J Biol Chem, 2002 Aug 16, 277(33), 29555 - 60 Epub 2002 May 31. Molecular cloning and characterization of a unique beta-glucosidase from Vibrio cholerae; Park JK et al.; We have recently reported the molecular cloning of a gene, gspK, in Vibrio cholerae that encodes a specific glucosamine kinase . We describe here the identification of bglA, a gene contiguous to gspK in a presumptive large chitin catabolic operon . BglA was molecularly cloned into Escherichia coli, and the protein BglA was overexpressed and purified to apparent homogeneity . BglA is 65 kDa (574 amino acids) with an N-terminal amino acid sequence predicted by the gene sequence, suggesting that the enzyme is cytoplasmic . The purified enzyme exhibited optimal activity with p-nitrophenyl beta-glucoside, cellobiose, and higher oligosaccharides of cellulose . No other glucosides or glycosides tested were hydrolyzed, including Glc-Glc disaccharides where the linkage is beta 1-->2, beta 1-->3, and beta 1-->6, respectively . The predicted BglA sequence bears little similarity to other proteins in the data banks . The Henrissat algorithm places BglA sequence in Family 9 of the glycosidases, suggesting it is an endoglucanase . However, the results summarized above suggested that BglA is an exoenzyme yielding Glc at each cleavage step . To resolve this apparent discrepancy, detailed kinetic studies were conducted with cellotetraose . Only exoglucanase activity was detected . The function of this enzyme in V . cholerae remains to be determined, especially because our strain of this organism does not utilize cellobiose. Appl Environ Microbiol, 2002 Jun, 68(6), 2901 - 9 Comparative phenotypic, molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates; Yeung PS et al.; Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes . However, since 1996, V . parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence . We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes . Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates . Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates . In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates . Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay . At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%) . A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells . Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V . parahaemolyticus O3:K6 strains. Croat Med J, 2002 Jun, 43(3), 346 - 9 Ilizarov technique in the treatment of chronic osteomyelitis caused by Vibrio alginolyticus; Barbarossa V et al.; We present a case of 26-year-old woman with posttraumatic chronic osteomyelitis caused by Vibrio alginolyticus, contracted after contamination of a tibial fracture with seawater . The patient underwent bone resection and bifocal osteosynthesis according to Ilizarov and was treated with a combination of ciprofloxacin and tetracycline . The patient responded in the same way to distraction osteogenesis as any other patient with chronic osteomyelitis with large defects. Microb Ecol, 2000 Apr, 39(3), 175 - 185 Role of Microcolony Formation in the Protistan Grazing Defense of the Aquatic Bacterium Pseudomonas sp . MWH1; Hahn MW et al.; A BSTRACTThe defense strategy of the aquatic bacterium Pseudomonas sp . MWH1 against flagellate grazing was investigated in chemostat and batch experiments . The influence of predation on the Pseudomonas population was studied in the absence and presence of a potential competitor ( Vibrio sp . CB5), as well as under starvation conditions and in a situation of unlimited growth . In the competition experiment the two bacterial strains were distinguished by immunofluorescence microscopy . When the Pseudomonas strain was cultured in the absence of the predator Ochromonas sp . DS, only mobile single cells were detectable . Grazing by this bacterivorous flagellate resulted in all experiments in the occurrence of a Pseudomonas subpopulation, which grew as floclike, suspended microcolonies . These microcolonies consisted of up to approximately 1,000 cells and were, because of their large size, protected against flagellate grazing . The microcolony subpopulation dominated the total Pseudomonas population in situations of high grazing pressure at a wide range of bacterial growth conditions . Thus, the formation of the microcolonies is interpreted as a successful grazing-defense strategy, which is effective under several growth conditions, allowing for the survival of the strain even when substrate depletion is combined with strong grazing pressure . Batch culture experiments demonstrated that the change in morphology of Pseudomonas sp . MWH1 is not controlled by growth rate, although no formation of microcolonies was observed after the addition of 0.2-&mgr;m-filtered flagellate cultures to Pseudomonas cultures, indicating that a chemical trigger released by the flagellate is not involved in the control of this defense mechanism. Vaccine, 2002 Jun 21, 20(21-22), 2720 - 6 Comparison of mucosal and systemic humoral immune responses after transcutaneous and oral immunization strategies; John M et al.; In order to compare the ability of transcutaneous and oral immunization strategies to induce mucosal and systemic immune responses, we inoculated mice transcutaneously with cholera toxin (CT) or the non-toxic B subunit of cholera toxin (CtxB), or orally with Peru2(pETR1), an attenuated vaccine strain of Vibrio cholerae expressing CtxB . In addition, we also evaluated dual immunization regimens (oral inoculation with transcutaneous boosting, and transcutaneous immunization with oral boosting) in an attempt to optimize induction of both mucosal and systemic immune responses . We found that transcutaneous immunization with purified CtxB or CT induces much more prominent systemic IgG anti-CtxB responses than does oral inoculation with a vaccine vector strain of V . cholerae expressing CtxB . In comparison, anti-CtxB IgA in serum, stool and bile were comparable in mice either transcutaneously or orally immunized . Overall, the most prominent systemic and mucosal anti-CtxB responses occurred in mice that were orally primed with Peru2(pETR1) and transcutaneously boosted with CT . Our results suggest that combination oral and transcutaneous immunization strategies may most prominently induce both mucosal and systemic humoral responses. Dis Aquat Organ, 2002 Apr 5, 48(3), 221 - 31 Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams; Allam B et al.; Brown ring disease (BRD) causes high mortalities in the introduced Manila clam Ruditapes philippinarum cultured in western Europe . The etiological agent of BRD, Vibrio tapetis, adheres to and disrupts the production of the periostracal lamina, causing the anomalous deposition of periostracum around the inner shell . Because the primary sign of BRD is found outside the soft tissues, the processes leading to death are not as obvious as those for internal pathogens . This study was designed to evaluate the pathogenicity of V . tapetis, in an attempt to help explain the mechanisms of mortality . We found high mortalities (up to 100%) for clams following the inoculation of V . tapetis into the extrapallial space (between mantle and inner shell) or the posterior adductor muscle of healthy R . philippinarum . Microscopy and immunological detection methods showed that the pathogen was rapidly eliminated from tissues and hemolymph of animals that survived the inoculation . In clams that died, the bacteria were found to have proliferated, resulting in severe tissue disruption . Bacteria were able to penetrate into tissues from the extrapallial space through the external epithelium of the mantle . In contrast, no mortalities were observed following injection of V . tapetis in the native European clam Ruditapes decussatus, which is resistant to BRD . This clam rapidly eliminated the bacterium from hemolymph and soft tissues . Clam mortality associated with BRD in the field is likely to result from the penetration of V . tapetis into the clam's extrapallial space through the disrupted periostracal lamina and from there into the soft tissues through the irritated mantle epithelium . Some bacteria also penetrate through the digestive epithelia . In either case, bacteria proliferate rapidly in the soft tissues, causing severe damage and subsequent death. J Membr Biol, 2002 May 1, 187(1), 65 - 70 Functional expression of the Vibrio parahaemolyticus Na+/galactose (vSGLT) cotransporter in Xenopus laevis oocytes; Leung DW et al.; We have successfully expressed a bacterial cotransporter in a functional form in the Xenopus laevis oocyte expression system . The goals were to compare the kinetics and selectivity of the cotransporter expressed in oocytes with those obtained in bacteria and in proteoliposomes, and to determine if it is possible to measure the electrical properties of the bacterial cotransporter expressed in oocytes . The Vibrio parahaemolyticus Na+/galactose cotransporter (vSGLT) expressed in oocytes has functional properties that are similar to those expressed in bacteria and those of the purified cotransporter reconstituted into liposomes . vSGLT is a Na+-dependent transporter that is saturable with Na+ (K(0.5)=17 mM) and D-galactose (K(0.5)=237 microM) and is sensitive to both D-fucose and phlorizin . In addition, vSGLT in oocytes shows a sugar specificity in the order of D-galactose >D-fucose > D-glucose, distinguishing it from the animal members of the Na+/glucose cotransporter family . The level of transport by vSGLT in oocytes is lower overall (V(max) approximately 10 pmol/oocyte/hour) compared to other plant and animal cotransporters (V(max) approximately 1000 pmol/oocyte/hour) . The low level of expression does not permit us to carry out electrophysiological studies of the bacterial cotransporter . This study shows the potential and unique advantages of utilizing a eukaryotic oocyte expression system to study bacterial cotransporters. Microb Ecol, 2001 Dec, 42(4), 540 - 548 Seasonal Abundance and Distribution of Vibrio cholerae in Coastal Waters Quantified by a 16S-23S Intergenic Spacer Probe; Jiang SC et al.; Vibrio cholerae is the causative agent of the severe diarrheal disease cholera and is indigenous to brackish waters . To advance our understanding of the ecology of this bacterium, we have developed a molecular probing method for detection of V . cholerae in coastal waters . Water samples from 7 locations in the Newport Bay watershed, California were sampled monthly for a whole year . V . cholerae concentrations were determined by membrane filtration-colony hybridization using an oligonucleotide probe targeting the 16S-23S intergenic spacer (ITS) region . In addition to V . cholerae concentrations, environmental parameters, including temperature, salinity, total bacterial direct counts, total viable counts, and chlorophyll a concentrations, were determined for each site . V . cholerae was detected year-round throughout the watershed . Regression analysis indicated that the concentration of V . cholerae inversely correlated with salinity (p <0.001) . The sampling sites located nearest to the Pacific Ocean had lower concentrations, whereas sites located along the brackish San Diego Creek (salinity 0-12 per thousand) routinely had higher concentrations . V . cholerae concentrations also correlated with temperature (p <0.01) in the watershed, with concentrations ranging from less than 1 CFU mL-1 to 2,930 CFU mL-1 of water . The results of this study indicate that the dynamics of V . cholerae is mainly influenced, out of the parameters measured, by the temperature and salinity of the environment . This information is valuable for understanding the ecology of V . cholerae. Microb Ecol, 2001 Dec, 42(4), 531 - 539 RecA Expression in Response to Solar UVR in the Marine Bacterium Vibrio natriegens; Booth MG et al.; Solar ultraviolet radiation may produce daily stress on marine and estuarine communities as cells are damaged and repair that damage . Reduction in the earth's stratospheric ozone layer has increased awareness of the potential effects that ultraviolet radiation may have in the environment, including how marine bacteria respond to changes in solar radiation . We examined the use of the bacterial RecA protein as an indicator of the potential of bacteria to repair DNA damage caused by solar UV irradiation using the marine bacterium Vibrio natriegens as a model . RecA is universally present in bacteria and is a regulator protein for the so-called Dark Repair Systems, which include excision repair, postreplication recombinational repair, and mutagenic or SOS repair . Solar UVB and UVA both reduced V . natriegens viability in seawater microcosms . After exposure to unfiltered solar radiation or radiation in which UVB was blocked, survival dropped below 1%, whereas visible light from which UVA and UVB had been filtered had no effect on survival . Using a RecA-specific antibody for detection, RecA protein was induced by solar radiation in a diel pattern in marine microcosms conducted in the Gulf of Mexico . Peak induction was observed at dusk each day . Although RecA expression was correlated with the formation of UVB-induced cyclobutyl pyrimidine dimers, longer wavelength UVA radiation also induced recA gene expression . Our results demonstrate that RecA-regulated, light-independent repair is an important component in the ability of marine bacteria to survive exposure to solar ultraviolet radiation and that RecA expression is a useful monitor of bacterial repair after exposure to solar UVR. Microb Ecol, 2002 Jul, 44(1), 10 - 8 Epub 2002 May 20. Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism; Nishiguchi MK; Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages {3, 8, 24} . The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont {1} . Infection by intraspecific V . fischeri symbionts exhibited preferential colonization over interspecific V . fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains . Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids . These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host. C R Biol, 2002 Mar, 325(3), 231 - 8 {Characterization of pathogenic bacteria of the cupped oyster Crassostrea gigas}; Waechter M et al.; The French mollusc production is mainly based on the Pacific cupped oyster, Crassostrea gigas . Since 1991, outbreaks of mass mortality of juveniles are reported during the summer period . These outbreaks are a major concern of oyster industry . Several studies have established given bacterial strains to be pathogenic for bivalve species, including oysters . Here we present a study of mortality outbreaks of C . gigas, as initiated in 1995 . In a first step, bacterial strains were isolated during mass mortality outbreak and were biochemically characterised . Among the isolated strains, some strains of Vibrio splendidus biovar II were found to be pathogenic by means of experimental challenge of oyster juveniles . In the second step, a genotypical identification of the pathogenic strain was undertaken, based on 16S RNA sequences and phylogenetic analysis . It confirmed that the pathogenetic strain belonged to Vibrio splendidus biovar II. Cell Tissue Res, 2002 Apr, 308(1), 97 - 102 Epub 2002 Mar 15. Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response; Sepulcre MP et al.; The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers . In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy . The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes . More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum . The results are discussed in relation to the role played by this cell type in fish immune responses. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 7066 - 71 Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli; Tauschek M et al.; Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals . ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown . We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11) . The genes for this pathway are absent from E . coli K-12, although examination of the K-12 genome suggests that it probably once possessed them . The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin . With this in mind, we determined whether the homologous pathway of E . coli H10407 played a role in the secretion of LT . To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination . LT secretion by E . coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells . This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC . Our findings have revealed the mechanism for the secretion of LT by ETEC, which previously was unknown, and provide further evidence of close biological similarities of the LT and cholera toxin. Infect Immun, 2002 Jun, 70(6), 3085 - 93 Quorum-sensing Escherichia coli regulator A: a regulator of the LysR family involved in the regulation of the locus of enterocyte effacement pathogenicity island in enterohemorrhagic E . coli; Sperandio V et al.; The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E . coli (EPEC) . The LEE comprises 41 open reading frames organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins . The first gene of LEE1 encodes the Ler regulator, which activates all the other genes within the LEE . We previously reported that the LEE genes were activated by quorum sensing through Ler (V . Sperandio, J . L . Mellies, W . Nguyen, S . Shin, and J . B . Kaper, Proc . Natl . Acad . Sci . USA 96:15196-15201, 1999) . In this study we report that a putative regulator in the E . coli genome is itself activated by quorum sensing . This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and E . coli K-12; and shares homology with the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively . We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing E . coli regulator A (QseA) . We observed that QseA activated transcription of ler and therefore of the other LEE genes . An EHEC qseA mutant had a striking reduction of type III secretion activity, which was complemented when qseA was provided in trans . Similar results were also observed with a qseA mutant of EPEC . The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing. J Appl Microbiol, 2002, 92(6), 1123 - 35 Occurrence of pathogenic vibrios in coastal areas of France; Hervio-Heath D et al.; AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments . METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar . Presumptive Vibrio colonies were isolated and identified using selected biochemical tests . Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V . parahaemolyticus and V . cholerae, respectively . In this study, V . alginolyticus (99 of 189) was the predominant species, followed by V . parahaemolyticus (41 of 189), V . vulnificus (20 of 189) and non-O1/non-O139 V . cholerae (three of 189) . All 20 V . vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water . The PCR amplification of the pR72H DNA fragment in 41 V . parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp . The latter, present in 24.4% of these isolates, had not previously been found in V . parahaemolyticus strains examined to date . Amplification of the trh gene in two of the isolates suggested these to be virulent strains . Three strains identified as V . cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains . CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters . Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification . SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp . in cultivated mussels and in the aquatic environment . The PCR can be used to detect pathogenic vibrios directly in environmental samples. J Appl Microbiol, 2002, 92(6), 1087 - 96 Relevance of incubation temperature for Vibrio salmonicida vaccine production; Colquhoun DJ et al.; AIMS: To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.) . METHODS AND RESULTS: The significance of sea temperature was investigated using historical clinical and oceanographic data . An upper threshold for disease of approx . 10 degrees C was established . The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media . The highest rates of cell division were identified at 15 degrees C on solid media and 10 degrees C in liquid media . Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay . A novel 76-kDa OMP produced in unshaken cultures at 10 degrees C was not found to stimulate a specific humoral response . CONCLUSIONS: Diagnostic agar plate-based incubation of suspected V . salmonicida should be carried out at 15 degrees C . High yield broth cultures for vaccine production should be incubated at 10 degrees C or lower . SIGNIFICANCE AND IMPACT OF THE STUDY: This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media . The evidence presented suggests that V . salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs. J Appl Microbiol, 2002, 92(6), 1066 - 77 An investigation into the changed physiological state of Vibrio bacteria as a survival mechanism in response to cold temperatures and studies on their sensitivity to heating and freezing; Johnston MD et al.; AIMS: To induce pathogenic Vibrio bacteria into a changed physiological state, in response to cold temperatures in sea water, and assess their sensitivity to heating and freezing, as compared with normal cells . METHODS AND RESULTS: Cells of exponential phase Vibrio vulnificus, V . cholerae and V . parahaemolyticus were washed and inoculated into flasks of sea water, which were stored at 20 and 4 degrees C . Cells stored at 20 degrees C could be recovered after 60 d on non-selective agar (heart infusion agar; HIA) and on the selective agar (thiosulphate citrate bile salts agar) which is used in most Vibrio detection methodology . At 4 degrees C cells became non-culturable on both agars over time . The non-culturable cells appeared to be metabolically active and maintained their membrane integrity, whilst undergoing a change in morphology from rod-shaped to coccoid cells . Resuscitation was possible, in some cases, by an upshift in temperature before plating and the addition of catalase to HIA plates was found to increase recovery . Studies were carried out to assess the sensitivity of the non-culturable cells to heating and freezing compared with the normal cells . Vibrio organisms, whether culturable or in the non-culturable form, were not inactivated by freezing to -20 degrees C . Heating studies showed that V . parahaemolyticus was very heat resistant at low temperatures . However, a pasteurization regime of 2 min at 70 degrees C was found to be effective against all three strains . Experiments showed that the non-culturable cells of all three strains were similar in their heat resistance or, in some cases, were more heat sensitive than cells in the normal form . CONCLUSIONS: Cells in the changed physiological form would not be detected in fish or seafood products by the current Vibrio detection methods . Freezing had no effect in reducing cell numbers . Vibrio parahaemolyticus was very heat resistant in the low temperature pasteurization studies . The higher pasteurization regime of 70 degrees C for 2 min was effective against all three pathogens . Non-culturable cells had similar heat sensitivity or were more heat sensitive than cells in the normal state . SIGNIFICANCE OF IMPACT OF THE STUDY: The study has highlighted a need for the development of better Vibrio detection methods . The low temperature pasteurization of oysters, which has been recommended in the USA, would not be adequate against the strain of V . parahaemolyticus used in this study . Heating regimes which were found to control cells in the normal form will also be effective for the control of the cells with changed physiology. J Invertebr Pathol, 2001 Nov, 78(4), 215 - 9 Differences in the susceptibility of American white shrimp larval substages (Litopenaeus vannamei) to four vibrio species; Aguirre-Guzman G et al.; The rapid expansion of commercial culture of penaeid shrimp is threatened by Vibrio diseases affecting survival and growth . These opportunistic microorganisms are considered part of the normal ecosystem of penaeid shrimp and cause diseases only under conditions that favor them over the host . Shrimp larvae show different susceptibility to these pathogenic agents . In the present work, we report on a comparative study of the susceptibility of all American white shrimp (Litopenaeus vannamei) larval substages to four potentially pathogenic Vibrio species (V . harveyi, V . parahaemolyticus, V . alginolyticus, and V . penaeicida) . Strains of these bacterial species were used to infect nauplii, protozoea I-III, mysis I-III, and postlarvae 1 by immersion challenge at 10(3), 10(5), or 10(7) cfu mL(-1) for 30 min . V . alginolyticus infection had no significant effect on survival rate, compared to control, in all shrimp larvae and at all doses tested . Shrimp larvae infected with V . alginolyticus showed a high survival rate compared to other Vibrio species at the three dose levels . V . penaeicida produced a significant mortality effect (P < 0.01) in all shrimp substages and only in postlarvae 1 at low infection dose (10(3) cfu mL(-1)) . V . harveyi and V . parahaemolyticus induced significant mortality rates (P < 0.01) only at high doses in shrimp larvae . In summary, shrimp larvae demonstrated an age susceptibility that depends on the Vibrio species and dose level. Eur J Med Chem, 2002 May, 37(5), 355 - 66 Novel substituted quinoxaline 1,4-dioxides with in vitro antimycobacterial and anticandida activity; Carta A et al.; Thirty-six 6(7)-substituted-3-methyl- or 3-halogenomethyl-2-phenylthio-phenylsulphonyl-chloro-quinoxaline 1,4-dioxides belonging to series 3-6 were synthesised and submitted to a preliminary in vitro evaluation for antimycobacterial, anticandida and antibacterial activities . Antitubercular screening showed a generally good activity of 3-methyl-2-phenylthioquinoxaline 1,4-dioxides (3d,e,h-j) against Mycobacterium tuberculosis, and exhibited MIC between 0.39 and 0.78 microg mL(-1) (rifampicin MIC=0.25 microg mL(-1)), whereas in compounds 4d,e, 5a,b,d,e,l and 6b-e,j,l MIC ranged between 1.56 and 6.25 microg mL(-1) . Results of the antibacterial and anticandida screening showed that 6e and 6l exhibited MIC=0.4 and 1.9 microg mL(-1), respectively, against Candida krusei (miconazole MIC=0.9 microg mL(-1)), and 4i, 5b,d, 6e, MIC=3.9 microg mL(-1) against Candida glabrata (miconazole MIC=0.4 microg mL(-1)), while compounds 3d,l, 5e,l, and 6b,d,e,l showed MIC=15.6 microg mL(-1) against Vibrio alginolyticus. FEMS Microbiol Lett, 2002 Apr 9, 209(2), 215 - 21 Construction and initial characterization of a luminescent Synechococcus sp . PCC 7942 Fe-dependent bioreporter; Durham KA et al.; A Synechococcus sp . PCC 7942 bioreporter strain capable of sensing bioavailable Fe was constructed by fusing the Fe-responsive isiAB promoter to the Vibrio harveyi luxAB genes . Monitoring luxAB-dependent luminescence through the growth curve demonstrated that in Fe-replete media, transcription from the isiAB promoter was induced transiently in the mid-exponential phase of growth . The initiation of transcription was the functional response to a 10-fold depletion of intracellular Fe to approximately 12 amol Fe per cell . Constitutive isiAB-dependent transcription was observed in Fe-depleted growth media . A dose-response relationship of the bioreporter was generated using trace metal-buffered Fraquil medium and was best represented by a sigmoidal curve having a linear component extending between pFe 21.1 (Fe3+=10(-21.1) M) and pFe 20.6 (Fe3+)=10(-20.6) M) . Initial field trials conducted using water sampled from Lake Erie demonstrate that the bioreporter can serve as a quantitative tool to assess Fe deficiency in natural freshwater environments. FEMS Microbiol Lett, 2002 Mar 19, 209(1), 31 - 7 Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus; Lee CY et al.; A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no . 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography . The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein . Additionally, the isoelectric point of this protein was 5.0 . The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively . Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions . These results indicated that protease A is a serine protease that requires metal . The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease . The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides . Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein . The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously . In conclusion, this is the first report of a serine protease purified directly from the supernatant of V . parahaemolyticus and identifying it as a potential virulence factor. Biomacromolecules, 2002 May-Jun, 3(3), 445 - 55 Biodegradation of lactic acid based polymers under controlled composting conditions and evaluation of the ecotoxicological impact; Tuominen J et al.; The biodegradability of lactic acid based polymers was studied under controlled composting conditions (CEN prEN 14046), and the quality of the compost was evaluated . Poly(lactic acids), poly(ester-urethanes), and poly(ester-amide) were synthesized and the effects of different structure units were investigated . The ecotoxicological impact of compost samples was evaluated by biotests, i.e., by the Flash test, measuring the inhibition of light production of Vibrio fischeri, and by plant growth tests with cress, radish, and barley . All the polymers biodegraded to over 90% of the positive control in 6 months, which is the limit set by the CEN standard . Toxicity was detected in poly(ester-urethane) samples where chain linking of lactic acid oligomers had been carried out with 1,6-hexamethylene diisocyanate (HMDI) . Both the Flash test and the plant growth tests indicated equal response to initial HMDI concentration in the polymer . All other polymers, including poly(ester-urethane) chain linked with 1,4-butane diisocyanate, showed no toxicological effect. Dis Aquat Organ, 2002 Mar 11, 48(2), 91 - 9 Clearing mechanisms of Vibrio vulnificus biotype I in the black tiger shrimp Penaeus monodon; Alday-Sanz V et al.; Vibrio species' infections are a common sequelae to environmental stress or other disease processes in shrimp, but the mechanism by which the shrimp eliminate the bacteria is poorly understood . In this study, the penetration, fate and the clearing of V . vulnificus were investigated in Penaeus monodon . A bacterial disease isolate from a shrimp farm was identified as V . vulnificus biotype I . Polyclonal antiserum was raised in rabbits against the bacterium and the specificity was verified by ELISA and immunoblot against a range of Vibrio spp . and other gram-negative bacteria . The bacteria were then administered to P . monodon juveniles by injection, immersion and oral intubation . An indirect immunoperoxidase technique was employed in a time course study to follow the bacteria and bacterial antigens in the tissue of the shrimp . Bacteria were cleared by a common route, regardless of the method of administration . Observations in immersion challenge were similar to a combination of those for oral and injection challenges . With immersion, bacteria entered the shrimp through damaged cuticle or via insertion points of cuticular setae . Shortly after entry, whole bacterial cells were observed in the haemolymph and connective tissue . They were either phagocytosed by haemocytes, or broken down outside host cells . Haemocytes containing bacterial cells or antigens (HCB) were observed in the connective tissue and haemolymph . HCB accumulated around the hepatopancreas, midgut, midgut-caecum, gills, heart and lymphoid organ . Free bacterial antigens also accumulated in the heart and lymphoid organ . Bacteria entering through the mouth by oral intubation or immersion were broken down so that only soluble or very fine particles entered the hepatopancreas . Bacterial antigens passed through the hepatopancreas into the haemolymph . Antigens were initially observed in the haemolymph sinuses and subsequently accumulated in the heart and lymphoid organ . Bacterial antigens were released from the shrimp, initially through the gills and subsequently through hepatopancreatic B-cells, branchial podocytes and sub-cuticular podocytes. Life Sci, 2002 Mar 8, 70(16), 1923 - 34 Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells; Rho HW et al.; Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection . The cytolysin, one of the important virulence determinants in V . vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability . In the present study, we investigated the cytotoxic mechanism of V . vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo . V . vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level . These cytotoxic effects of V . vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase . These results indicate that the elevation of cytosolic free Ca2+ induced by V . vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death. J Am Acad Dermatol, 2002 May, 46(5 Suppl), S144 - 5 Vibrio vulnificus septicemia and leg ulcer; Patel VJ et al.; Vibrio vulnificus is a gram-negative bacteria that can cause septicemia, wound infection, or a self-limiting diarrhea . This infection typically presents as an extremely virulent infection in patients with underlying liver disease 1 to 2 days after exposure . We report a case of V vulnificus septicemia, cellulitis, and leg ulceration in a patient who had symptoms develop after exposure to brackish water (19 days before admission) or after ingestion of raw oysters (10 days before admission) . The longest incubation period previously reported is 6 days . The diagnosis was made from identification of the bacteria from blood cultures . No organisms were seen or grown in culture from the skin biopsy specimen, which showed epidermal necrosis and dermal and subcutaneous neutrophilic abscess . We review 13 cases of V vulnificus septicemia and leg ulcers and their approximate incubation time. East Afr Med J, 2001 Mar, 78(3), 124 - 7 Characterisation of Vibrio parahaemolyticus isolated from fish in Kenya; Kagiko MM et al.; BACKGROUND: Acute gastroenteritis associated with fish has been reported since 1951 but is gaining importance with increase in fish consumption in Kenya . The causative agent is Vibrio parahaemolyticus . The importance of this organism is increasing due to the rise in the incidence of outbreaks of food poisoning related to it . OBJECTIVE: To isolate and characterise local strains of Vibrio parahaemolyticus from sea and fresh-water fish . DESIGN: A prospective study . SETTING: Three lakes, a river, a dam and the Kenyan coastline . SUBJECTS: Water and fish samples collected from the study sites . MAIN OUTCOME MEASURES: Isolation of Vibrio parahaemolyticus on glucose-salt-teepol broth (GTSB), and triphenyltetrazolium chloride soya tryptone (TSAT) and several biochemical media, testing the pathogenicity for the isolates by Kanagawa phenomenon and testing the plasmids profiles, coagglutination sensitivity to antimicrobial substance using standard methods . RESULTS: Twenty nine isolates (4%) were obtained from a total of 666 samples screened, twenty seven of which were isolated from 62 coastal samples . They were Kanagawa negative although their plasmid profiles and sensitivity to antimicrobials varied . CONCLUSION: There is need to recognise V . parahaemolyticus as a potential problem due to the increase in consumption of fish as an alternative source of protein. Chemosphere, 2002 Feb, 46(7), 987 - 97 Time-dependent toxicity in the long-term inhibition assay with Vibrio fischeri; Froehner K et al.; The significance of the duration of exposure for the detection of toxicity was evaluated in a 24 h long-term bioluminescence inhibition assay with Vibrio fischeri . Bioluminescence was measured over the time course of 24 h using microplates . The undisturbed luminescence of controls in this assay exhibited characteristic dynamics: a decrease within a period of 12 h with minimal luminescence followed by a continuous increase of luminescence beyond the starting value . To evaluate the toxic influence of compounds chosen to reflect immediate and delayed toxicity to V . fischeri, the bioluminescence was measured for 24 h at 30 min intervals . Luminescence inhibition patterns were recorded for subdinoseb, pentachlorophenol and 2,4,5-trichlorophenol) and for substances causing delayed toxicity (chloramphenicol, nalidixic acid and phosphomycin) . The toxic influence of substances with immediate toxicity was observed directly after application, whereas the toxicity patterns of substances with delayed toxicity developed specifically over the time according to the different involved mechanisms of action . It is concluded that knowledge about time to toxicity in bioassays is necessary in order to identify suitable test endpoints as well as to recognize potential hazards related to time-dependent toxicity. Carbohydr Res, 2002 Apr 30, 337(9), 813 - 7 Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6; Bergstrom N et al.; The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy . The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof . The O-polysaccharide had the following tetrasaccharide repeating unit . Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue. Wilderness Environ Med, 1995 May, 6(2), 167 - 72 Intestinal coinfection with numerous Giardia trophozoites and Vibrio cholerae in hospitalized children with watery diarrhea; Zerpa R et al.; During the recent cholera epidemic which affected Peru and other Latin American countries, fresh stool samples of 100 hospitalized children were assessed February through April 1991 . The children had been admitted because of profuse watery diarrhea . The microbiologic study of wet mount preparations showed curved bacteria suspicious for cholera agent and confirmed afterward to be Vibrio cholerae 01 scrotype Inaba . In 30% of such cases, besides the curved bacteria, a strikingly large number of trophozoites of Giardia intestinalis were observed . The same samples studied by permanent stains confirmed the presence of Giardia trophozoites . To our knowledge, the association of Giardia with serious epidemic cholera infection has not been previously described . All patients were admitted because of severe dehydration requiring intravenous fluid replacement, which might suggest a synergistic role for the above-noted coinfection . Adequately designed case-control studies are needed to ascertain the frequency and the pathophysiologic and clinical significance of such an unusual association. J Toxicol Environ Health A, 2002 Apr 26, 65(8), 589 - 602 Circulating lysozyme and hepatic CYP1A activities during a chronic dietary exposure to tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures in channel catfish, Ictalurus punctatus; Burton JE et al.; Exposure to planar congeners of halogenated aromatic hydrocarbons (HAHs) leads to a myriad of toxicities, including developmental, reproductive, and immunotoxic effects . Halogenated aromatic hydrocarbons possessing structural similarity to TCDD, such as 3,3',4,4',5-pentachlorobiphenyl (PCB-126), are more prevalent in the environment than TCDD, and they elicit similar toxic effects, primarily through the cytosolic aromatic hydrocarbon receptor (AhR) . While polychlorinated biphenyls (PCBs) are ubiquitous pollutants, they rarely exist alone in the environment . The aquatic biocide tributyltin (TBT) is also a widespread environmental contaminant, and numerous studies indicate that it has reproductive, developmental, and immunotoxic effects in a variety of organisms . Unlike planar HAHs, TBT is not associated with any known cellular receptor . The induction of cytochrome P-4501A (CYP1A) activity in most vertebrates is a classical physiological response to planar HAH exposure . TBT has been shown to inhibit the induction of cytochrome P-4.50s at high doses . Recent studies demonstrate that low levels of TBT potentiate PCB-126-associated CYP1A induction in channel catfish, Ictalurus punctatus, and in rodents following intraperitoneal injections . In this study, the effects of dietary exposures to TBT, PCB-126, and mixtures of the two on channel catfish hepatic CYP1A activity, as well as plasma lysozyme activity, were examined . Circulating lysozyme, a marker of proinflammatory responses, was monitored to determine the relative specificity of treatments for CYP1A induction . Plasma lysozyme levels were examined along with hepatic CYP1A protein induction and EROD activity following exposure to nominal doses of 1 and 100 ppb dietary TBT, PCB-126, or mixtures of the two . As expected, the highest level of PCB-126 induced CYP1A, and TBT did not . In mixtures, the low level of TBTpotentiated the ability of the high PCB-126 dose to induce CYP1A . Plasma lysozyme levels were suppressed by both concentrations of TBT and by the low concentrations of PCB- 126 during the initial phase of the response to Vibrio anguillarum . The normal and expected pattern of initial increases in circulating lysozyme levels following immunization, with subsequent return to baseline level, was disrupted by TBT . High levels of PCB-126 potentiated the lysozyme response . As seen with CYP1A activities, exposures to mixtures of TBT and PCB-126 resulted in a potentiation of plasma lysozyme levels . The data show that dietary TBT modulates PCB- 126-induced CYP1A activities and that these mixtures may have potent proinflammatory properties as well. J Med Microbiol, 2002 May, 51(5), 392 - 8 A molecular and phenotypic study of Vibrio cholerae in Iran; Pourshafie M et al.; Vibrio cholerae is again the subject of attention on account of the current increase in the world-wide incidence of cholera . In this study, 200 clinical isolates of V . cholerae serotypes O1 and non-O1, non-O139, were collected from different provinces in Iran . The isolates were subjected to biochemical analysis, antibiogram, PCR of toxin genes, plasmid profile, ribotyping and pulsed-field gel electrophoresis (PFGE) . The analysis of plasmid content showed that 33-96% of V . cholerae isolated from different provinces carry a large plasmid . PCR analysis of V . cholerae O1 showed that the genes encoding cholera toxin (ctx), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin (zot) were present in 55-97% of isolates in different provinces . Restriction fragment length polymorphism (RFLP) of BglI-digested DNA probed with five oligonucleotides revealed three different ribotype patterns in isolates of V . cholerae O1 . The ribotype pattern B21 of V . cholerae O1 El Tor was found to be the predominant pattern in the isolates studied . V . cholerae non-O1, non-O139 isolates showed a single ribotype pattern . PFGE analysis also showed 10 different patterns amongst the isolates, 9 of which were in V . cholerae O1 . Overall, the analysis of polymorphism of ribotypes and PFGE patterns of the isolates showed that the provinces in Iran were affected by a limited number of clones of V . cholerae O1 and non-O1, non-O139 strains. FEMS Immunol Med Microbiol, 2002 Mar 25, 33(1), 35 - 40 Minor pilin subunits are conserved in Vibrio cholerae type IV pili; Toma C et al.; The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined . The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP . PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V . cholerae strains, with the exception of the previously reported major pilin subunit . Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them . Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili . The results suggested that MshB and MshO are minor components of the pilus fiber . Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive. Med Trop (Mars), 2001, 61(6), 513 - 20 {Cholera update at the dawn of the millenium}; Boutin JP et al.; Cholera is an ancestral disease belonging to the mythology of numerous societies . In the last two centuries, seven pandemias have been recorded, in which the spatial and temporal modalities of disease transmission are related to the major technical revolutions of the period . The now ongoing seventh pandemia is by far the longest and most widespread with specific features that raise new challenges and hopes . The authors present the situation at the dawn of the third millennium based on a review of current epidemiological, clinical, therapeutic, diagnostic and vaccinal data . This update shows that the field is progressing and may indeed be standing on the verge of significant breakthroughs for management of the disease and vibrion endemicity. Environ Toxicol, 2002, 17(2), 93 - 7 Toxicity of different industrial effluents in Taiwan: a comparison of the sensitivity of Daphnia similis and Microtox; Liu MC et al.; Industrial effluents are known to exhibit toxicity toward different aquatic organisms . In Taiwan management of these discharges still relies on chemical and physical and physical characteristics of water, although various standard method for assessing aquatic toxicity have been proposed by the Taiwan Environmental Protection Administration . In this study we examined the toxicity and compared the sensitivity of different types of industrial effluents using two proposed toxicity tests: the Daphnia similis acute toxicity test and the Microtox acute assay (Vibrio fischeri) . Results showed that electroplating effluents were the most toxic of all the effluents tested, followed by acrylonitrile manufacturing, pulp/paper, and tannery effluents . The EC50 of an electroplating effluent for D . similis and V . fischeri (15 min) was as low as, respectively, 2.9% and 3.9% of the whole effluent . The other effluents were not acutely toxic to either organism tested . However, the tests exhibited different sensitivity toward various discharges . Only the electroplating and acrylonitrile manufacturing effluents had effects on both organisms . These results indicate the importance of the incorporation of aquatic toxicity tests into the management scheme for treated wastewaters. Appl Environ Microbiol, 2002 May, 68(5), 2519 - 28 Alterations in Vibrio fischeri motility correlate with a delay in symbiosis initiation and are associated with additional symbiotic colonization defects; Millikan DS et al.; Motility is required for Vibrio fischeri cells to interact with and specifically colonize the light-emitting organ of their host, the squid Euprymna scolopes . To investigate the influence of motility on the expression of the symbiotic phenotype, we isolated mutants of the squid symbiont V . fischeri ES114 that had altered migration abilities . Spontaneous hyperswimmer (HS) mutants, which migrated more rapidly in soft agar and were hyperflagellated relative to the wild type, were isolated and grouped into three phenotypic classes . All of the HS strains tested, regardless of class, were delayed in symbiosis initiation . This result suggested that the hypermotile phenotype alone contributes to an inability to colonize squid normally . Class III HS strains showed the greatest colonization defect: they colonized squid to a level that was only 0.1 to 10% that achieved by ES114 . In addition, class III strains were defective in two capabilities, hemagglutination and luminescence, that have been previously described as colonization factors in V . fischeri . Class II and III mutants also share a mucoid colony morphology; however, class II mutants can colonize E . scolopes to a level that was 40% of that achieved by ES114 . Thus, the mucoid phenotype alone does not contribute to the greater defect exhibited by class III strains . When squid were exposed to ES114 and any one of the HS mutant strains as a coinoculation, the parent strain dominated the resulting symbiotic light-organ population . To further investigate the colonization defects of the HS strains, we used confocal laser-scanning microscopy to visualize V . fischeri cells in their initial interaction with E . scolopes tissue . Compared to ES114, HS strains from all three classes were delayed in two behaviors involved in colonization: (i) aggregation on host-derived mucus structures and (ii) migration to the crypts . These results suggest that, while motility is required to initiate colonization, the presence of multiple flagella may actually interfere with normal aggregation and attachment behavior . Furthermore, the pleiotropic nature of class III HS strains provides evidence that motility is coregulated with other symbiotic determinants in V . fischeri. J Org Chem, 2002 May 3, 67(9), 2848 - 53 Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity; Shin JS et al.; An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity . Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure . The result suggested a two-binding site model consisting of two pockets, one large (L) and the other small (S) . The difference in the size of each binding pocket and strong repulsion for a carboxylate in the S pocket were key determinants to control its substrate specificity and stereoselectivity . The L pocket showed dual recognition mode for both hydrophobic and carboxyl groups as observed in the side-chain pockets of aspartate aminotransferase and aromatic aminotransferase . Comparison of the model with those of other aminotransferases revealed that the L and S pockets corresponded to carboxylate trap and side-chain pocket, respectively . The active site model successfully explains the observed substrate specificity as well as the stereoselectivity of the APA. Mol Microbiol, 2002 Apr, 44(2), 533 - 47 Binding site requirements of the virulence gene regulator AphB: differential affinities for the Vibrio cholerae classical and El Tor tcpPH promoters; Kovacikova G et al.; The differential expression of virulence genes be-tween the two disease-causing biotypes of Vibrio cholerae, classical and El Tor, is primarily due to a single basepair change in the tcpPH promoter, which strongly influences the ability of the LysR regulator AphB to activate transcription in response to environmental conditions . We show here that this single basepair change influences virulence gene expression by dramatically altering the affinity of AphB for its recognition site in the tcpPH promoter . AphB binds greater than 10-fold more efficiently to a wild-type classical tcpPH promoter fragment with an A at -65 relative to a wild-type El Tor fragment that has a G at this position . As this single basepair change is located within the left arm of the LysR recognition motif (5'-TGCAA-N7-TTGCA), which extends from -69 to -53, a systematic mutagenesis of the other positions within this site was carried out to assess their influence on AphB binding in vitro and transcriptional activation in vivo . This analysis revealed that the left and right arms of the interrupted dyad display a high degree of symmetry with respect to their role in AphB binding . The right promoter proximal arm also plays a role in transcriptional activation that is distinct from its role in AphB binding . A second AphB binding site (5'-TGCAA-N7-TGTCA) was identified upstream of the aphB gene itself, which extends from +17 to +33 relative to the start of transcription and functions in autorepression . Although the sequences of the AphB binding sites at the tcpPH and aphB promoters are highly conserved, important differences exist in the way that AphB functions at each of these sites. Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 6316 - 21 Epub 2002 Apr 23. YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae, localizes in codependent fashion with FtsL to the division site; Buddelmeijer N et al.; YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae . In E . coli the ygbQ gene was discovered as a result of a computer search of the E . coli genome designed to find potential interacting partners for cell division protein FtsL . In V . cholerae, ygbQ was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter . The role of YgbQ in cell division is supported by the following . Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected . YgbQ localizes to the constriction site in wild-type E . coli cells . Localization of E . coli YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site . Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in E . coli . Our results show colocalization of proteins to the bacterial midcell in E . coli and raise the possibility that these proteins interact in a coiled-coil structure. Curr Opin Infect Dis, 2000 Oct, 13(5), 523 - 529 Role of the neuroendocrine system in pathogenesis of gastroenteritis; Turvill JL et al.; The concept of neuroendocrine modulation of infectious gastroenteritis adds another dimension to the pathophysiology of diarrhoeal diseases . Furthermore it opens up new avenues for therapeutic intervention . Until now, most interest has been directed at enterotoxin-producing bacteria, notably Vibrio cholerae and the enterotoxigenic Escherichia coli . However, more recently neuroendocrine recruitment has been implicated by other pathogens . The roles of vasoactive intestinal peptide, 5-hydroxytryptamine, tachykinins, nitric oxide and opioids are explored in this review . In addition new insights in the contradictory galanin story are discussed. Antimicrob Agents Chemother, 2002 May, 46(5), 1550 - 2 New Mg2+-dependent oxytetracycline resistance determinant tet 34 in Vibrio isolates from marine fish intestinal contents; Nonaka L et al.; A new oxytetracycline (OTC) resistance (Otc(r)) determinant, Tet 34, was cloned from chromosomal DNA of Vibrio sp . no . 6 isolated from intestinal contents of cultured yellowtail (Seriola quinqueradiata) . The transformant, containing cloned Tet 34, could grow in broth containing 25 microg of drug per ml with 10 mM MgCl2 . Tet 34 encoded an open reading frame (ORF) 154 amino acids long . The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which act in purine nucleotide salvage synthesis . Mg2+ binding site residues and the active site were highly conserved in XPRT and the ORF of Tet 34 . The results suggest that Tet 34 encodes a new Mg2+-dependent Otc(r) mechanism. Infect Immun, 2002 May, 70(5), 2441 - 53 Evidence for the emergence of non-O1 and non-O139 Vibrio cholerae strains with pathogenic potential by exchange of O-antigen biosynthesis regions; Li M et al.; The novel epidemic strain Vibrio cholerae O139 Bengal originated from a seventh-pandemic O1 El Tor strain by antigenic shift resulting from homologous recombination-mediated exchange of O-antigen biosynthesis (wb*) clusters . Conservation of the genetic organization of wb* regions seen in other serogroups raised the possibility of the existence of pathogenic non-O1 and non-O139 V . cholerae strains that emerged by similar events . To test this hypothesis, 300 V . cholerae isolates of non-O1 and non-O139 serogroups were screened for the presence of virulence genes and an epidemic genetic background by DNA dot blotting, IS1004 fingerprinting, and restriction fragment length polymorphism (RFLP) analysis . We found four non-O1 strains (serogroups O27, O37, O53, and O65) with an O1 genetic backbone suggesting exchange of wb* clusters . DNA sequence analysis of the O37 wb* region revealed that a novel approximately 23.4-kb gene cluster had replaced all but the approximately 4.2-kb right junction of the 22-kb O1 wbe region . In sharp contrast to the backbones, the virulence regions of the four strains were quite heterogeneous; the O53 and O65 strains had the El Tor vibrio pathogenicity island (VPI) cluster, the O37 strain had the classical VPI cluster, and the O27 strain had a novel VPI cluster . Two of the four strains carried CTXphi; the O27 strain possessed a CTXphi with a recently reported immune specificity (rstR-4** allele) and a novel ctxB allele, and the O37 strain had an El Tor CTXphi (rstR(ET) allele) and novel ctxAB alleles . Although the O53 and O65 strains lacked the ctxAB genes, they carried a pre-CTXphi (i.e., rstR(cla)) . Identification of non-O1 and non-O139 serogroups with pathogenic potential in epidemic genetic backgrounds means that attention should be paid to possible future epidemics caused by these serogroups and to the need for new, rapid vaccine development strategies. Infect Immun, 2002 May, 70(5), 2419 - 33 Comparative and genetic analyses of the putative Vibrio cholerae lipopolysaccharide core oligosaccharide biosynthesis (wav) gene cluster; Nesper J et al.; We identified five different putative wav gene cluster types, which are responsible for the synthesis of the core oligosaccharide (OS) region of Vibrio cholerae lipopolysaccharide . Preliminary evidence that the genes encoded by this cluster are involved in core OS biosynthesis came from analysis of the recently released O1 El Tor V . cholerae genome sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of O1 El Tor mutant strains defective in three genes (waaF, waaL, and wavB) . Investigations of 38 different V . cholerae strains by Southern blotting, PCR, and sequencing analyses showed that the O1 El Tor wav gene cluster type is prevalent among clinical isolates of different serogroups associated with cholera and environmental O1 strains . In contrast, we found differences in the wav gene contents of 19 unrelated non-O1, non-O139 environmental and human isolates not associated with cholera . These strains contained four new wav gene cluster types that differ from each other in distinct gene loci, providing evidence for horizontal transfer of wav genes and for limited structural diversity of the core OS among V . cholerae isolates . Our results show genetic diversity in the core OS biosynthesis gene cluster and predominance of the type 1 wav gene locus in strains associated with clinical cholera, suggesting that a specific core OS structure could contribute to V . cholerae virulence. Int J Infect Dis, 2001, 5(4), 214 - 9 Are the environmental niches of Vibrio cholerae O139 different from those of Vibrio cholerae O1 El Tor? Ali M, Emch M, Yunus M, Sack RB. BACKGROUND: Vibrio cholerae are known to be normal inhabitants of surface water . However, the environmental niches of the different strains of cholera are not well known, and therefore, populations at risk for cholera outbreaks cannot be clearly identified . METHODS: This study identifies environmental risk factors for cholera caused by V . cholerae O1 El Tor and O139 and environmental niches of the two strains present in Matlab, a cholera endemic area of Bangladesh . The study year was 1993, the year that the O139 strain first appeared in the study area . Patients who had either strain of cholera identified in a laboratory were included in the study . A geographic information system was used to map the household locations of the patients, to describe the human sanitary environment and population density, and to address potential anthropogenic and environmental risk factors of the disease . Spatial point pattern and exploratory spatial data analysis techniques were used to define the environmental niches of the two cholera strains . RESULTS: The study suggests the niches of O1 El Tor and O139 strains of V . cholerae appear to be similar, based on common environmental risk factors . CONCLUSIONS: The results of this study support a theory that O1 El Tor could possibly be replaced by the newer O139 strain in the future. Mol Microbiol, 2002 Mar, 43(6), 1657 - 69 Bacterial resistance evolution by recruitment of super-integron gene cassettes; Rowe-Magnus DA et al.; The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates . The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution . Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species . SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes . However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli . We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI . We show that MRIs can randomly recruit genes directly from the cache of SI cassettes . By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V . cholerae SI and its precise insertion into the MRI . The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency . These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events. Mol Microbiol, 2002 Mar, 43(6), 1577 - 89 ToxR interferes with CRP-dependent transcriptional activation of ompT in Vibrio cholerae; Li CC et al.; In pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus . The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR . In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase . Deletion of the crp gene completely abolishes OmpT expression . Comparison of ompT transcription levels in the isogenic crp-, toxR- and crp-toxR- mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP-dependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase . To provide further insights into the molecular mecha-nism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression . Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase (RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from -310 of the ompT promoter via DNA looping. Mol Microbiol, 2002 Mar, 43(6), 1471 - 91 Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae; Merrell DS et al.; Despite over 100 years of study, the intestinal pathogen Vibrio cholerae still causes epidemic disease in areas of the world where there is poor sanitation . While cholera toxin and the toxin-coregulated pilus (TCP) are known to be essential for full virulence, the role that other factors play has remained ill-defined . Herein, we describe a large-scale signature-tagged mutagenesis (STM) screen utilizing 100 pools of 96 mutants each to identify factors involved in colonization of the infant mouse small intestine . A total of 164 mutants representing transposition events into 95 different open reading frames were shown to be recovered at greatly reduced numbers from the infant mouse model . Analysis of the sites of insertion revealed multiple independent mutations within the rfb gene cluster, needed for synthesis of lipopolysaccharide (LPS), and the tcp gene cluster, needed for synthesis of the TCP . More importantly, in addition to these previously known colonization factors, we identified many genes whose activity in colonization was not previously appreciated . These can be divided into a number of functional groups, which include production of factors involved in metabolic activities, regulation of cellular processes, transport, adaptation to stress and unknown functions . In addition, we describe the reiterative use of STM, whereby colonization-defective mutants were assembled into virulence-attenuated pools (VAPs), which were used to begin to reveal roles that the identified virulence factors play in the infection process . Nine new factors were shown to be crucial for the V . cholerae acid tolerance response, which has previously been hypothesized to be important for epidemic spread of cholera . Competition assays of these nine acid tolerance response (ATR)-defective mutants revealed that mutations in gshB, hepA and recO result in a 1000-fold reduction in colonization. J Food Prot, 2002 Apr, 65(4), 670 - 2 Misidentification of Vibrio cholerae O155 isolated from imported shrimp as O serogroup O139 due to cross-agglutination with commercial O139 antisera; Dalsgaard A et al.; Fish and shellfish products imported into Denmark are routinely analyzed for pathogenic Vibrio spp., particularly Vibrio cholerae, if products originate from subtropical or tropical areas . A V . cholerae strain that agglutinated commercial O139 antiserum but not the O1, Inaba, or Ogawa antisera was isolated from imported raw frozen shrimp . The toxigenicity of the strain was analyzed, and the results of a polymerase chain reaction showed that the V . cholerae strain did not contain the virulence genes ctx, tcpA, and zot, which are normally found in V . cholerae O1 and O139 . The strain was resistant to colistin and spectinomycin . The high susceptibility of the strain to antimicrobial agents was confirmed by the lack of an SXT element, a self-transmissible, chromosomal genetic element that is normally present in 0139 strains and encodes resistance to sulfonamides, trimethoprim, and streptomycin . The strain contained two plasmids, in contrast to other O139 strains, which normally do not contain plasmids . The characteristics of the strain led to further agglutination testing with other antisera that are not commercially available, and the strain was found to agglutinate O155 antiserum in repeated testing . Manufacturers of 0139 antiserum should be aware of the closely related O antigens of the O139, O22, and O155 serogroups and should be aware that their commercial diagnostic O139 antiserum must be absorbed to remove cross-reacting agglutinins of O22 and O155 strains. Mol Plant Microbe Interact, 2002 Mar, 15(3), 262 - 8 Transformation and transposon mutagenesis of Leifsonia xyli subsp . xyli, causal organism of ratoon stunting disease of sugarcane; Brumbley SM et al.; Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp . xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally . Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km . The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively . It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E . coli strain and purify the DNA by anion exchange . To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics . Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623 . All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes . Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites . The cosmid cloning vector pLAFR5-km was stably maintained in Lxx . The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx. Zh Mikrobiol Epidemiol Immunobiol, 2002 Jan-Feb, (1), 72 - 3 {Estimation of diagnostic value of the microtest systems for biochemical identification of vibrios groups and their typing according to Heiberg's method}; Abdullaeva ZG; Diagnostic value determination of microtest systems for biochemical identification of vibrios and their groups identification according to Heiberg's was made with the use of 260 collection strains of V . cholerae O1 and non-O1, as well as microorganisms belonging to other genera and families . The newly developed microtest systems were found to have a number of advantages over the traditional test tube method being more economic, ensuring rapid identification, standard and reproducible results. J Bacteriol, 2002 May, 184(9), 2429 - 38 Rhodospirillum centenum utilizes separate motor and switch components to control lateral and polar flagellum rotation; McClain J et al.; Rhodospirillum centenum is a purple photosynthetic bacterium that is capable of differentiating from vibrioid swimming cells that contain a single polar flagellum into rod-shaped swarming cells that have a polar flagellum plus numerous lateral flagella . Microscopic studies have demonstrated that the polar flagellum is constitutively present and that the lateral flagella are found only when the cells are grown on solidified or viscous medium . In this study, we demonstrated that R . centenum contains two sets of motor and switch genes, one set for the lateral flagella and the other for the polar flagellum . Electron microscopic analysis indicated that polar and lateral flagellum-specific FliG, FliM, and FliN switch proteins are necessary for assembly of the respective flagella . In contrast, separate polar and lateral MotA and MotB motor subunits are shown to be required for motility but are not needed for the synthesis of polar and lateral flagella . Phylogenetic analysis indicates that the polar and lateral FliG, FliM, and FliN switch proteins are closely related and most likely arose as a gene duplication event . However, phylogenetic analysis of the MotA and MotB motor subunits suggests that the polar flagellum may have obtained a set of motor genes through a lateral transfer event. Immunol Lett, 2002 May 1, 81(3), 217 - 21 Zonula occludens toxin (Zot) interferes with the induction of nasal tolerance to gliadin; Rossi M et al.; Both nasal and oral administration of soluble protein antigens (Ags) induce tolerance, a phenomenon that has hampered mucosal vaccine design . To produce active immunity the use of adjuvants co-administered with soluble Ags is required . Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) were found to be powerful mucosal adjuvants, but they are not suitable for clinical use because of their associated toxicity . Therefore, there is the need to develop alternative strategies to deliver Ag in order to induce immunoprotection . Among these innovative tools, a new toxin, Zonula occludens toxin (Zot), produced by phages in toxigenic strains of Vibrio cholerae, has been recently exploited for its adjuvant activity at the mucosal level . The present study was undertaken to further highlight the adjuvant properties of Zot . The ability of Zot to induce a mucosal response to gliadin was demonstrated per serum antibody production . In our established model of systemic tolerance to gliadin, induced by its nasal administration, we found a reduced production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) upon administration of gliadin alone . This immune suppression was reverted in mice receiving gliadin together with Zot . As previously shown, the down-regulation of Th1-like cytokines was found to be associated to a suppression of the T-cell proliferation, while such a suppression was completely reverted by Zot co-administration . In conclusion, these data confirm Zot as a good mucosal adjuvant, considering its ability to interfere with the suppression of specific cell mediated immunity, probably as a result of the increased dose and/or altered processing of Ag at mucosal level. Cell Mol Biol Lett, 2002, 7(1), 167 - 9 Electron paramagnetic resonance characterization of the exopolysaccharide layer produced by bacteria; Stopar D et al.; In order to establish a method to characterize the structural heterogeneity of the bacterial surface, research was conducted with a combination of experiments based on electron paramagnetic resonance (EPR) concentration-imaging (CI) and the modeling of translational diffusion with local restrictions . The benefits and drawbacks of this approach are discussed for the Vibrio sp . exopolysaccharide (EPS) layer. Microbiol Immunol, 2002, 46(2), 59 - 66 Molecular ecology of toxigenic Vibrio cholerae; Faruque SM et al.; Toxigenic Vibrio cholerae is the etiological agent of cholera, an acute dehydrating diarrhea that occurs in epidemic form in many developing countries . Although V . cholerae is a human pathogen, aquatic ecosystems are major habitats of Vibrio species, which includes both pathogenic and nonpathogenic strains that vary in their virulence gene content . V . cholerae belonging to the 01 and 0139 serogroups is commonly known to carry a set of virulence genes necessary for pathogenesis in humans . Recent studies have indicated that virulence genes or their homologues are also dispersed among environmental strains of V . cholerae belonging to diverse serogroups, which appear to constitute an environmental reservoir of virulence genes . Although the definitive roles of the virulence-associated factors in the environment, and the environmental selection pressures for V . cholerae-carrying virulence genes or their homologues is not clear, the potential for origination of new epidemic strains from environmental progenitors seems real . It is likely that the aquatic environment harbors different virulence-associated genes scattered among environmental vibrios, which possess a lower virulence potential than the epidemic strains . The ecosystem comprising the aquatic environment, V . cholerae, genetic elements mediating gene transfer, and the mammalian host appears to support the clustering of critical virulence genes in a proper combination leading to the origination of new V . cholerae strains with epidemic potential. Anaesth Intensive Care, 2002 Feb, 30(1), 77 - 81 Vibrio vulnificus septicaemia; Sie MY et al.; Vibrio vulnificus is an opportunistic pathogen capable of causing a fulminant septicaemia in susceptible patients . Underlying chronic diseases such as liver impairment and immunosuppression are important factors contributing to the severity of the infection and outcome . Early suspicion and diagnosis with appropriate antibiotic therapy is important as delay can adversely affect outcome . For those who develop tissue necrotizing fasciitis, early surgical debridement is recommended to allow better penetration of antibiotics and also to reduce the severity of the septicaemia . Mortality is quoted as between 50% and 90% . Current antibiotic recommendations are intravenous ceftazidime 2 g tds and doxycycline 100 mg od. Bioorg Med Chem, 2002 Jun, 10(6), 1761 - 6 Study on quantitative structure-toxicity relationships of benzene derivatives acting by narcosis; Khadikar PV et al.; Hydrophobicity (logP) as well as quantiative structure-toxicity relationships (QSTRs) of some benzene derivatives acting by narcosis have been established based on narcotic mechanisms of action and toxicity data to the fathead minnow, Daphnia magna and Vibrio fischeri using information-theoretic topological index (Id) . Excellent results are obtained in multiparametric regression upon introduction of dummy parameters (indicator variables) . Consistent increase in R(2)(A) values indicated that inspite of collinarity between Id and one of the indicator variables (I(3-6)) the proposed models are statistically significant. Mol Cell Biochem, 2002 Jan, 229(1-2), 119 - 24 Cloning, functional expression in Escherichia coli and primary characterization of a new Na+/H+ antiporter, NhaD, of Vibrio cholerae; Dzioba J et al.; Vibrio cholerae is the infectious agent of the deadly diarrheal disease, cholera . Na+ ion homeostasis is believed to play a key role in both physiology and pathogenicity of this bacterium . However, molecular mechanisms of sodium exchange in V . cholerae are still poorly understood . In the present work a gene encoding an unusual Na+/H+ antiporter, nhaD, was identified in the V . cholerae genome . nhaD was cloned from Vibrio cholerae and expressed in Escherichia coli . The antiporter functioned in an E . coli nhaAnhaB mutant strain to confer resistance to LiCl and NaCl . When assayed in inside-out subbacterial vesicles, V . cholerae NhaD demonstrated high affinity for Na+ ions (1.1 mM Na+ was required for the half-maximal response at the pH-optimum) . The most striking feature of Vc-NhaD is a unique pH-profile of its activity with a sharp maximum at pH 8.0, different from that of any bacterial sodium-proton antiporter described so far . The difference is rationalized as being the result of a His to Arg substitution in a putative pH sensing residue. Bioorg Med Chem Lett, 2002 Apr 22, 12(8), 1153 - 7 New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing; Reverchon S et al.; A series of 22 novel synthetic N-acyl-homoserine lactone analogues has been evaluated for both their inducing activity and their ability to competitively inhibit the action of 3-oxo-hexanoyl-L-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri . In the newly synthesized analogues, the extremity of the acyl chain was modified by introducing ramified alkyl, cycloalkyl or aryl substituents at the C-4 position . Most of the analogues bearing either acyclic or cyclic alkyl substituents showed inducing activity . In contrast, the phenyl substituted analogues displayed significant antagonist activity . We hypothesized that the antagonist activity of the phenyl compounds may result from the interaction between the aryl group and aromatic amino acids of the LuxR receptor, preventing it from adopting the active dimeric form. FEMS Immunol Med Microbiol, 2002 Feb 18, 32(3), 187 - 9 N-acetyl-D-glucosamine specific hemagglutinin receptor of Vibrio cholerae O1 in chicken erythrocyte membranes; Sasmal D et al.; N-Acetyl-D-glucosamine specific cell-associated hemagglutinin (HA)/lectin, previously purified from a strain of Vibrio cholerae O1, had been established as an adhesin molecule of V . cholerae O1 cells . This communication records the isolation and purification of the glycoprotein receptor of the N-acetyl-D-glucosamine specific HA of the V . cholerae O1 strain from chicken erythrocyte membranes . The most salient feature of this study is that the pretreatment of partially purified glycoprotein with purified HA could completely inhibit the hemagglutinating activity of the V . cholerae O1 strain with chicken erythrocytes. FEMS Microbiol Lett, 2002 Feb 19, 208(1), 83 - 7 Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan; Alam MJ et al.; Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples . About 95% of the samples were positive for V . parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics) . Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique . No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure . The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors. FEMS Microbiol Lett, 2002 Feb 19, 208(1), 77 - 81 Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds; Miyoshi S et al.; Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme . This assimilation of iron bound to TPPS was demonstrated to be a common property of V . vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources . Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source . TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS . When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium . In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge . Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V . vulnificus. Arch Cardiol Mex, 2002 Jan-Mar, 72(1), 29 - 35 {Early arterial reflexion and ventricular extrasystole . A novel mechanism detected with sphygmokinetocardiography}; Sanchez Torres G et al.; ANTECEDENT: Through sphygmokynetocardiography (SKCG) an exploratory method that records an electrocardiographic signal, a carotid pulse (CP), and two vibriograms (kinetocardiograms) of the left ventricle (LV) recorded in the left hemithorax (anterior kinetocardiogram, AKC) and the subcostal region of left abdomen (posterior kinetocardiogram of PKC, vibrations transmitted through the hemidiaphragm) we observed a systolic precocious reflection wave (Rw) in the CP and prolongation of LV ejection time (LVET) measured in AKC or in PKC of the previous sinusal pre-extrasystolic beat (PEB) vs control beats (CB) in cases with ventricular extrasystoles (VEs) . OBJECTIVE: To demonstrate whether the intervals just mentioned are associated with ventricular extrasystoles . METHOD: Sixty cases: 30 with VEs, group A, and 30 without arrhythmia, group B, were studied through SKCG . The LVET and the arterial reflection index or ARI = Ta-rw/LEVT, Ta-rw = time between initial ventricular impulse to reflexive wave, were measured . RESULTS: Demography was similar in both groups . PEB had a longer LVET than the CB (291 +/- 41 vs 279 +/- 39, p < 0.01) and ARI was shorter (0.36 +/- 0.17 vs 0.58 +/- 0.21, p < 0.001) . CONCLUSIONS: 1) Distention of the LV due to Rw, possibly through the well-known experimental mechanism of electromechanic feedback, is believed to underlie the arrhythmia . 2) The observation has important clinical implications. Microbiology, 2002 Apr, 148(Pt 4), 1233 - 9 Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio; Moreno C et al.; Analysis of the 16S rDNAs obtained from cultures of single colonies of either type collection strains or environmental strains of the genus Vibrio revealed the presence of polymorphism in every one of the strains examined . Polymorphism was detected by visualization of heteroduplexes produced after 16S rDNA PCR amplification, a procedure that allows for the screening of a large number of isolates . Amplified 16S rDNAs obtained from both Vibrio parahaemolyticus and an environmental strain were cloned . Their nucleotide sequences revealed differences of up to 2% among 16S rDNAs from the same strain . Polymorphic sites were concentrated in a recognized variable stem-loop of bacterial 16S rRNA that contained in some cases up to 83% of the total mismatches observed . Most of the substitutions present in the stem-loop region showed compensating base covariation . The accumulation of so many compensating changes in the stem-loop region implies that the divergence of the different versions of this stem-loop is relatively ancient . This divergence could be the result of either a selection process or a lateral transfer of independently evolved genes. Microbiology, 2002 Apr, 148(Pt 4), 1119 - 27 Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover; Manefield M et al.; N-acyl-L-homoserine lactones (AHLs) are co-regulatory ligands required for control of the expression of genes encoding virulence traits in many Gram-negative bacterial species . Recent studies have indicated that AHLs modulate the cellular concentrations of LuxR-type regulatory proteins by binding and fortifying these proteins against proteolytic degradation (Zhu & Winans, 2001 ) . Halogenated furanones produced by the macroalga Delisea pulchra inhibit AHL-dependent gene expression . This study assayed for an in vivo interaction between a tritiated halogenated furanone and the LuxR protein of Vibrio fischeri overproduced in Escherichia coli . Whilst a stable interaction between the algal metabolite and the bacterial protein was not found, it was noted by Western analysis that the half-life of the protein is reduced up to 100-fold in the presence of halogenated furanones . This suggests that halogenated furanones modulate LuxR activity but act to destabilize, rather than protect, the AHL-dependent transcriptional activator . The furanone-dependent reduction in the cellular concentration of the LuxR protein was associated with a reduction in expression of a plasmid encoded P(luxI)-gfp(ASV) fusion suggesting that the reduction in LuxR concentration is the mechanism by which furanones control expression of AHL-dependent phenotypes . The mode of action by which halogenated furanones reduce cellular concentrations of the LuxR protein remains to be characterized. Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 657 - 64 Thioalkalivibrio thiocyanoxidans sp . nov . and Thioalkalivibrio paradoxus sp . nov., novel alkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria capable of growth on thiocyanate, from soda lakes; Sorokin DY et al.; Nine strains of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria able to grow with thiocyanate (SCN-) as the sole energy and nitrogen source were isolated from soda lakes in South-East Siberia, Kenya and Egypt after enrichment on sodium carbonate minerals buffered at pH 10 with thiocyanate as the substrate . The isolates fell into two groups that were substantially different in terms of cell morphology, growth parameters and the ability to oxidize carbon disulfide . The bacteria were able to oxidize sulfide, polysulfide, sulfur and tetrathionate, as well as thiocyanate . Two isolates belonged to an extremely halotolerant type growing in the presence of up to 4 M Na+ . Cyanate (CNO-) was the main nitrogen-containing intermediate during thiocyanate degradation in both groups . According to DNA-DNA hybridization data and phylogenetic analysis, both groups of isolates belong to a recently described genus of haloalkaliphilic sulfur-oxidizing bacteria, i.e . Thioalkalivibrio, belonging to the gamma-Proteobacteria, in which where they represent two new species . The species name Thioalkalivibrio paradoxus (type strain ARh 1T = DSM 13531T = JCM 11367T) is proposed for the group with barrel-shaped cells, and the species name Thioalkalivibrio thiocyanoxidans (type strain ARh 2T, DSM 13532T = JCM 11368T) is proposed for the group with vibrio-shaped cells . The diagnosis of the genus Thioalkalivibrio is amended according to the new data. Fish Shellfish Immunol, 2002 Mar, 12(3), 283 - 5 Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut (Hippoglossus hippoglossus L.); Bowden TJ et al.; Atlantic halibut (Hippoglossus hippoglossus L.) is a potentially important new species to cold-water aquaculture . Development of a viable industrial farming technique has been hampered by continued pathogen problems within the rearing cycle and there are several reports that indicated how susceptible juvenile halibut are to bacterial and viral diseases . Interest has been expressed, within the industry, over the possibility of vaccinating suitably sized animals to protect against the more common aquaculture pathogens . Vibrio spp . are of particular concern due to their ubiquitous nature and the relatively frequent occurrence of these pathogens within marine aquaculture . We have previously investigated the susceptibility of Atlantic halibut to infection by Vibrio anguillarum and the efficacy of intraperitoneal injected delivery of a commercial vaccine in protecting against the disease . Given the very high rate of protection offered by immunisation we wanted to investigate the effect of alternate routes of administration on the efficacy of the vaccine. Fish Shellfish Immunol, 2002 Mar, 12(3), 273 - 81 Production and characterisation of monoclonal anti-idiotype antibody to Vibrio anguillarum; Yongjuan X et al.; Seven monoclonal anti-idiotype antibodies (mab2) were raised against mouse monoclonal antibody (mab1) 4A6 . Identification of subclass showed that 1H5, 1D1, 2B12 and 2F12 belonged to IgG2b, 2H12 and 1H12 to IgG2a and lE10 to IgG3 . The titres of these mab2 ascitic fluids ranged from 1 x 10(-4)-1 x 10(-6) . The capacity of the mab2 to inhibit the binding between the corresponding rabbit antiserum and Vibrio anguillarum was investigated with the competitive inhibition ELISA . The results showed that mab2 1D1, 1E10, 1H5 and 1H12 were able to inhibit this binding . Another experiment demonstrated that mab2 1D1, 1E10 and 1H5 might induce Balb/c mice to produce Ab3 and these Ab3 competed the same antigen epitopes with Ab1 . These results indicate that mab2 1D1, 1E10 and 1H5 are likely to represent an internal image of V . anguillarum and may thus be described as Ab2-beta anti-idiotype antibodies . In protection experiments, Japanese flounders vaccinated with mab21D1, 1E10 and 1H5 showed significantly enhanced survival from challenge with V . anguillarum . Thus . mab21D1, 1E10 and 1H5 may have use as idiotype vaccines for fish in aquaculture. Fish Shellfish Immunol, 2002 Mar, 12(3), 229 - 42 The acute phase response of rainbow trout (Oncorhynchus mykiss) plasma proteins to viral, bacterial and fungal inflammatory agents; Gerwick L et al.; The innate arm of the immune system responds to inflammatory stimuli by the activation of phagocytes, and by altered levels of several plasma proteins . These changes in plasma proteins comprise a major component of the acute phase response, which is thought to be an adaptive response that contributes to regaining homeostasis after tissue injury or infection . In this study, rainbow trout (Oncorhynchus mykiss) were injected with a variety of potential inflammatory agents, and changes in the concentrations of plasma proteins were sought in polyacrylamide gels in which plasma proteins had been electrophoresed . Bacteria, viruses and yeast all induced changes in plasma protein profiles . Increases were first evident 2 days after injections, and most were evident within 1 week . The greatest number of changes occurred after injection with a Vibrio bacterin emulsified in Freund's incomplete adjuvant . While some proteins increased and others decreased following several treatments, other proteins changed only in response to injections of viruses or viral proteins, and others changed in response to bacterial components . Some proteins that increased after yeast injection decreased after injection of viral components . The partial amino acid sequence of one increased protein identified it as haptoglobin. EMBO J, 2002 Apr 2, 21(7), 1864 - 72 Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli; Ho TQ et al.; Targeting of DNA molecules to specific subcellular positions is essential for efficient segregation, but the mechanisms underlying these processes are poorly understood . In Escherichia coli, several plasmids belonging to different incompatibility groups (F, P1 and RK2) localize preferentially near the midcell and quartercell positions . Here we compare the relative positions of these three plasmids using fluorescence in situ hybridization . When plasmids F and P1 were localized simultaneously using differentially labeled probes, the majority of foci (approximately 75%) were well separated from each other . Similar results were found when we compared the subcellular localization of F with RK2, and RK2 with P1: regardless of the number of foci per cell or growth conditions, most of the foci (70-80%) were not in close proximity to one another . We also localized RK2 in Pseudomonas aeruginosa and Vibrio cholerae, and found that plasmid RK2 localization is conserved across bacterial species . Our results suggest that each plasmid has its own unique subcellular address, implying a mechanism for the stable co-existence of plasmids in which subcellular targeting plays a major role. Trans R Soc Trop Med Hyg, 2002 Jan-Feb, 96(1), 39 - 40 Characteristics of a cholera outbreak, patterns of Vibrio cholerae and antibiotic susceptibility testing in rural Malawi; Zachariah R et al.; The cumulative cholera attack rate in an epidemic in Malawi in 1999/2000 was 59/100,000 population, case-fatality rate 4%, and 98% of all cases presenting to health facilities required intravenous therapy . Microbiological studies showed high resistance of Vibrio cholerae to commonly recommended antibiotics, predominant Ogawa serotypes and no O139 isolates. EXS, 2002, (92), 237 - 45 The use of physiological data to corroborate cospeciation events in symbiosis; Nishiguchi MK; The symbiotic association between sepiolid squids (Family Sepiolidae) and luminous bacteria (Genus Vibrio) provides an unusually tractable model to study the evolution and speciation of mutualistic partnerships . Both host and symbiont can be cultured separately, providing a new avenue to test phylogenetic congruence through molecular and physiological techniques . Combining both molecular and morphological data as well as measuring the degree of infectivity between closely related pairs can help decipher not only patterns of co-speciation between these tightly linked associations, but can also shed new light on the evolution of specificity and recognition among animal-bacterial associations. J Clin Microbiol, 2002 Apr, 40(4), 1395 - 9 Reactivity of convalescent-phase hemolytic-uremic syndrome patient sera with the megaplasmid-encoded TagA protein of Shiga toxigenic Escherichia coli O157; Paton AW et al.; A cosmid library of Shiga toxigenic Escherichia coli (STEC) O157:H7 strain EDL933 DNA was screened for clones capable of reacting with convalescent-phase serum from a patient with hemolytic-uremic syndrome (HUS), in an attempt to identify candidate virulence genes . One of the immunoreactive clones contained a portion of the large plasmid pO157, and the immunoreactive gene product was identified as TagA . The function of this 898-amino-acid protein is unknown, but it exhibits 42% amino acid sequence identity and 63% similarity to a 312-amino-acid region of a ToxR-regulated lipoprotein of Vibrio cholerae . Antibodies to E . coli O157 TagA were detected in sera from other HUS patients with O157 STEC infection but not in those from patients whose illnesses were caused by other STEC types or in healthy controls . These data demonstrate that TagA is expressed in vivo and provide circumstantial evidence for a role in the pathogenesis of the disease . The tagA gene is present only in STEC strains belonging to serogroup O157, and so antibodies to TagA are a potentially useful serological marker for infections due to such strains. J Infect Dis, 2002 Apr 1, 185(7), 950 - 62 Epub 2002 Mar 19. Induction of protective immunity by synthetic Vibrio cholerae hexasaccharide derived from V . cholerae O1 Ogawa lipopolysaccharide bound to a protein carrier; Chernyak A et al.; Synthetic antigens that mimic the terminal hexasaccharide epitope of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, were conjugated to bovine serum albumin (BSA) . Conjugates with carbohydrate-to-carrier molar ratios of 15.5:1, 9.2:1, and 4.6:1 were tested for immunogenicity and efficacy in mice . The role of preimmunity to BSA and the use of adjuvant in the generation of the serologic response to the O-specific polysaccharide and protection against virulent V . cholerae was examined . Preimmunity to BSA did not affect the anti-Ogawa titers but seemed to enhance the protective capacity of antiserum . All 3 conjugates were immunogenic, but adjuvant was effective at inducing higher and earlier antibody responses . In tertiary serum samples, a correlation was found between vibriocidal activity and protection . The protective capacity of antiserum was evident in serum from mice immunized with all conjugates, but it was highest in the groups that received the conjugate with the lowest level of substitution . Further studies are required to increase understanding of the reason for differential protection. Ecotoxicol Environ Saf, 2001 Nov, 50(3), 189 - 95 Effect of dust on the viability of Vibrio fischeri in the Microtox Test; Park K et al.; The standard Microtox test involving the bioluminescent bacterium Vibrio fischeri is a frequently used ecotoxicological bioassay whose EC50, values have been correlated to acute toxicity parameters of vertebrates, to irritancy measures, and to cytotoxicity indices . The aims were to explore the dependence of light output on viable cell number, with the latter estimated with the naked eye using a colorimetric tetrazolium salt method, the effects of dust on the bioluminescence and cell viability, how the viability of the cells is affected after spills, and how spills can be sampled . The lower limit of the linear dynamic range of the light-emitting bacterium was first defined to be 3.7 x 10(7) cells/ mL, compared with 37 x 10(7) cells/mL in the Microtox assay . The effects of dust were then explored in the working range by the method of standard additions by adding 5-, 10-, and 20-mg amounts of Standard Reference Material Urban Dust 1649a . This simulated dust samples collected by a cordless vacuum technique involving a filter cassette . A mass of 20 mg dust totally inhibited the Microtox test at all times (5, 15, and 30 min) . Masses of 5 and 10 mg dust lowered the luminescence significantly by 20 and 64%, respectively, after 30 min . However, the viability test was totally inhibited by 5 mg of dust . A spectrophotometric modification of the viability test using a wavelength of 508 nm was developed that was twice as sensitive as the naked eye test, and was as sensitive as the Microtox test . Mechanical shock involved with spilling and sampling bacterial reagent on hard surfaces killed the luminescent bacteria as shown by inhibition of luminescence . The optimum filter cassette for Microtox reagent collection was a 25-mm 1.00-microm PTFE filter in a 25-mm Delrin holder operated at 4.0 L/min, with a Tygon sampling probe. J Bacteriol, 2002 Apr, 184(8), 2305 - 9 Type 4 pilus biogenesis and type II-mediated protein secretion by Vibrio cholerae occur independently of the TonB-facilitated proton motive force; Bose N et al.; In Vibrio cholerae, elaboration of toxin-coregulated pilus and protein secretion by the extracellular protein secretion apparatus occurred in the absence of both TonB systems . In contrast, the cognate putative ATPases were required for each process and could not substitute for each other. J Bacteriol, 2002 Apr, 184(8), 2225 - 34 The extracellular transport signal of the Vibrio cholerae endochitinase (ChiA) is a structural motif located between amino acids 75 and 555; Folster JP et al.; ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin . To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kan(r)), a chiA-deficient but secretion-competent mutant of V . cholerae . Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera . To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered . Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555 . A mutant ChiA comprised of only those amino acids was secreted by wild-type V . cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V . cholerae . Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal. Microbiol Immunol, 2002, 46(1), 47 - 50 Characterization of the outermembrane proteins of Vibrio cholerae expressed in in vivo culture; Nakasone N et al.; Two outer membrane proteins (Omps) of Vibrio cholerae O1, expressed in the intestine (in vivo) but not in culture media (in vitro), were investigated . The molecular masses of those proteins were 116 kDa and 15 kDa, and they were not associated with iron-regulated proteins . Convalescent cholera patients' sera reacted with the 15 kDa protein but not with the 116 kDa protein . The N-terminal amino acid sequence of the 15 kDa protein was homologous to V . cholerae OmpT . Anti-serum to the 15 kDa protein caused agglutination of the organisms grown in the intestine, but not