Infect Immun, 2000 Dec, 68(12), 6857 - 64 Sequence analysis of TnphoA insertion sites in Vibrio cholerae mutants defective in rugose polysaccharide production; Ali A et al.; Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions . In order to understand the genetic basis of rugosity, we isolated TnphoA-induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique . One of the TnphoA insertions was mapped to the first gene of the vps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster . Three insertions were mapped to a previously unknown hlyA-like gene, also in the vps region . Five other insertions were found in loci unlinked to the vps region: (i) in the epsD gene (encodes the "secretin" of the extracellular protein secretion apparatus), (ii) in a hydG-like gene (encodes a sigma(54)-dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli, (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E . coli (encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy-D-xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation) . These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites. Infect Immun, 2000 Dec, 68(12), 6691 - 6 Vibrio cholerae requires rpoS for efficient intestinal colonization; Merrell DS et al.; Vibrio cholerae is a facultative intestinal pathogen that lives in aquatic environments, often in association with planktonic species . In the suckling mouse, oral inoculation with V . cholerae leads to intestinal colonization and symptoms of diarrheal disease . Results reported here indicate a role for the alternative sigma factor, RpoS, in intestinal colonization in this model of cholera . We constructed within rpoS multiple independent mutations which consistently resulted in a fivefold decrease in colonization ability as assessed by competition assays . These mutations had no detectable effect on the in vitro growth of V . cholerae in a rich medium . The occurrence of spontaneous suppressor mutations potentially required for viability of rpoS strains was ruled out by determination of the frequency of insertional inactivation of rpoS in comparison to two other nonessential loci . Finally, both the in vitro and in vivo mutant phenotypes of rpoS strains were fully complemented by providing rpoS in trans or by allelic reversion, indicating that the observed decrease in colonization fitness was indeed due to the loss of functional RpoS. Yakugaku Zasshi, 2000 Oct, 120(10), 825 - 37 {Recent advances in tetrodotoxin research}; Matsui T et al.; One century has passed since fugu toxin was named tetrodotoxin (TTX) by Tahara . Chemical problems such as crystallization of tetrodotoxin and subsequent structure determination were solved by research groups headed by Tsuda, Hirata, Woodward, and Mosher . The International Symposium on the Chemistry of Natural Products in Kyoto (1964) was well known as symposium which the structure of TTX was internationally clarified . Since the first isolation of toxin from taricha torosa (imori) as natural source except for fugu fishes, distribution of toxin in nature has been widely investigated . And, it was proved that toxin is not produced by fugu fishes, but rather is formed by sea bacteria (30 sp.) such as Alteromonas sp, Vibrio sp, Shewanella . However, it seems to be difficult to explain the tetrodotoxin accumulation at high concentration in fugu by only toxin production by bacteria . TTX analogues were isolated from natural origins such as crabs, fish, annelids, and algae . Based on the structure of these toxin analogues, the biosynthesis of toxin and the structure-activity relationship (Na+ channel) were proposed by Yasumoto-Yamashita . The findings of wide distribution of toxin in nature may be attributed to development of highly sensitive detection method for toxin . The interesting proposal for the biosynthesis and the structure activity, and the detection method for toxin are outlined in this review. J Cell Sci, 2000 Dec, 113 Pt 24, 4435 - 40 Human zonulin, a potential modulator of intestinal tight junctions; Wang W et al.; Intercellular tight junctions are dynamic structures involved in vectorial transport of water and electrolytes across the intestinal epithelium . Zonula occludens toxin derived from Vibrio cholerae interacts with a specific intestinal epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tight junction permeability . We postulated that this toxin may mimic the effect of a functionally and immunologically related endogenous modulator of intestinal tight junctions . Affinity-purified anti-zonula occludens toxin antibodies and the Ussing chamber assay were used to screen for one or more mammalian zonula occludens toxin analogues in both fetal and adult human intestine . A novel protein, zonulin, was identified that induces tight junction disassembly in non-human primate intestinal epithelia mounted in Ussing chambers . Comparison of amino acids in the active zonula occludens toxin fragment and zonulin permitted the identification of the putative receptor binding domain within the N-terminal region of the two proteins . Zonulin likely plays a pivotal role in tight junction regulation during developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory/autoimmune disorders. Fish Shellfish Immunol, 2000 Oct, 10(7), 611 - 22 Modulation of the chemiluminescence response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes; Ordas MC et al.; The influence of several factors on the chemiluminescence (CL) activity of haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) was studied . Haemocytes were stimulated in vitro with different concentrations of zymosan, phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) (adding superoxide dismutase, SOD, to the zymosan-stimulated haemocytes in order to test the specificity of the reaction) . The in vitro effect of the clam pathogens Vibrio tapetis (bacteria) and a Perkinsus atlanticus-like protozoan tentatively named Pseudoperkinsus taapetis on the mussel haemocytes CL response was also assessed . To study the in vivo stimulation of haemocytes, mussels were inoculated with zymosan and the CL response of their haemocytes was subsequently measured . Zymosan added in vitro produced the highest CL response, although PMA also enhanced the CL emission and, in addition, increased the zymosan-stimulated CL . LPS and V . tapetis did not activate haemocytes . SOD significantly decreased the CL emission in zymosan-stimulated haemocytes . P . tapetis cells, as well as their extracellular products, inhibited the CL response to zymosan . Haemocytes from mussels injected with zymosan showed lower levels of stimulation than in vitro treated cells, and CL increased with time after injection. Emerg Infect Dis, 2000 Nov-Dec, 6(6), 631 - 6 Molecular evidence of clonal Vibrio parahaemolyticus pandemic strains; Chowdhury NR et al.; The upsurge in worldwide incidence of Vibrio parahaemolyticus infection in the last 5 years has been attributed to the recent appearance of three serotypes with pandemic potential: O3:K6, O4:K68, and O1:K untypeable (KUT) . Thirty-five strains of these serotypes, isolated from different countries over 4 years, were characterized by ribotyping and pulsed-field gel electrophoresis to determine their origin . The ribotypes of the strains of these serotypes were indistinguishable, except for a Japanese tdh- negative O3:K6 strain and a U.S . clinical O3:K6 isolate, which had slightly different profiles . The migration patterns of the NotI-digest of the total DNA of the strains were similar, and only slight variations were observed between the serotypes . By contrast, the O3:K6 and O1:KUT strains isolated before 1995 and strains of other serotypes had markedly different profiles . The O4:K68 and O1:KUT strains most likely originated from the pandemic O3:K6 clone. J Bacteriol, 2000 Dec, 182(23), 6762 - 73 The overlapping angB and angG genes are encoded within the trans-acting factor region of the virulence plasmid in Vibrio anguillarum: essential role in siderophore biosynthesis; Welch TJ et al.; Products encoded in the trans-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of Vibrio anguillarum . Here we identify angB, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis . We demonstrate that a 287-amino-acid polypeptide, encoded by angB and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2, 3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate . Complementation of various angB mutations provided evidence that an additional, overlapping gene exists at this locus . This second gene, designated angG, also has an essential biosynthetic function . The angG gene directs the expression of three polypeptides when overexpressed in Escherichia coli, all of which are translated in the same frame as AngB . The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule . Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the angB and angG genes. J Bacteriol, 2000 Dec, 182(23), 6694 - 7 NorM of vibrio parahaemolyticus is an Na(+)-driven multidrug efflux pump; Morita Y et al.; NorM of Vibrio parahaemolyticus apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins . Based on the following experimental results, we conclude that NorM is an Na(+)-driven Na(+)/drug antiporter . (i) Energy-dependent ethidium efflux from cells possessing NorM was observed in the presence of Na(+) but not of K(+) . (ii) An artificially imposed, inwardly directed Na(+) gradient elicited ethidium efflux from cells . (iii) The addition of ethidium to cells loaded with Na(+) elicited Na(+) efflux . Thus, NorM is an Na(+)/drug antiporting multidrug efflux pump, the first to be found in the biological world . Judging from the similarity of the NorM sequence to those of putative proteins in sequence databases, it seems that Na(+)/drug antiporters are present not only in V . parahaemolyticus but also in a wide range of other organisms. J Bacteriol, 2000 Dec, 182(23), 6687 - 93 Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features; Ogawa N et al.; Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules . Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V . cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis . The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically . As in E . coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX) . However, unlike in E . coli, PPX activity was not detected in cell extracts of wild-type V . cholerae . The ppk null mutant of V . cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment. Med J Malaysia, 2000 Mar, 55(1), 143 - 5 Tetracycline resistant cholera in Kelantan; Ranjit K et al.; Sensitivity testing on Vibrio cholerae isolates during an epidemic in 1998 in Kelantan identified strains resistant to tetracycline . This prompted a change in the usual management of cholera in Kelantan . The antibiotic of choice was changed from tetracycline to erythromycin. FEMS Microbiol Lett, 2000 Nov 15, 192(2), 231 - 6 A unique and common restriction fragment pattern of the nucleotide sequences homologous to the genome of vf33, a filamentous bacteriophage, in pandemic strains of Vibrio parahaemolyticus O3:K6 O4:K68, and O1:K untypeable; Chang B et al.; Vf33 is a filamentous bacteriophage isolated from Vibrio parahaemolyticus . We performed Southern blot hybridization analysis to examine the distribution of Vf33-related genetic elements in the pandemic strains (O3:K6 strains isolated between 1995 and 1997, O4:K68 and O1:K untypeable strains isolated between 1997 and 1999) of V . parahaemolyticus . Nucleotide sequences homologous to the Vf33 DNA were detected in all 57 test strains including pandemic and non-pandemic strains . However, the profiles of hybridization, including the restriction fragment length polymorphism, with nine Vf33-derived DNA probes exhibited by the pandemic strains were identical and were different from those by the non-pandemic strains . The results support the hypothesis that the pandemic strains are clonal, and suggest a possibility that they have acquired (a) new gene(s) via a Vf33-like filamentous phage. J Clin Microbiol, 2000 Nov, 38(11), 4249 - 53 Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba from the prevailing O1 Ogawa serotype strains in India; Garg P et al.; The toxigenic Inaba serotype of Vibrio cholerae O1 biotype El Tor reappeared in India in 1998 and 1999, almost 10 years after its last dominance in Calcutta in 1989 . Extensive molecular characterization by ribotyping, restriction fragment length polymorphism, and pulsed-field gel electrophoresis indicated that recent Inaba strains are remarkably different from the earlier Inaba strains but are very similar to the prevailing V . cholerae O1 Ogawa El Tor biotype strains . The antibiograms of the Inaba strains were also similar to those of the recent V . cholerae Ogawa strains . These V . cholerae O1 Inaba strains appear to have evolved from the currently prevailing Ogawa strains and are likely to dominate in the coming years. J Clin Microbiol, 2000 Nov, 38(11), 4145 - 51 Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW; Nandi B et al.; The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes . PCR amplification results showed that all (100%) of the 254 V . cholerae strains tested were positive for ompW and 229 ( approximately 98%) of 233 were positive for toxR . None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay . These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay . While the V . cholerae strains reacted with ompW probe, only one (V . mimicus) out of 60 other bacterial strains tested showed weak recognition . In contrast, several strains belonging to other Vibrio species (e.g., V . mimicus, V . splendidus, V . alginolyticus, V . fluvialis, V . proteolyticus, V . aestuarianus, V . salmonicida, V . furnissii, and V . parahaemolyticus) showed weak to strong reactivity to the toxR probe . Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V . cholerae strains belonging to different biotypes and/or serogroups . All of these results suggest that the ompW gene can be targeted for the species-specific identification of V . cholerae strains . The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V . cholerae strains of clinical as well as environmental origin. Epidemiol Infect, 2000 Aug, 125(1), 27 - 34 Detection of virulence associated genes, haemolysin and protease amongst Vibrio cholerae isolated in Malaysia; Iyer L et al.; Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease . The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR . Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively . Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease . Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes . All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin . One strain which had only cep but none of the toxin genes may be a true environmental isolate . The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors . These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic. Epidemiol Infect, 2000 Aug, 125(1), 17 - 25 Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3:K6 and O4:K68) in Thailand and India; Chowdhury NR et al.; Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar . Strains belonging to both these serovars have been reported to possess pandemic potential . The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness . The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene . Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint . Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand . Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions . The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns. Antibiot Khimioter, 2000, 45(9), 7 - 12 {Experimental resistance of Vibrio cholerae el tor to nalidixic acid and fluoroquinolones}; Ryzhko IV et al.; It was shown that sensitivity of Vibrio cholerae eltor P-5879 to tetracycline, levomycetin, furazolidone, trimethoprim/sulfamethoxazole, aminoglycosides, beta-lactams, rifampicin, quinolones in vitro correlated with drugs efficacy in the treatment of experimental cholera of albino mice . Mutants of V . cholerae eltor P-5879 Nalr resistant to nalidixic acid (MIC 160-200 mg/l) formed with frequency 10(-9)-110(-8) had no cross resistance to fluoroquinolones . But the efficacy of ofloxacin, lomefloxacin, norfloxacin against these mutants in vivo reduced, though it was not changed in vitro . Mutants of V . cholerae eltor P-5879 resistant to fluoroquinolones and selected after culturing in the presence of the drugs had cross resistance to all quinolones studied . Infection caused by Cpfr mutant could not be treated with nalidixic acid and fluoroquinolones, therapeutic efficacy of rifampicin and beta-lactams, also reduced though sensitivity in vitro was not changed . The results of investigation proves the necessity of quinolones use for cholerae treatment as it is recommended for other severe enteric infections. Appl Environ Microbiol, 2000 Nov, 66(11), 4649 - 54 Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998); DePaola A et al.; Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York . Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V . parahaemolyticus in environmental shellfish samples . V . parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas . However, only two samples (one each from Washington and Texas) were found to harbor total V . parahaemolyticus densities exceeding the level of concern of 10,000 g(-1) . Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g(-1)) . Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7 degrees C) and V . parahaemolyticus levels (100 to 1,000 g(-1)) during the summer . Salinity varied from 14.9 to 29.3 ppt . A slight but significant (P < 0.05) negative correlation (-0.25) was observed between V . parahaemolyticus density and salinity . Based on our data, findings of more than 10,000 g(-1) total V . parahaemolyticus or >10 g(-1) tdh- and/or trh-positive V . parahaemolyticus in environmental oysters should be considered extraordinary. Appl Environ Microbiol, 2000 Nov, 66(11), 4634 - 40 Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts; Eilers H et al.; Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy . A metabolic inhibitor of cell division (nalidixic acid {NA}) was added to substrate-amended (S+) and unamended (S-) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls . In addition, starvation survival experiments were performed on selected isolates . Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction . Vibrio spp . and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S-, respectively, and this trend was even magnified by the addition of NA . These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments . Uncultured dominant gamma-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either . Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp . did not . The life strategy of rapidly enriched culturable gamma-proteobacteria could thus be described as a "feast and famine" existence involving different activation levels of substrate concentration. Am J Hum Genet, 2000 Dec, 67(6), 1422 - 7 Epub 2000 Oct 30. Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion; Hogenauer C et al.; To explain the very high frequency of cystic fibrosis (CF) mutations in most populations of European descent, it has been proposed that CF heterozygotes have a survival advantage when infected with Vibrio cholerae or Escherichia coli, the toxins of which induce diarrhea by stimulation of active intestinal chloride secretion . Two assumptions underlie this hypothesis: (1) chloride conductance by the CF transmembrane conductance regulator (CFTR) is the rate-limiting step for active intestinal chloride secretion at all levels of expression, from approximately zero in patients with CF to normal levels in people who are not carriers of a mutation; and (2) heterozygotes have smaller amounts of functional intestinal CFTR than do people who are not carriers, and heterozygotes therefore secrete less chloride when exposed to secretagogues . The authors used an intestinal perfusion technique to measure in vivo basal and prostaglandin-stimulated jejunal chloride secretion in normal subjects, CF heterozygotes, and patients with CF . Patients with CF had essentially no active chloride secretion in the basal state, and secretion was not stimulated by a prostaglandin analogue . However, CF heterozygotes secreted chloride at the same rate as did people without a CF mutation . If heterozygotes are assumed to have less-than-normal intestinal CFTR function, these results mean that CFTR expression is not rate limiting for active chloride secretion in heterozygotes . The results do not support the theory that the very high frequency of CF mutations is due to a survival advantage that is conferred on heterozygotes who contract diarrheal illnesses mediated by intestinal hypersecretion of chloride. J Appl Microbiol, 2000 Oct, 89(4), 702 - 9 A new bacteriophage, VHML, isolated from a toxin-producing strain of Vibrio harveyi in tropical Australia; Oakey HJ et al.; Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans . Despite their importance, their modes of virulence have yet to be fully elucidated . Here, we present a previously unreported bacteriophage extracted from a toxin-producing strain of V . harveyi isolated from moribund prawn larvae in tropical Australia . Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double-stranded linear DNA) . We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like) . VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V . harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested) . The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed. J Appl Microbiol, 2000 Oct, 89(4), 599 - 606 An indirect immunofluorescent antibody technique for detection and enumeration of Vibrio vulnificus serovar E (biotype 2): development and applications; Marco-Noales E et al.; The applications of an indirect fluorescent antibody technique (IFAT), developed to detect and enumerate the pathogenic bacterium Vibrio vulnificus serovar E from water and clinical samples, are described . This technique proved accurate for detecting V . vulnificus, even under starvation conditions and in the non-culturable state, and could differentiate this species from other bacteria which share the same habitats . The IFAT was successfully used to diagnose vibriosis from naturally- and artificially-infected eels . The overall data suggest that applying this technique properly in environmental and epidemiological/epizootiological studies could significantly increase our knowledge of this bacterium. Biol Pharm Bull, 2000 Oct, 23(10), 1235 - 8 Bactericidal activity of lemon juice and lemon derivatives against Vibrio cholerae; de Castillo MC et al.; Food products can be possible vectors of the agent responsible for cholera epidemics, because some of these products allow Vibrio cholerae O1 to develop to concentrations above the dangerous level . This study deals with the behaviour of essential oils, natural and concentrated lemon juice and fresh and dehydrated lemon peel against V . cholerae O1 biotype Eltor serotype Inaba tox+ . Our aim was to evaluate whether these products, used at different dilutions, exhibit bactericidal or bacteriostatic activity against the microorganism, when present at concentrations of 10(2), 10(4), 10(6) and 10(8) colony forming units (CFU) ml(-1), and after different exposure times . 10(8) CFU ml(-1) was considered an infectious dose . Concentrated lemon juice and essential oils inhibited V . cholerae completely at all studied dilutions and exposure times . Fresh lemon peel and dehydrated lemon peel partially inhibited growth of V . cholerae . Freshly squeezed lemon juice, diluted to 10(-2), showed complete inhibition of V . cholerae at a concentration of 10(8) CFU ml(-1) after 5 min of exposure time; a dilution of 2 x 10(-3) produced inhibition after 15 min and a dilution of 10(-3) after 30 min . It can be concluded that lemon, a natural product which is easily obtained, acts as a biocide against V . cholerae, and is, therefore, an efficient decontaminant, harmless to humans. FEMS Microbiol Lett, 2000 Nov 1, 192(1), 73 - 7 Structural variation in the 16S-23S rRNA intergenic spacers of Vibrio parahaemolyticus; Maeda T et al.; The 16S-23S rRNA intergenic spacers (IGS) of Vibrio parahaemolyticus were PCR-amplified and cloned with pT7Blue T vector . A total of six clones were isolated dependent on size difference . The clones were different with respect to both the number and the composition of the tRNA genes included, and were designated IGS-0, IGS-E, IGS-IA, IGS-AE, IGS-EKV and IGS-EKAV . IGS-EKAV included the cluster of tRNA(Glu)-tRNA(Lys)-tRNA(Ala)-tRNA(Val); IGS-EKV, tRNA(Glu)-tRNA(Lys)-tRNA(Val); IGS-AE, tRNA(Ala)-tRNA(Glu); IGS-IA, tRNA(Ile)-tRNA(Ala); and IGS-E, tRNA(Glu) . IGS-0 had no tRNA gene . Some similarities were found in the nucleotide sequence of the non-coding regions flanked by the tRNA genes . The structure difference found in the spacers is meaningful for elucidating the evolutionary line of each ribosomal RNA operon and the profile is applicable as a molecular marker of the bacterium. Protein Expr Purif, 2000 Oct, 20(1), 87 - 94 Purification and characterization of a luxO promoter binding protein LuxT from Vibrio harveyi; Lin YH et al.; Bioluminescence in the marine bacterium Vibrio harveyi is cell density dependent and is regulated by small molecules (autoinducers) excreted by the bacteria . The autoinducer signals are relayed to a central regulator, LuxO, which acts in its phosphorylated form as a repressor of the lux operon at the early stages of cell growth . We report in these studies the purification to homogeneity of a luxO DNA binding protein (LuxT) from V . harveyi after five major chromatography steps, including a highly effective DNA affinity chromatography step and reverse-phase HPLC . Regeneration of binding activity was accomplished after HPLC and SDS-PAGE by renaturation of LuxT from guanidine hydrochloride . It was also demonstrated that the functional LuxT was a dimer of 17 kDa that bound tightly (K(d) = 2 nM) to the luxO promoter . The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the Protein Data Bank, indicating that LuxT is a new V . harveyi lux regulatory protein . Infect Immun, 2000 Nov, 68(11), 6487 - 92 Association of protease activity in Vibrio cholerae vaccine strains with decreases in transcellular epithelial resistance of polarized T84 intestinal epithelial cells; Mel SF et al.; Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells . This decrease correlates with the presence of hemagglutinin/protease but not with the presence of other potential accessory toxins or proteases . These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute. Infect Immun, 2000 Nov, 68(11), 6411 - 8 Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638; Valle E et al.; In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers . In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety . In so doing, the thyA gene from V . cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele . Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium . The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model . However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638 . Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate. Infect Immun, 2000 Nov, 68(11), 6391 - 7 Construction of a Vibrio cholerae vaccine candidate using transposon delivery and FLP recombinase-mediated excision; Chiang SL et al.; Recent efforts to develop a vaccine against the diarrheal disease cholera have focused on the use of live attenuated strains of the causative organism, Vibrio cholerae . The Ogawa lipopolysaccharide phenotype is expressed by many epidemic strains, and motility defects reduce the risk of reactive diarrhea in vaccine recipients . We therefore converted a motile Inaba(+) vaccine candidate, Peru-2, to a nonmotile Ogawa(+) phenotype using a mariner-based transposon carrying rfbT, the gene required for expression of the Ogawa phenotype . Analysis of 22 nonmotile Peru-2 mutants showed that two were Ogawa(+), and both of these strains had insertions in the flgE gene . It was possible to convert these strains to antibiotic sensitivity by introducing a recombinase that acts on sites flanking the antibiotic marker on the transposon . The resulting strains are competent for colonization in infant mice and may therefore be suitable as vaccine candidates for use either independently or in a combination with strains of different biotypes and serotypes. EMBO J, 2000 Oct 16, 19(20), 5315 - 23 In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin; Fullner KJ et al.; Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure . Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA . Here we investigate the association of rtxA with the cell-rounding effect . We find that V . cholerae exports a large toxin, RTX (repeats-in-toxin) toxin, to culture supernatant fluids and that this toxin is responsible for cell rounding . Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore-forming toxins . Rather, RTX toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers . This RTX toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the toxin target . Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins. J Biol Chem, 2001 Jan 26, 276(4), 2816 - 23 Epub 2000 Oct 16. Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution; Kobori T et al.; The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution . This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH . The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain . The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site . On the basis of the crystal structure of NfsA and its alignment with the V . harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding . The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group. Mol Microbiol, 2000 Oct, 38(1), 67 - 84 The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter; Krukonis ES et al.; ToxR is required in Vibrio cholerae for transcriptional activation of the toxT gene, the protein product of which activates numerous genes involved in virulence . Although ToxR cannot activate the toxT promoter in Escherichia coli, the products of the tcpPH operon are shown here to activate the toxT promoter, and co-expression with ToxRS enhances activation . An identical pattern was seen in a DeltatcpPDeltatoxR strain of V . cholerae when TcpPH or ToxRS was expressed from plasmids . Although overexpression of the TcpP/H proteins in V . cholerae partially complemented both a DeltatoxR strain and a DeltatcpPDeltatoxR double mutant for toxin production and toxT-lacZ activation, the presence of ToxR greatly increased their expression . Analysis of a toxT-lacZ promoter deletion series demonstrated that TcpP was able to interact functionally with the toxT promoter downstream of the ToxR binding site . This was confirmed using electrophoretic mobility shift assays of this toxT promoter deletion series and DNase I footprinting analysis, which showed that TcpP interacts with the promoter region from -51 to -32, whereas ToxR protected a region from -100 to -69 . In addition, membranes containing endogenous levels of ToxR bound more readily to the toxT promoter than did membranes containing only TcpP . Characterization of a number of tcpP substitution mutants revealed one derivative (TcpP-H93L) that, when overexpressed, was markedly defective for toxT activation, cholera toxin and TcpA (toxin co-regulated pilus) production and DNA binding; however, toxT activation by TcpP-H93L was restored in the presence of ToxR, suggesting that ToxR can provide the promoter recognition function for toxT activation . Two additional mutant derivatives, TcpP-W68L and TcpP-R86A, failed to activate toxT or direct toxin and TcpA production in the presence or absence of ToxR . Both TcpP-W68L and TcpP-R86A, like TcpP-H93L, were defective for DNA binding . Finally, a ToxR mutant derivative, ToxR-G80S, served to separate the different roles of ToxR on different promoters . Although ToxR-G80S was inefficient at activating the ompU promoter in V . cholerae (ompU encodes an outer membrane porin regulated by ToxR), it was fully capable of activating the toxT promoter . These data suggest that ToxR is not a direct activator in the toxT expression system but, instead, enhances the activity of TcpP, perhaps by recruiting it to the toxT promoter under conditions in which expression levels of TcpP are too low for it to activate toxT efficiently on its own. J Bacteriol, 2000 Nov, 182(21), 6264 - 7 Characterization of the DNA- and metal-binding properties of Vibrio anguillarum fur reveals conservation of a structural Zn(2+) ion; Zheleznova EE et al.; The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions . Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals . We found that V . anguillarum Fur binds the iron box in the presence of Mn(2+), Co(2+), Cd(2+), and to a lesser extent Ni(2+) but, unlike E . coli Fur, not in the presence of Zn(2+) . We also found that V . anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding. Microbiology, 2000 Oct, 146 ( Pt 10), 2613 - 26 Genetic relationships between clinical and environmental Vibrio cholerae isolates based on multilocus enzyme electrophoresis; Farfan M et al.; A total of 107 isolates of Vibrio cholerae, including 29 strains belonging to serogroup O139, were studied using multilocus enzyme electrophoresis (MLEE) to determine allelic variation in 15 housekeeping enzyme loci . All loci were polymorphic and 99 electrophoretic types (ETs) were identified from the total sample . No significant clustering of isolates was detected in the dendrogram generated from a matrix of coefficients of distances with respect to serogroup, biotype or country of isolation . The mean genetic diversity of this V . cholerae population (H:=0.50) was higher than reported previously . Linkage disequilibrium analysis of the MLEE data showed a clonal structure for the entire population, but not in some of the population subgroups studied . This suggests an epidemic population structure . The results showed that the O139 strains were not clustered in a unique ET, in contrast to previous MLEE studies . This higher genetic variation of the O139 serogroup is concordant with ribotyping studies . The results also confirm that the O139 and O1 ElTor isolates are genetically more closely related to each other than to all the other subpopulations of V . cholerae studied. Microbiology, 2000 Oct, 146 ( Pt 10), 2605 - 12 The Vibrio cholerae O1 chromosomal integron; Clark CA et al.; Until the discovery of the Vibrio cholerae repeat (VCR), the gene capture and expression systems termed integrons had been typically associated with antibiotic-resistance gene cassettes with usually less than five genes in an array . A method is described for the cloning of the ends of large cassette arrays . Conserved restriction sites within VCRs facilitated the mapping by Southern hybridization and cloning of the 5' end of the VCR array, and using appropriate fragments it was possible to develop a physical map of the region of the V . cholerae chromosome . Sequence determination of the predicted beginning of this region revealed intI4, a member of the integron family of integrases . Comparison of these sequences from El Tor, Classical and serotype O134 V . cholerae strains identified the 3' end of the attI site, thereby defining the class 4 integron in one of the V . cholerae chromosomes, and providing the first evidence for integron-like site-specific recombination within V . cholerae . Conduction assays demonstrated IntI1-mediated recombination between VCRs . Restriction mapping places the sequences of intI4 and 26 VCR gene cassettes in arrays within a 120 kb region of the V . cholerae O1 strain 569B genome . This region contains an estimated 150 VCR gene cassettes, dwarfing previously described arrays . Southern analysis of genomic DNA from strains of Vibrio anguillarum, Vibrio mimicus and a number of V . cholerae serotypes revealed fragments that hybridized with VCR-specific probes but showed a high degree of restriction fragment length polymorphism . These data facilitate the identification of part of a new class 5 integron from V . mimicus. J Appl Microbiol, 2000 Sep, 89(3), 539 - 46 Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure; Theron J et al.; A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated . The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V . cholerae . The PCR procedure coupled with an enrichment culture detected as few as four V . cholerae organisms in pure culture . Treated sewage, surface, ground and drinking water samples were seeded with V . cholerae and following enrichment, a detection limit of as few as 1 V . cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates . The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V . cholerae in environmental water samples. J Med Chem, 2000 Oct 5, 43(20), 3624 - 31 Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme; Parkinson GN et al.; The FMN-dependent flavoprotein nitroreductase from Escherichia coli B (NTR) is used in cancer chemotherapy to activate a range of prodrugs . The crystal structure of this enzyme has been determined, using molecular replacement methods and refined at 2.06 A resolution . The recombinant 24-kDa enzyme was crystallized in the tetragonal space group P4(1)2(1)2, with unit cell dimensions of a = b = 57.74 A and c = 275.51 A and two molecules in the asymmetric unit . The structure has a final R factor of 20.3% (R(free) = 26.7%), for all data between the resolution ranges of 10-2.06 A, and includes 4453 protein atoms, 230 water molecules, and 2 flavin mononucleotide (FMN) molecules . The functional unit is a homodimer, which forms the asymmetric unit in the crystal structure . The tertiary structures of these two monomers and their subunit interactions are nearly identical . The molecular replacement search model, the crystal structure of the major NAD(P)H:FMN oxidoreductase of Vibrio fisheri (FRase 1), was selected on the basis of its high sequence identity to that of NTR . The final superposition of these two enzymes revealed a very similar overall fold, with variation in the structures focused around surface loops and helices near the FMN cofactor . Helix G is implicated in substrate specificity and is better resolved in the present NTR structure than in the previously reported FRase 1 structure . The FMN binding pocket is also well-resolved, showing the presence of two channels leading into the active site . The amino acid side chains and main chain atoms interacting with the FMN are well-ordered . The structure of the substrate binding pocket has been used to examine substrate specificity and enzyme kinetics for prodrugs used in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT). Fish Shellfish Immunol, 2000 Aug, 10(6), 489 - 504 Isolation and characterisation of a serum lectin from blue gourami, Trichogaster trichopterus (Pallus); Fock WL et al.; A novel lectin, designated BGL, has been purified from the serum of blue gourami, Trichogaster trichopterus, with the use of (NH4)2SO4 fractionation, affinity chromatography and gel filtration chromatography . Electrophoretic analyses and mass spectrometric study of purified BGL showed that the lectin is composed of two isoforms with native molecular masses estimated to be 65 and 66 kDa, and two subunits of 32 and 34 kDa on SDS-PAGE under non-reducing conditions . Upon reduction with 20 mM dithiothreitol (DTT), BGL showed two close bands of 27 and 29 kDa . After isoelectric focusing, the lectin focused as close double bands at pH 5.6 . The N-termini of both isoforms share the same sequence (HGEENRXGPR) and show no significant homology with any known proteins . The BGL agglutinating activity is specifically inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine, and to a lesser degree by D-(+)-mannose, but not by D-(+)-galactose, D-(+)-glucose, maltose or N-acetyl-D-mannosamine . Haemagglutination assay showed that BGL is more specific for rabbit than mouse, chicken, rat or guinea pig erythrocytes, and haemagglutination was Ca2+-dependent . In addition, BGL could agglutinate a range of micro-organisms and yeast cells, with the exception of some fish pathogens, such as Aeromonas hydrophila (strains: PPD 134/91 and PPD 11/90) and Vibrio harveyi (strain: W618) . Localisation of BGL by fluorescein isothiocyanate (FITC)-labelled antibodies revealed that the lectin is associated with the cell surface of fish leukocytes. J Clin Microbiol, 2000 Oct, 38(10), 3774 - 9 Class 1 integron-borne, multiple-antibiotic resistance encoded by a 150-kilobase conjugative plasmid in epidemic vibrio cholerae O1 strains isolated in Guinea-Bissau; Dalsgaard A et al.; In the 1996-1997 cholera epidemic in Guinea-Bissau, surveillance for antimicrobial resistance showed the emergence of a multidrug-resistant strain of Vibrio cholerae O1 during the course of the epidemic . The strain was resistant to ampicillin, erythromycin, tetracycline, furazolidone, aminoglycosides, trimethoprim, and sulfamethoxazole . Concomitant with the emergence of this strain, we observed a resurgence in the number of registered cholera cases as well as an increase in the case fatality rate from 1.0% before the emergence of the multiple-drug-resistant strain to 5.3% after the emergence of the strain . Our study shows that the strain contained a 150-kb conjugative multiple-antibiotic resistance plasmid with class 1 integron-borne gene cassettes encoding resistance to trimethoprim (dhfrXII) and aminoglycosides {ant(3")-1a}) . The finding of transferable resistance to almost all of the antibiotics commonly used to treat cholera is of great public health concern . Studies should be carried out to determine to what extent the strain or its resistance genes have been spread to other areas where cholera is endemic. J Health Popul Nutr, 2000 Jun, 18(1), 44 - 8 Production of heat-labile enterotoxin by strains of Aeromonas veronii bv veronii; Singh DV; Three isolates of Aeromonas veronii bv veronii (2 environmental, one blood infection) were examined to see if they produce any enterotoxin and, if so, to determine its characteristics . Two isolates caused fluid accumulation in the initial rabbit ileal loop tests . The other strain did so after a single passage through the rabbit ileal loop . All the isolates showed gradual enhancement of fluid secretion after each subsequent passage . Inocula of 1 x 10(4) viable cells and 0.25 mL of culture filtrate caused fluid accumulation comparable to those of toxigenic Vibrio cholerae 569B . The enterotoxic activity was inactivated at higher temperature, and showed biological activity over a wide range of pH . The only histopathological change observed was depletion of mucous from goblet cells . The findings of the study indicate that strains of A . veronii bv veronii produce a heat-labile, pH-stable diarrhoeagenic factor without causing any damage to the intestinal mucosa. Biochim Biophys Acta, 2000 Jul 26, 1475(3), 231 - 7 Terminal glycosylation of bovine uroplakin III, one of the major integral-membrane glycoproteins of mammalian bladder; Malagolini N et al.; Uroplakin III (UPIII) is one of the major transmembrane glycoproteins exposed at the luminal face of mammalian bladder . We investigated the terminal glycosylation of bovine UPIII in order to ascertain whether it contains the alpha 2,3-sialylated sequence thus potentially serving as a receptor for uropathogenic Escherichia coli expressing type S adhesins . We report the occurrence of sialic acid in alpha 2,3- and alpha 2,6-linkage to galactose in bovine UPIII glycans as evidenced by the sensitivity of UPIII to both Vibrio cholera and Newcastle disease virus neuraminidase and by the colocalization of UPIII antigen and material detected by lectins of Sambucus nigra and Maackia amurensis on the luminal face of the bladder . We also present evidence that UPIII glycans are capped by Gal-alpha 1,3-Gal epitope . Consistently, alpha 2,3- and alpha 2, 6-sialyltransferase, as well as alpha 1,3-galactosyltransferase were found to be present in the cells detached from the luminal side of bovine bladder, which are responsible for the UPIII biosynthesis . The putative role of UPIII sialylated glycans in enhancing the uropathogenicity of E . coli expressing type S adhesins is discussed. J Biotechnol, 2000 Sep 29, 83(1-2), 115 - 23 Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili; Eko FO et al.; Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance . Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP) . Immunoblotting confirmed the TCP status of these V . cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria . Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP . Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays . These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM) . Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup. J Biotechnol, 2000 Sep 29, 83(1-2), 57 - 66 Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells; Haslberger AG et al.; Bacterial ghosts have been shown to be an innovative system to prepare vaccines of various bacteria with all features of the intact bacterial cell envelopes, especially all antigenic epitopes, but also to target recombinant proteins inserted in the cell envelopes of the ghost preparations to specific antigen presenting cells . To investigate the activation of the antigen presenting cell by bacterial ghosts in more detail we studied the uptake of bacterial ghosts in dendritic porcine cells and RAW macrophages and the induction of inflammatory mediators or mediators directing the immune response in THP-1 human macrophage cell line . The synthesis of inflammatory macrophage mediators such as TNFalpha in the THP1 cell line was stimulated by a hundred-fold higher dose of ghosts from Vibrio cholerae than the corresponding LPS using ELISA-analysis . These results confirm in vivo experiments indicating no toxic effects of ghosts in rabbits even after intravenous administration in doses stimulating significant humoral responses . We were also able to see a significant activation of IL-12 indicated by the analysis of IL-12(p70) synthesis and IL-12(p40) mRNA accumulation . This interleukine is of special importance in the activation of cellular TH1 immune responses . A rapid uptake of bacterial ghosts in macrophages within 10-30 min could be confirmed by electron microscopy . As antigen presentation is especially effective in porcine dendritic cells (DC) and even a low capacity of antigen uptake is sufficient for an induction of immune responses we investigated uptake and activation of bacterial ghosts by DC . DC are known to be phagocytic in specific immature stages . We found a significant uptake of bacterial ghosts from Actinobacillus pleuropneumoniae (App) and V . cholerae conjugated with FITC (fluorescinisothiocyanate) within 2 h . These data suggest that bacterial ghosts effectively stimulate monocytes and macrophages for the induction of TH1 directed immune responses and dendritic cells treated with bacterial ghosts may serve as a promising vehicle for active immunization and immunotherapy in situ. J Nat Toxins, 2000 Aug, 9(3), 281 - 97 Cholera toxin and related enterotoxins: a cell biological and immunological perspective; de Haan L et al.; Cholera toxin (Ctx) from Vibrio cholerae and the closely related Escherichia coli heat-labile enterotoxin (Etx) are the primary virulence factors responsible for causing cholera and traveller's diarrhea, respectively . Studies on the mode of action of these toxins on gut epithelial cells have revealed important insights into the mechanisms of toxin uptake and trafficking in eukaryotic cells . However, of perhaps even greater fascination have been the discoveries that Ctx and Etx exhibit remarkable immunological properties . When either of these toxins is administered via mucosal routes, it triggers a potent mucosal and systemic anti-toxin immune response . By contrast, local or systemic immunization with other soluble protein antigens usually stimulates only a meagre immune response, or results in a state of immunological tolerance . Even more striking are the findings that when Ctx or Etx are mixed with heterologous antigens, they function as adjuvants, leading to stimulation of mucosal responses to the admixed antigen, and the abrogation of oral tolerance . In addition, recent observations have shown that the receptor-binding component of these toxins can down-regulate inflammatory diseases associated with the induction of autoimmune disorders such as rheumatoid arthritis, diabetes, and multiple sclerosis . While the underlying mechanisms responsible for these remarkable properties have yet to be resolved, it is clear that the toxins' ability to bind to cell surface receptors plays an important role in their potent immunogenicity, adjuvanticity, and immunotherapeutic properties . This review provides an overview of the latest developments within the Ctx/Etx field, with a special emphasis on the cell entry mechanisms and immunomodulatory action of Ctx/Etx and their component subunits. Infect Immun, 2000 Oct, 68(10), 6077 - 81 Enterotoxin-specific immunoglobulin E responses in humans after infection or vaccination with diarrhea-causing enteropathogens; Qadri F et al.; Cholera toxin (CT)-specific antibody responses of the immunoglobulin E (IgE) isotype in the sera of adult patients suffering from infection with either Vibrio cholerae O1, V . cholerae O139, or enterotoxigenic Escherichia coli (ETEC) were analyzed and compared with those in the sera of volunteers immunized with a bivalent B subunit O1/O139 whole-cell cholera vaccine . A significant IgE response to CT was observed in 90% of the patients with V . cholerae O1 infection (18 of 20; P = <0.001) and 95% of the patients with V . cholerae O139 infection (19 of 20; P = <0.001) . Similarly, the majority of the patients with ETEC diarrhea (83%; 13 of 15) showed a positive IgE response to CT . Eight of 10 North American volunteers (80%) orally challenged with V . cholerae O1 showed CT-specific IgE responses (P = 0.004) . In contrast, Swedish volunteers immunized with the oral cholera vaccine showed no IgE responses to CT (P value not significant) . During the study period, total IgE levels in the sera of the diarrheal patients, the North American volunteers, and the Swedish cholera vaccinees alike remained unchanged . However, the total IgE levels in the sera of patients and healthy Bangladeshi controls were on average 89-fold higher than those in the sera of the healthy Swedish volunteers and 34-fold higher than those in the sera of the North American volunteers. Infect Immun, 2000 Oct, 68(10), 6062 - 5 Purification and characterization of a cytotonic protein expressed In vitro by the live cholera vaccine candidate CVD 103-HgR; Sathyamoorthy V et al.; Cholera vaccines developed by the deletion of CTX genes from Vibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees . A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467-470, 1988) . Five fractionation steps yielded electrophoretically pure S-CEP with an M(r) of 79,000 . A partially purified preparation caused fluid accumulation in the sealed infant mouse model . The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V . cholerae. Infect Immun, 2000 Oct, 68(10), 5785 - 93 Pathogenesis of infection by clinical and environmental strains of Vibrio vulnificus in iron-dextran-treated mice; Starks AM et al.; Vibrio vulnificus is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico . In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality . V . vulnificus-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage . In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease . All six V . vulnificus strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains . At lethal doses, all strains caused s . c . skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 10(8) CFU/g, especially in perivascular regions . These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses . In non-iron dextran-treated mice, strains required 10(5)-fold-higher inocula to cause an identical disease process as with iron dextran treatment . These results demonstrate that s.c . inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by V . vulnificus. Microb Drug Resist, 2000 Summer, 6(2), 171 - 2 Antibiotic- and metal-resistant strains of Vibrio parahaemolyticus isolated from shrimps; Bhattacharya M et al.; Enteropathogenic strains of Vibrio parahaemolyticus were isolated from shrimps, Penaeus monodon collected from the region of the Deltaic Sundarbans (West Bengal, India) . About 63% of the isolated strains were resistant to ampicillin, cephalexin, and kanamycin . However, all these strains were sensitive to nitrofurantoin, nalidixic acid, tetracycline, and norfloxacin . The isolated strains were resistant to Ni2+} (75%), Cu2+ (87%), and Co2+ (37%), but all the strains were resistant against Cd2+, Zn2+, and Pb2+ at 10 mM concentration. Comp Biochem Physiol A Mol Integr Physiol, 2000 Aug, 126(4), 471 - 80 Negotiations between animals and bacteria: the 'diplomacy' of the squid-vibrio symbiosis; McFall-Ngai MJ; A shared characteristic among animals is their propensity to form stable, beneficial relationships with prokaryotes . Usually these associations occur in the form of consortia, i.e . a diverse assemblage of bacteria interacting with a single animal host . These complex communities, while common, have been difficult to characterize . The two-partner symbiosis between the squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri offers the opportunity to study the interaction between animal and bacterial cells, because both partners can be cultured in the laboratory and the symbiosis can be manipulated experimentally . This system is being used to characterize the mechanisms by which animals establish, develop and maintain stable alliances with bacteria . This review summarizes the progress to date on the development of this model. Clin Infect Dis, 2000 Aug, 31(2), 561 - 5 Epub 2000 Sep 07. Cholera vaccines; Ryan ET et al.; Cholera causes significant morbidity and mortality worldwide . For travelers, the risk of developing cholera per month of stay in a developing country is approximately 0.001%-0.01%, and cholera may present as traveler's diarrhea . In the United States, only a poorly tolerated, marginally effective, parenterally administered, phenol-inactivated vaccine is available . Outside the United States, 2 additional vaccines are commercially available: an oral killed whole cell-cholera toxin recombinant B subunit vaccine (WC-rBS) and an oral live attenuated Vibrio cholerae vaccine (CVD 103-HgR) . These oral vaccines are well tolerated . In field trials, WC-rBS provides 80%-85% protection from cholera caused by V . cholerae serogroup O1 for at least 6 months . In volunteer studies, CVD 103-HgR provides 62%-100% protection against cholera caused by V . cholerae for at least 6 months . No commercially available cholera vaccine protects against disease caused by V . cholerae serogroup O139 . New cholera vaccines are being developed. Clin Chem Lab Med, 2000 Jun, 38(6), 483 - 7 Integrons: an antibiotic resistance gene capture and expression system; Ploy MC et al.; Bacteria can transfer genetic information to provide themselves with protection against most antibiotics . The acquisition of resistance gene arrays involves genetic mobile elements like plasmids and transposons . Another class of genetic structures, termed integrons, have been described and contain one or more gene cassettes located at a specific site . Integrons are defined by an intl gene encoding an integrase, a recombination site attl and a strong promoter . At least six classes of integrons have been determined according to their intl gene . Classes 1, 2 and 3 are the most studied and are largely implicated in the dissemination of antibiotic resistance . A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC . Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase . However, insertion can occur, albeit rarely, at non-specific sites leading to a stable situation for the cassette . Cassettes are transcribed from the common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes . Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described . Integrons seem to have a major role in the spread of multidrug resistance in gram-negative bacteria but integrons in gram-positive bacteria were described recently . Moreover, the finding of super-integrons with gene-cassettes coding for other determinants (biochemical functions, virulence factors) in Vibrio isolates dating from 1888 suggests the likely implication of this multicomponent cassette-integron system in bacterial genome evolution before the antibiotic era and to a greater extent than initially believed. J Bacteriol, 2000 Oct, 182(19), 5530 - 8 Molecular analyses of a putative CTXphi precursor and evidence for independent acquisition of distinct CTX(phi)s by toxigenic Vibrio cholerae; Boyd EF et al.; The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi, a filamentous phage that infects Vibrio cholerae . To study the evolutionary history of CTXphi, we examined genome diversity in CTX(phi)s derived from a variety of epidemic and nonepidemic Vibrio sp . natural isolates . Among these were three V . cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes . These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXphi replicative form, with the exception of missing ctxAB . Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5' of ctxA, as well as the ctxAB promoter region and coding sequences . These findings are consistent with the hypothesis that a CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences . To assess the evolutionary relationships among additional CTX(phi)s, two CTXphi-encoded genes, orfU and zot, were sequenced from 13 V . cholerae and 4 V . mimicus isolates . Comparative nucleotide sequence analyses revealed that the CTX(phi)s derived from classical and El Tor V . cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences) . These findings suggest that nontoxigenic precursors of the two V . cholerae O1 biotypes independently acquired distinct CTX(phi)s. J Bacteriol, 2000 Oct, 182(19), 5513 - 20 Relation of capsular polysaccharide production and colonial cell organization to colony morphology in Vibrio parahaemolyticus; Enos-Berlage JL et al.; Vibrio parahaemolyticus is a ubiquitous, gram-negative marine bacterium that undergoes phase variation between opaque and translucent colony morphologies . The purpose of this study was to determine the factor(s) responsible for the opaque and translucent phenotypes and to examine cell organization within both colony types . Examination of thin sections of ruthenium red-stained bacterial cells by electron microscopy revealed a thick, electron-dense layer surrounding the opaque cells that was absent in preparations from translucent strains . Extracellular polysaccharide (EPS) material was extracted from both opaque and translucent strains, and the opaque strain was shown to produce abundant levels of polysaccharide, in contrast to the translucent strain . Compositional analysis of the EPS identified four major sugars: glucose, galactose, fucose, and N-acetylglucosamine . Confocal scanning laser microscopy was used to investigate cell organization within opaque and translucent colonies . Cells within both types of colonies exhibited striking organization; rod-shaped cells were aligned parallel to one another and perpendicular to the agar surface throughout the depth of the colony . Cells within translucent colonies appeared more tightly packed than cells in opaque colonies . In addition, a dramatic difference in the structural integrity of these two colony types was observed . When colonies were perturbed, the cell organization of the translucent colonies was completely disrupted while the organization of the opaque colonies was maintained . To our knowledge, this study represents the first description of how cells are organized in the interior of a viable bacterial colony . We propose that the copious amount of EPS produced by the opaque strain fills the intercellular space within the colony, resulting in increased structural integrity and the opaque phenotype. J Bacteriol, 2000 Oct, 182(19), 5342 - 50 Regulation of vibrio cholerae genes required for acid tolerance by a member of the "ToxR-like" family of transcriptional regulators; Merrell DS et al.; The ability of the intestinal pathogen Vibrio cholerae to undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence . An essential component of the ATR is CadA-mediated lysine decarboxylation . CadA is encoded by the acid- and infection-induced gene cadA . Herein, cadA is shown to be the second gene in an operon with cadB, encoding a lysine/cadaverine antiporter . cadC, which is 5' of cadB, encodes an acid-responsive, positive transcriptional regulator of cadBA . Unlike in Escherichia coli, V . cholerae cadB and cadA are also transcribed monocistronically . Of note, bicistronic cadBA is transcribed at low constitutive levels in an acid- and CadC-independent manner . CadC represents a new member of the "ToxR-like" family of transcriptional regulators in V . cholerae and, in addition, exhibits extensive amino acid and functional similarity to E . coli CadC . The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors. Epidemiol Infect, 2000 Jun, 124(3), 489 - 95 Vibrio gastroenteritis in the US Gulf of Mexico region: the role of raw oysters; Altekruse SF et al.; We examined clinical and epidemiological features of 575 laboratory-confirmed cases of vibrio gastroenteritis in Alabama, Florida, Louisiana, and Texas from 1988 to 1997 (the US Gulf of Mexico Regional Vibrio Surveillance System) . Illnesses occurred year round, with peaks in spring and autumn . Illnesses lasted a median of 7 days and included fever in half of patients and bloody stools in 25% of patients with relevant information . Seventy-two percent of patients reported no underlying illnesses . In the week before onset, 236 (53%) of 445 patients for whom data were available ate raw oysters, generally at a restaurant or bar . Educational efforts should address the risk of vibrio gastroenteritis for raw oyster consumers, including healthy individuals . Further studies should examine environmental conditions affecting vibrio counts on seafood and processing technologies to enhance the safety of raw oysters. Epidemiol Infect, 2000 Jun, 124(3), 393 - 9 Expanding multiple antibiotic resistance among clinical strains of Vibrio cholerae isolated from 1992-7 in Calcutta, India; Garg P et al.; Antimicrobial susceptibilities of Vibrio cholerae strains isolated from cholera patients admitted to the Infectious Diseases Hospital, Calcutta, India for 6 years were analysed to determine the changing trends; 840 V . cholerae strains isolated in 1992-1997 were included in this study . Among V . cholerae serogoup O1 and O139, ampicillin resistance increased from 1992 (35 and 70%, respectively) to 1997 (both serogroups 100%) . Resistance to furazolidone and streptomycin was constantly high among V . cholerae O1 strains with gradual increase in resistance to other drugs such as ciprofloxacin, co-trimoxazole, neomycin and nalidixic acid . V . cholerae O139 strains exhibited susceptibilities to furazolidone and streptomycin comparable with those of O1 strains . However, after initial increase in resistance to chloramphenicol and co-trimoxazole, all the V . cholerae O139 strains became susceptible to these two drugs from 1995 onwards . Both V . cholerae O1 and O139 remained largely susceptible to gentamicin and tetracycline . V . cholerae non-O1, non-O139 strains, in contrast, exhibited high levels of resistance to virtually every class of antimicrobial agents tested in this study especially from 1995 . Kruskal-Wallis one-way analysis showed that V . cholerae O1 Ogawa serogroup exhibited significant yearly increase in resistance to nine antibiotics followed by non-O1 non-O139 and O139 strains to six antibiotics and two antibiotics respectively . Interesting observation encountered in this study was the dissipation of some of the resistant patterns commonly found among V . cholerae non-O1 non-O139 or O1 serogroups to the O139 serogroup and vice versa during the succeeding years. FEMS Microbiol Lett, 2000 Sep 1, 190(1), 87 - 91 Comparison of global transcription responses allows identification of Vibrio cholerae genes differentially expressed following infection; Das S et al.; Comparison of global transcription profiles of Vibrio cholerae grown in vitro and in vivo revealed that 20% of the genome was repressed and about 5% was induced under in vivo conditions . Hybridization with the cloned genes revealed that the virulence genes ctx, toxR, toxT and tcpA were induced under in vivo conditions . Dissection of two in vivo induced cosmids identified another set of three genes homologous to che Y1 involved in motility and chemotaxis, pnuC encoding the major component of the nicotinamide mononucleotide transport system and icmF belonging to a cassette involved in multiplication inside host cells . These results demonstrate that the global transcription profile approach might be a powerful method for identification of differentially expressed transcripts under in vivo conditions. J Infect Dis, 2000 Oct, 182(4), 1199 - 206 Epub 2000 Sep 08. Albendazole treatment of children with ascariasis enhances the vibriocidal antibody response to the live attenuated oral cholera vaccine CVD 103-HgR; Cooper PJ et al.; Because concurrent infections with geohelminth parasites might impair the immune response to oral vaccines, we studied the vibriocidal antibody response to the oral cholera vaccine CVD 103-HgR in children infected with Ascaris lumbricoides and investigated the effect of albendazole pretreatment on the postvaccination response . Children with ascariasis were randomized to receive either 2 sequential doses of 400 mg of albendazole or placebo . After the second dose, CVD 103-HgR was given, and serum vibriocidal antibody levels were measured before and 10 days after vaccination . Postvaccination rates of seroconversion were greater in the treatment group that received albendazole (P=.06) . Significantly greater rates of seroconversion and geometric mean titer were observed in the albendazole group in subjects with non-O ABO blood groups . A significant association was observed between vibriocidal seroconversion rates and treatment group, suggesting that A . lumbricoides infections impair the immune response to oral cholera vaccine, particularly in subjects of non-O blood groups. J Infect Dis, 2000 Oct, 182(4), 1161 - 8 Epub 2000 Aug 24. The O139 serogroup of Vibrio cholerae comprises diverse clones of epidemic and nonepidemic strains derived from multiple V . cholerae O1 or non-O1 progenitors; Faruque SM et al.; Sixty-four representative strains of Vibrio cholerae O139 were analyzed, to re-examine the origin of this serogroup . Ribotyping differentiated the strains into 3 HindIII and 7 BglI ribotypes . One HindIII and 5 BglI ribotypes were shared by all toxigenic O139 strains . Of 6 nontoxigenic O139 strains, 3 shared ribotypes with the toxigenic strains, carried genes encoding toxin coregulated pilus, and were susceptible to the cholera toxin-converting bacteriophage CTXPhi . The remaining 3 strains belonged to 2 different ribotypes distinct from toxigenic O139 strains and were resistant to CTXPhi and JA-1, an O139-specific lytic bacteriophage . Polymerase chain reaction amplicons corresponding to the gmhD gene carried by these 3 strains also differed from those of the toxigenic O139 strains but were identical to those of 15 environmental non-O1-non-O139 strains . Thus, the O139 antigen is present in different lineages, and this serogroup appears to comprise epidemic and nonepidemic strains derived separately from different progenitors. J Appl Microbiol, 2000 Aug, 89(2), 261 - 6 Potentially pathogenic vibrios in brackish waters and mussels; Maugeri TL et al.; Water and mussel samples were collected from two brackish lakes, used as mussel farms, at different times of the year, for the quantitative analysis of Vibrio spp . and for the isolation of potentially pathogenic species . The isolates underwent cultural and biochemical tests selected for rapid identification . Glucose oxidizing-fermenting and O/129 sensitive strains were distinguished on the basis of the following tests: sucrose and cellobiose utilization, sulphatase activity and polymyxin B resistance performed, respectively, on TCBS, CPC and SPS media . Responses to the presence of beta-galactosidase, salt requirement and growth on triple sugar iron medium were also added . A total of 125 from 152 isolates were referred to the species Vibrio fluvialis (55 strains), V . alginolyticus (40), V . parahaemolyticus (11), V . vulnificus (10) and V . mimicus (9) . The remaining 27 isolates were not identified . The isolation of potentially pathogenic vibrios from cultivated mussels is a risk for health of people consuming raw seafood . Therefore, a long-term monitoring programme should also include the search for these bacterial species. Biochem J, 2000 Sep 15, 350 Pt 3, 671 - 6 Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus; Zhang ZZ et al.; An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide . Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli . Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro . Using L-Leu-p-nitroanilide as the substrate, the kinetic parameters (k(cat) and K(m)) of the pro-aminopeptidase and processed aminopeptidases were analysed . The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region . In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region. J Clin Microbiol, 2000 Sep, 38(9), 3518 - 9 A cytotoxin-producing strain of Vibrio cholerae non-O1, non-O139 as a cause of cholera and bacteremia after consumption of raw clams; Namdari H et al.; We report a case of a cholera-like gastroenteritis subsequent with bacteremia in a healthy man following consumption of raw clams . Although we failed to recover the organism from the patient's stool culture, his blood culture was positive for a non-cholera toxin-producing yet cytotoxin-producing non-O1 and non-O139 Vibrio cholerae. SAR QSAR Environ Res, 2000, 11(3-4), 301 - 12 Structure-toxicity relationships for aliphatic compounds encompassing a variety of mechanisms of toxic action to Vibrio fischeri; Croni MT et al.; QSARs based upon the logarithm of the octanol-water partition coefficient, log P, and energy of the lowest unoccupied molecular orbital, ELUMO were developed to model the toxicity of aliphatic compounds to the marine bacterium Vibrio fischeri . Statistically robust, hydrophobic-dependent QSARs were found for chloroalcohols and haloacetonitriles . Modelling of the toxicity of the haloesters and the diones required the use of terms to describe both hydrophobicity and electrophilicity . The differences in intercepts, slopes, and fit of these models suggest different electrophilic mechanisms occur between classes, as well as within the diones and haloesters . In order to model globally the toxicity of aliphatic compounds to V . fischeri, all the data determined in this study were combined with those determined previously for alkanones, alkanals, and alkenals . A highly predictive two-parameter QSAR {pT15 = 0.760(log P) - 0.625(ELUMO) - 0.466; n = 63, s = 0.462, r2 = 0.846, F = 171, Pr > F = 0.0001} was developed for the combined data that models across classes and is independent of mechanisms of action . The toxicity of these compounds to V . fischeri compares well to the toxicity (50% population growth inhibition) to the ciliate Tetrahymena pyriformis (r2 = 0.850). Rozhl Chir, 2000 Jun, 79(6), 252 - 3 {A rare case of necrotizing fasciitis and subsequent sepsis with multiorgan failure caused by Vibrio vulnificus}; Hanek P et al.; The authors report a rare case of necrotizing fasciitis, sepsis caused by a strain of Vibrio vulnificus rare in Czech Republic. FEMS Immunol Med Microbiol, 2000 Sep, 29(1), 53 - 7 Identification and characterization of pseudomonas aeruginosa PA-IIL lectin gene and protein compared to PA-IL; Gilboa-Garber N et al.; Using the 33 N-terminal amino acids of the fucose/mannose binding lectin PA-IIL of Pseudomonas aeruginosa ATCC 33347 in a tblastn search of P . aeruginos PAOI genomic sequence in GenBank revealed a single open reading frame encoding a 114-amino acid protein (excluding initiator methionine) perfectly matching that amino acid sequence . Following its stop codon there is a GC-rich sequence having a perfect dyad symmetry promoting formation of a hairpin loop structure, potentially enabling rho-independent transcription termination . Upstream of the putative ribosomal binding site there are sequences resembling Vibrio fischeri luxIbox . consistent with autoinduction of this gene, The predicted PA-IIL molecular mass, confirmed by mass spectrometry, is 11,732 Da . Its pI is 3.88 . The C-terminal domain is particularly hydrophobic, implying possible embedding in the cell membrane . PA-IIL is similar to P . aeruginosa PA-IL lectin in some amino acids and potential glycosylation sites but lacks cysteine, methionine and histidine . Despite their relations in functions and regulation.,their genes are widely separated (by about 867.5 kb). Appl Environ Microbiol, 2000 Sep, 66(9), 4091 - 7 Alterations in the proteome of the Euprymna scolopes light organ in response to symbiotic Vibrio fischeri; Doino Lemus J et al.; During the onset of the cooperative association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria . This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria . The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h . In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis . Specifically, V . fischeri-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs . Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria . Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes . In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with V . fischeri cells accelerates certain developmental processes of the symbiotic organ . These data suggest that V . fischeri-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome. Appl Environ Microbiol, 2000 Sep, 66(9), 4022 - 8 Virulence genes in environmental strains of Vibrio cholerae; Chakraborty S et al.; The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression . The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups . In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V . cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India . PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V . cholerae strains . Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V . cholerae O1 . Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili . Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits . Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V . cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V . cholerae. Appl Environ Microbiol, 2000 Sep, 66(9), 3981 - 6 Characteristics of Vibrio parahaemolyticus O3:K6 from Asia; Wong HC et al.; A variety of serovars of the food-borne pathogen Vibrio parahaemolyticus normally cause infection . Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan . For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics . After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups . The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns . Pattern I1 was the major one for strains from Japan, Korea, and Taiwan . All recently isolated O3:K6 strains carried the thermostable direct hemolysin (tdh) gene . No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses . Results in this study confirmed that the recently isolated O3:K6 strains of V . parahaemolyticus are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains. J Med Microbiol, 2000 Sep, 49(9), 801 - 10 Mechanisms of chloride secretion induced by thermostable direct haemolysin of Vibrio parahaemolyticus in human colonic tissue and a human intestinal epithelial cell line; Takahashi A et al.; Thermostable direct haemolysin (TDH) produced by Vibrio parahaemolyticus is thought to play an important role in the severe diarrhoea caused by this organism . This study investigated the enterotoxicity of TDH for human intestinal cells . Addition of TDH to the mucosal side of human colonic tissue in Ussing chambers caused increased short circuit currents (Isc), a process that was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Ca2+ -activated chloride (Cl-) channels . With human colonic epithelial (Caco-2) cells, high Isc and intracellular Ca2+ concentrations ({Ca2+}in) were detected after the addition of TDH to the apical side of the cell monolayer . The Isc decreased with the addition of DIDS, but not with glybenclamide, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or gadolinium chloride . No Isc increase with TDH was observed when the Cl- in the medium was replaced by gluconate or when Ca2+ was depleted . Similarly, TDH did not raise {Ca2+}in after depletion of extracellular Ca2+ . R7, a mutant form of TDH, reduced the effects of TDH on Isc and {Ca2+}in, as did protein kinase C (PKC) inhibitors . Thus, TDH increases Cl- secretion in human colonic epithelial cells, apparently through mechanisms involving cell binding and Ca2+ influx, followed by elevation of {Ca2+}in associated with PKC phosphorylation. Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 704 - 8 Mutation of the nucleophilic elbow of the Lux-specific thioesterase from Vibrio harveyi; Li J et al.; Myristoyl-ACP thioesterase (LuxD) from Vibrio harveyi causes the slow release of fatty acids for reduction into the aldehyde substrate required for the bacterial bioluminescence reaction . The active site Ser nucleophile (S(114)) of the LuxD thioesterase is in a gamma-turn with a sequence (AXS(114)XS) quite different from the standard motif of GXSXG found in almost all (thio) esterases and lipases . The presence of an Arg residue (R(118)) in the first turn of the helix after the gamma-turn also distinguishes LuxD from other enzymes . Mutation of R(118) to Leu inactivated the enzyme and prevented acylation of the Ser(114) nucleophile, while even a conservative replacement with Lys resulted in over 75% loss of the same functions, suggesting that R(118) helps maintain the configuration of the active site . In contrast, replacement of S(116) with Gly but not Ala stimulated the esterase and deacylation rates by over threefold . Purification of the S116G mutant to homogeneity and analyses of its intrinsic fluorescence on acylation with myristoyl-CoA clearly demonstrated that this mutant was much more active than wild-type LuxD . The presence of S(116) rather than the expected Gly residue in the gamma-turn containing the Ser nucleophile may function so that release of fatty acids from LuxD is restricted allowing a more efficient delivery of fatty acids to the luminescent system . Biologicals, 2000 Sep, 28(3), 149 - 54 Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine; Studer E et al.; Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR . This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance . In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches . A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed . To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V . cholerae 569B cells and tested by PCR . The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit) . This number is below the infective dose of wild-type Vibrio cholerae . In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol . Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10231 - 5 Establishment of an animal-bacterial association: recruiting symbiotic vibrios from the environment; Nyholm SV et al.; While most animal-bacterial symbioses are reestablished each successive generation, the mechanisms by which the host and its potential microbial partners ensure tissue colonization remain largely undescribed . We used the model association between the squid Euprymna scolopes and Vibrio fischeri to examine this process . This light organ symbiosis is initiated when V . fischeri cells present in the surrounding seawater enter pores on the surface of the nascent organ and colonize deep epithelia-lined crypts . We discovered that when newly hatched squid were experimentally exposed to natural seawater, the animals responded by secreting a viscous material from the pores of the organ . Animals maintained in filtered seawater produced no secretions unless Gram-negative bacteria, either living or dead, were reintroduced . The viscous material bound only lectins that are specific for either N-acetylneuraminic acid or N-acetylgalactosamine, suggesting that it was composed of a mucus-containing matrix . Complex ciliated fields on the surface of the organ produced water currents that focused the matrix into a mass that was tethered to, and suspended above, the light organ pores . When V . fischeri cells were introduced into the seawater surrounding the squid, the bacteria were drawn into its fluid-filled body cavity during ventilation and were captured in the matrix . After residing as an aggregate for several hours, the symbionts migrated into the pores and colonized the crypt epithelia . This mode of infection may be an example of a widespread strategy by which aquatic hosts increase the likelihood of successful colonization by rarely encountered symbionts. J Invertebr Pathol, 2000 Jul, 76(1), 63 - 9 Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis; Allam B et al.; In a recent study, we demonstrated the presence of defense factors, competent hemocytes and high enzymatic activities (peptidases, hydrolases, lytic, etc.), in the extrapallial fluid, located between the mantle and the shell, of the Manila clam, Ruditapes philippinarum . In Europe, this species is affected by brown ring disease, an epizootic disease caused by the bacterium Vibrio tapetis . The present work focused on the effect of the development of the disease on cellular and humoral defense parameters in the hemolymph and the extrapallial fluid of experimentally infected clams . Results indicate significant changes in total and dead hemocyte counts, as well as modifications in lysozyme activity and protein content, in the hemolymph and extrapallial fluid of challenged animals . Hemocyte counts and lysozyme activity increased significantly in the hemolymph, but particularly in the extrapallial fluid, where the highest values were observed . A healing (recalcification) process was observed 7 weeks following challenge, suggesting defense system efficiency at neutralizing the pathogen . These results are discussed with emphasis on the role of extrapallial fluids in the defense process against invading microorganisms. J Invertebr Pathol, 2000 Jul, 76(1), 1 - 5 Hepatopancreatic alterations in Litopenaeus vannamei (Boone, 1939) (Crustacea: Decapoda: Penaeidae) experimentally infected with a Vibrio alginolyticus strain; Esteve M et al.; As in other countries, the presence of disease has affected and limited the development of shrimp culture in Venezuela . Vibriosis is one of the most prevalent, causing high mortality not only in larval cultures but also in shrimp production . The present work shows the histological changes in hepatopancreas of Litopenaeus vannamei experimentally induced by a strain of Vibrio alginolyticus . The shrimps were infected by the immersion technique, being exposed to a concentration of 5.2 x 10(7) bacteria/ml . As soon as their behavior indicated a moribund condition, the animals were sacrificed, and their hepatopancreata were fixed and processed for histological observation . Light microscopical observations revealed loss of the acinar structure of the digestive gland and sloughing off of cellular lining because of cytolisis . The acinar cellular content was dispersed within the lumina and among the acini . Clusters of hemocytes, in the majority loaded with bacteria, were observed under the connective tissue capsule surrounding the hepatopancreatic lobes and in the hemal spaces of the connective tissue sheath surrounding the tubules. J Bacteriol, 2000 Sep, 182(18), 5097 - 104 Characterization of vibrio cholerae O1 antigen as the bacteriophage K139 receptor and identification of IS1004 insertions aborting O1 antigen biosynthesis; Nesper J et al.; Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae . In this study we have determined the phage adsorption site on the bacterial cell surface . Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor . In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139 . Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide . By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection . Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region) . Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster . Treating one wbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance . Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation . This strain had a significant reduction in its ability to colonize the mouse small intestine. J Bacteriol, 2000 Sep, 182(18), 5070 - 5 Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state; Bogosian G et al.; The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C . The number of CFU was measured by plate count methods on rich media . The total cell numbers were determined by direct microscopic count methods . The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged . Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates . Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation . At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present . Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells . The cells that grew recovered hydrogen peroxide resistance . When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered . Warming has been reported to "resuscitate" nonculturable cells . Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells. Nature, 2000 Aug 3, 406(6795), 477 - 83 DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae; Heidelberg JF et al.; Here we determine the complete genomic sequence of the gram negative, gamma-Proteobacterium Vibrio cholerae El Tor N16961 to be 4,033,460 base pairs (bp) . The genome consists of two circular chromosomes of 2,961,146 bp and 1,072,314 bp that together encode 3,885 open reading frames . The vast majority of recognizable genes for essential cell functions (such as DNA replication, transcription, translation and cell-wall biosynthesis) and pathogenicity (for example, toxins, surface antigens and adhesins) are located on the large chromosome . In contrast, the small chromosome contains a larger fraction (59%) of hypothetical genes compared with the large chromosome (42%), and also contains many more genes that appear to have origins other than the gamma-Proteobacteria . The small chromosome also carries a gene capture system (the integron island) and host 'addiction' genes that are typically found on plasmids; thus, the small chromosome may have originally been a megaplasmid that was captured by an ancestral Vibrio species . The V . cholerae genomic sequence provides a starting point for understanding how a free-living, environmental organism emerged to become a significant human bacterial pathogen. Infect Immun, 2000 Sep, 68(9), 5435 - 8 Cl(-) secretion in colonic epithelial cells induced by the vibrio parahaemolyticus hemolytic toxin related to thermostable direct hemolysin; Takahashi A et al.; A hemolytic toxin related to thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), produced by Kanagawa-phenomenon-negative Vibrio parahaemolyticus is suspected of playing an important, but yet-to-be-elucidated role in diarrhea caused by this organism . In cultured human colonic epithelial cells, TRH increases Cl(-) secretion, followed by elevation of intracellular calcium. Infect Immun, 2000 Sep, 68(9), 5096 - 106 Construction and phenotypic evaluation of a Vibrio vulnificus vvpE mutant for elastolytic protease; Jeong KC et al.; Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters . Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater . Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V . vulnificus infections . Among the putative virulence factors is an elastolytic metalloprotease . We cloned and sequenced the vvpE gene encoding an elastase of V . vulnificus ATCC 29307 . The functions of the elastase were assessed by constructing vvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice . Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene . In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V . vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice . Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro . Therefore, it appears that elastase is less important in the pathogenesis of V . vulnificus than would have been predicted by examining the effects of administering purified proteins to animals . However, V . vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors. Infect Immun, 2000 Sep, 68(9), 5037 - 43 Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V . cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice; Kossaczka Z et al.; Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide {CPS}) is associated with immunity to the homologous pathogen . The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates) . Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V . cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G . Adipic acid dihydrazide was used as a linker . When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V . cholerae O139 . Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity . These results indicate that the conjugates elicited IgG with vibriocidal activity . Conjugates also elicited high levels of serum diphtheria toxin IgG . Convalescent sera from 20 cholera patients infected with V . cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to < or =50 . Treatment with 2-ME reduced the titers of 17 of 20 patients to < or =50 . These data show that, like infection with V . cholerae O1, infection with V . chol