicated that (i) each genomospecies is serologically heterogeneous and individual serogroups can be found in more than one species, (ii) most type or reference strains for each hybridization group are not serologically representative of the genomospecies at large, (iii) serogroups O:11, O:34, and O:16 predominate clinically (48%), supporting previous studies indicating their importance in human infections, and (iv) most A . trota strains do not express the O139 antigen of Vibrio cholerae . The collective results suggest that both species and serogroup designations are important factors in establishing which isolates can cause human infections when they are acquired from nonclinical sources (foods, animals, and the environment).

Epidemiol Infect, 1996 Aug, 117(1), 51 - 8
Comparison of pulsed-field gel electrophoresis and ribotyping for subtyping of Vibrio cholerae O139 isolated in Thailand; Dalsgaard A et al.; Pulsed-field gel electrophoresis (PFGE) of Cpo I-digested genomic DNA and ribotyping (Bgl I) were applied to 60 Vibrio cholerae strains including 48 V . cholerae O139 from Thailand to compare their value in differentiating strains of the present V . cholerae O139 epidemic . PFGE patterns were divided into groups A and B representing five and four subtypes, respectively, while ribotyping showed four different patterns . PFGE group B subtypes were only presented among O139 isolates from Thailand, whereas four O139 strains from Bangladesh and India showed identical PFGE group A subtypes observed in O139 isolates from Thailand . Two nontoxigenic O139 isolates from Thailand showed different and unique PFGE types as did five V . cholerae non-O139 isolates containing a gene virulence complex found in V . cholerae O139 . These results indicate that PFGE (Cpo I) can resolve recent evolutionary divergence within V . cholerae O139 and offers a useful supplementary tool for following the progressing V . cholerae O139 epidemic.

Epidemiol Infect, 1996 Aug, 117(1), 43 - 9
Resurgence of cholera in Hong Kong; Lee SH et al.; Cholera is one of the three diseases subject to the International Health Regulations . After a period of over 30 years, the seventh pandemic of cholera, which started in South East Asia in 1961, still shows no sign of a decline . On the contrary, it has increased its severity and invaded many other countries in Africa and Latin America . In the last two years, there has been a recrudescence of the disease in South East Asia and Western Pacific Regions . The discovery of a new strain of Vibrio cholerae 0139 in these regions is causing concern in view of its potential to cause major epidemics and higher mortality . Hong Kong had two intensive outbreaks of cholera in the last two years . The cause of these outbreaks was not clear, but adverse environmental conditions and increasing pollution of coastal waters have been implicated . The spread of cholera knows no geographical boundaries . There is a need for intensified efforts among health authorities in the affected areas to prevent the international spread of the disease.

Microbiology, 1996 Aug, 142 ( Pt 8), 2181 - 6
Attachment of Vibrio alginolyticus to chitin mediated by chitin-binding proteins; Pruzzo C et al.; Vibrio alginolyticus is the only culturable vibrio associated with the chitinaceous carapace of the copepod Tigriopus fulvus (Fisher 1860) living in Ligurian coastal rock pools (Tyrrhenian Sea) . The characteristics of the interaction between chitin particles and V . alginolyticus were studied by analysing strains isolated both from the copepod surface and from rock-pool water . The highest degree of attachment to chitin was observed at 20 degrees C, in the presence of 3% NaCl . Bacterial treatment with N-acetylglucosamine and pronase E caused a reduction in attachment of 52-62% and 77-94%, respectively . Chitin pretreatment with either wheat germ agglutinin or membrane proteins (MPs) from V . alginolyticus caused a reduction in attachment, of 50-57% and 53-70%, respectively . No inhibition was observed when bacteria were pretreated with D-glucose, D-fucose or D-fructose, or when chitin was pretreated with concanavalin A and Escherichia coli DH5 alpha MPs . V . alginolyticus MPs able to bind chitin were isolated and analysed by SDS-PAGE . Four chitin-binding proteins were visualized in all tested strains (53, 35, 20 and 14 kDa); in vivo these peptides may efficiently mediate V . alginolyticus attachment to chitin-containing substrates.

Microbiology, 1996 Aug, 142 ( Pt 8), 2165 - 74
A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae; Kovach ME et al.; A large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites . Adjacent to the attL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases . The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity) . Genomic analysis of numerous V . cholerae strains (O1, non-O1 and O139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase . The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V . cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.

J Bacteriol, 1996 Aug, 178(16), 5024 - 6
Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus; Atsumi T et al.; By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system . Pof+ Laf- cells could not swim in the high-viscosity environment (ca . 200 cP) in which Pof- Laf+ cells swam at 20 microns/s . The Pof- Laf+ cells swam at about 20 microns/s at normal viscosity (1 cP) without the viscous agent, and the speed increased to 40 microns/s at about 5 cP and then decreased gradually as the viscosity was increased further . These results show the functional difference between polar and lateral flagella in viscous environments.

J Bacteriol, 1996 Aug, 178(16), 4901 - 8
Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT; Ostling J et al.; The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp . strain S14 is subjected to carbon and energy starvation . By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp . strain S14 . The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp . S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins . However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation . We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp . strain S14 . It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp . S14 spoT mutant strain (csrS1) . Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival . On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell . This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied . The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.

Infect Immun, 1996 Aug, 64(8), 3369 - 73
Relative significance of mannose-sensitive hemagglutinin and toxin-coregulated pili in colonization of infant mice by Vibrio cholerae El Tor; Attridge SR et al.; A previously described in-frame deletion in mshA--the gene encoding the structural subunit of the mannose-sensitive hemagglutinin pilus--has been introduced into the chromosome of three El Tor O1 strains of Vibrio cholerae . None of the deltamshA mutants showed significant attenuation or loss of colonization potential in the infant mouse cholera model . A second mutation, created by insertion of a kanamycin resistance cartridge into deltamshA, also failed to affect in vivo behavior . In contrast, strains carrying mutations in tcpA (encoding the monomer of the toxin-coregulated pilus {TCP}) were markedly attenuated and showed dramatically impaired colonization . This result was in line with those of previous studies . Protection tests performed with antibodies to TCP and to MshA showed that only the former were able to confer immunity against El Tor O1 challenge in this model . Studies with mutants constructed from two O139 strains similarly suggest that TCP but not mannose-sensitive hemagglutinin pili are critical for colonization by strains of this serogroup.

Infect Immun, 1996 Aug, 64(8), 3101 - 8
Purification and characterization of an extracellular secretogenic non-membrane-damaging cytotoxin produced by clinical strains of Vibrio cholerae non-O1; Saha PK et al.; Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage . This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin . Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY . Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5 . NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold . The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin . Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop . Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum . Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region . Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V . cholerae non-O1 strains which produce this cytotoxin.

Infect Immun, 1996 Aug, 64(8), 2968 - 73
Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1; Ikigai H et al.; El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes . Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa . ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone . Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization . The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium . Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane . The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm . The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy . Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

J Bacteriol, 1996 Aug, 178(15), 4731 - 3
Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution; Zenno S et al.; NfsB is an oxygen-insensitive nitroreductase of Escherichia coli with significant amino acid sequence homology to the major flavin reductase (FRase I) of Vibrio fischeri . Here, we show that NfsB is convertible to an FRase I-like flavin reductase three times as active as the authentic FRase I by a single amino acid substitution in the least-conserved region.

J Bacteriol, 1996 Aug, 178(15), 4508 - 14
Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase; Zenno S et al.; We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E . coli map to be 19 min . We also purified its gene product, NfsA, to homogeneity . It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide . Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B . Lei, M . Liu, S . Huang, and S.-C . Tu, J . Bacteriol . 176:3552-3558, 1994), an observation supporting the notion that E . coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution . Although no appreciable sequence similarity was detected between two E . coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB . NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.

Appl Environ Microbiol, 1996 Aug, 62(8), 3023 - 5
Production of thermostable direct hemolysin by Vibrio parahaemolyticus enhanced by conjugated bile acids; Osawa R et al.; The effects of conjugated bile acids, glycocholic acid, and taurocholic acid (TC) on production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus were determined by a reversed passive latex agglutination assay against TDH . The amount of TDH excreted in growth medium containing either glycocholic acid or taurocholic acid (5 mM/liter) was, on a per-cell basis, 4- to 16-fold greater than that excreted in medium without the bile acids . The amounts of TDH released from lysed cells grown with the bile acids (5 mM/liter) were 4- to 32-fold greater than those from lysed cells grown without, suggesting that the bile acids enhanced synthesis of TDH within bacterial cells . These data imply that the conjugated bile acids play a key role in the pathogenicity of V . parahaemolyticus.

Appl Environ Microbiol, 1996 Aug, 62(8), 2806 - 10
Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2; Fouz B et al.; The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation . In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed . Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium . Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface . The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples . Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding . The expression of these proteins was not affected by iron levels . In addition, V . vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb . In conclusion, the overall data suggest that the eel pathogen V . vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.

Curr Microbiol, 1996 Aug, 33(2), 129 - 32
Isolation of Vibrio harveyi from diseased kuruma prawns Penaeus japonicus; Liu PC et al.; Outbreaks of high mortality among the cultured kuruma prawn Penaeus japonicus without overt gross signs occurred during August and December of 1994 in I-Lan, Taiwan . Eleven luminous bacterial strains were isolated from the hepatopancreas of moribund prawns from five different farms by use of tryptic soy agar (TSA, supplemented with 2% NaCl) and/or thiosulfate citrate bile salt sucrose (TCBS) agar . These strains, together with our two previously unpublished isolates, were characterized and identified to be Vibrio harveyi in comparison with two ATCC Type strains and one strain previously isolated from the tiger prawn, P . monodon.

Biochim Biophys Acta, 1996 Jul 31, 1275(3), 157 - 60
Cloning and sequencing of nhaB gene encoding an Na+/H+ antiporter from Vibrio alginolyticus; Nakamura T et al.; A gene has been cloned from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, TO114, defective in three Na+/H+ antiport genes (nhaA, nhaB, chaA) . The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 528 amino acid sequence and molecular mass of 57212 Da . This gene has 62% identity to nhaB gene at the DNA level from Escherichia coli and the deduced amino acid sequence is 67% identical with E . coli NhaB . This gene is presumably the V . alginolyticus nhaB gene and will be named nhaBv.

MMWR Morb Mortal Wkly Rep, 1996 Jul 26, 45(29), 621 - 4
Vibrio vulnificus infections associated with eating raw oysters--Los Angeles, 1996; Reported cholera in the United States et al.; Foodborne and Diarrheal Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333 USAOBJECTIVE: To describe US cholera surveillance data from 1992 to 1994 and the domestic impact of the epidemics of Vibrio cholerae O1 in Latin America and V cholerae O139 in Asia . DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of surveillance data from all cases of cholera reported to the Centers for Disease Control and Prevention (CDC) from January 1, 1992, through December 31, 1994, in the United States and its territories . MAIN OUTCOME MEASURES: Clinical, epidemiologic, and laboratory surveillance data . RESULTS: From 1992 through 1994, 160 cases of cholera were reported to CDC by 20 states and 1 territory . This is a marked increase: only 136 cases were reported from 1965 through 1991 . Outbreaks affecting 75 passengers on an airplane from Latin America and 5 passengers on a cruise ship in Southeast Asia accounted for 50 percent of cases . Vibrio cholerae O139 caused 6 cases (4 percent) . The proportion of V cholerae O1 isolates resistant to at least 1 antimicrobial agent rose from 3 percent in 1992 to 93 percent in 1994 . Of 158 patients whose location of exposure was known, 151 (96 percent) acquired infection abroad (125 in Latin America, 26 in Asia) . Of 105 persons whose reason for travel was known, 31 (30 percent) were US residents who had returned to their country of origin to visit family or friends, and 65 (62 percent) were non-US residents visiting the United States from cholera-affected countries . The cholera rate among persons arriving in the United States from cholera-affected regions was 0.27 case per 100000 air travelers, not substantially increased from earlier estimates . CONCLUSIONS: Cholera has increased in the United States since 1991, reflecting global changes in cholera epidemiology, and is now primarily travel associated and antimicrobial resistant . Most travelers were not traditional tourists; reaching them with prevention measures may be difficult . The risk of cholera to the individual traveler remains extremely low.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7991 - 5
Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression; DiRita VJ et al.; The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature . Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium . Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT . Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium . In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR . An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype . These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains.

Ugeskr Laeger, 1996 Jul 22, 158(30), 4291 - 4
{Vibrio vulnificus infections in Denmark during the summer of 1994}; Bruun BG et al.; The clinical manifestations and epidemiological data of 11 patients infected with Vibrio vulnificus found in Denmark during the unusually warm summer of 1994 are reported . All patients had been exposed to seawater prior to illness, but none had consumed seafood . Nine patients, including four with bacteraemia, developed skin manifestations of various degrees of severity . One patient died of septic shock despite surgery and treatment with relevant antibiotics . Four patients contracted the disease while fishing . High seawater temperature increases the risk of V . vulnificus infections even in temperate climates such as the Danish . Exposure to seawater, including handling of fresh seafood, during warm periods carries a risk of infection with V . vulnificus.

Carbohydr Res, 1996 Jul 19, 288, 85 - 98
Synthesis of four glycosides of a disaccharide fragment representing the terminus of the O-polysaccharide of Vibrio cholerae O:1, serotype Inaba, bearing aglycons suitable for linking to proteins; Ogawa Y et al.; Methyl 4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside was converted into the crystalline 2-(trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside . Debenzoylation of the latter, followed by glycosylation of the resulting 2-hydroxy derivative with 2-O-acetyl-4-azido-4,6-dideoxy-alpha-D-mannopyranosyl chloride, gave the 2-(trimethylsilyl)ethyl glycoside of the corresponding disaccharide (8) . Deacetylation of 8, followed by reduction of the resulting 4-azido-2-hydroxy derivative with H2S, gave the corresponding amine 10 . The latter was treated with 4-O-benzyl-3-deoxy-L-glycero-tetronic acid to give, after debenzylation and acetylation, the fully protected 2-(trimethylsilyl)ethyl alpha-glycoside of the disaccharide fragment of the O-PS of Vibrio cholerae O:1, serotype Inaba (13) . Compound 13 was transformed into the corresponding 1-trichloroacetimidate which was treated, separately, with methyl 6-hydroxy-hexanoate and 2-(2-methoxycarbonylethylthio)ethanol, to give two analogs of 13 possessing a differing linkage arm, namely the methyl esters 16 and 17 . Each of 16 and 17 was treated with aqueous sodium hydroxide, followed by a cation-exchange resin, to give the two corresponding carboxylic acids (19 and 22) . Alternately, treatment of 16 and 17 with hydrazine hydrate gave the acid hydrazides 20 and 23.

FEMS Microbiol Lett, 1996 Jul 15, 141(1), 19 - 23
Isolation of heme-binding proteins from Vibrio anguillarum using affinity chromatography; Mazoy R et al.; Using affinity chromatography techniques, several hemin- and hemoglobin-binding proteins of 97, 56 and 39 kDa were isolated from cell envelopes of Vibrio anguillarum strain H775-3 (serotype O1) and of 56, 46 and 37 kDa from strain RV22 (serotype O2) . All these proteins were isolated under iron-rich as well as iron-poor conditions . Proteins of 39 kDa in H775-3 and of 37 kDa in RV22 isolated by hemin affinity could also bind biotinylated hemoglobin after being transferred to nitrocellulose, which suggests that they could be the common receptors for the heme group in V . anguillarum.

J Biol Chem, 1996 Jul 5, 271(27), 16300 - 9
Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies; Lullau E et al.; The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms . We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms . Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms . LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not . Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm . Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 x 10(8)M-1) . Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen . KA falls to 6.6 x 10(5) M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Jul-Aug, (4), 38 - 41
{A DNA analysis of Vibrio cholerae strains by the polymerase chain reaction}; Onishchenkpo GG et al.; The method for the analysis of cholera toxin gene in V . cholerae strains was developed on the basis of polymerase chain reaction (PCR) . This specific and highly sensitive method using primers affecting the site of the DNA of the operon of cholera toxin gene made it possible to identify one copy of V . cholerae genome . For the first time the content of cholera toxin gene in 4 V . cholerae (eltor) strains, obtained from the clinical material of cholera patients in Tajikistan and Dagestan, was shown with the use of PCR.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Jul-Aug, (4), 23 - 6
{The variability of the biological characteristics of Vibrio cholerae isolated from environmental objects}; Khaitovich AB; The study of the biological characteristics used for the identification of the species, biovar and serovar of V.cholerae O1 isolated from environmental objects on the territory of 8 regions of Ukraine for the period of 1974-1993 revealed the tendency towards an increase in the number of altered cultures . Phage sensitivity (60.7%) and capacity for agglutination with cholera species- and type-specific sera (24.6%) proved to be the most variable properties . Resistance to polymyxin (4.8%), the absence of hemagglutination with chick red blood cells (8.7%), the absence of diastatic activity (28.9%) were also detected . The study established that the temporal, ecosystemic and territorial geographical dependence could be observed in the character of variability of V.cholerae O1 . The data thus obtained indicate the expediency of monitoring the dynamics of the properties of V.cholerae O1 in order to establish the relationship between its variability and the epidemic process.

Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 3 - 6
{Comparative study of the synthesis and specificity of enterotoxin from Vibrio cholerae O139 serotype using monoclonal antibodies}; Fedorova VA et al.; Strains of V . cholerae serovar 0139 showed a higher intensity of multiplication and lesser nutritive requirements and produced 2-5 times higher amounts of enterotoxin during in-depth culturing than cholera vibrios of groups O1 and non-O1 . R-forms of V . cholerae were characterized by the highest production of toxin . Dot-immunoanalysis and immunoblotting with monoclonal antibodies demonstrated the identity of cholera toxin and enterotoxin of V . cholerae serovar O139 . The authors come to a conclusion that selective efficacy of the developed vaccines against diarrhea caused by V . cholerae serovar O139 depends on the presence of homologous O-antigen in their composition.

Int J Food Microbiol, 1996 Jul, 30(3), 385 - 90
Effect of diluents on the enumeration of Vibrio vulnificus; Azanza PV et al.; Peptone (0.1%) solution containing 3% NaCl (PS) was a more suitable diluent than phosphate buffered saline (PBS) solution for the enumeration of Vibrio vulnificus in both broth cultures and oyster homogenates . PBS caused significant underestimation of the viable population of the species by plate counts on either selective or non-selective media . Dilution in PS is recommended in methods for the enumeration of V . vulnificus.

Vet Microbiol, 1996 Jul, 51(1-2), 137 - 49
Application of a modified disc diffusion technique to antimicrobial susceptibility testing of Vibrio anguillarum and Aeromonas salmonicida clinical isolates; Guerin-Faublee V et al.; Two techniques for antimicrobial susceptibility testing of Vibrio anguillarum and Aeromonas salmonicida strains were compared . The first method was the reference test that determines Minimal Inhibitory Concentrations (MIC); the second was a modified diffusion test that measures the Inhibitory Concentrations in Diffusion (ICD) by carrying out the diffusion test with five discs of differing contents . ICD measurement was not applicable for the susceptibility testing of oxytetracycline and sulfadimethoxine . On the other hand, a good correlation between the MICs and the ICDs was observed for oxolinic acid, sarafloxacin, chloramphenicol and trimethoprim . Moreover, the ICD values were close to those obtained for the MIC values . A . salmonicida resistant strains were detected by ICD determination . Thus, ICD could be used instead of MIC for oxolinic acid, sarafloxacin, trimethoprim and chloramphenicol susceptibility testings . The ICD technique is easy to carry out and is not dependent on the growth characteristics of bacteria.

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