J Appl Microbiol, 1998 Feb, 84(2), 227 - 33 Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae; Ringo E et al.; Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot (Scophthalmus maximus) larvae exposed to V . pelagius and/or Aer . caviae . The results demonstrated that this method is suitable for detection of V . pelagius and Aer . caviae in water samples and larvae at population levels higher than 10(3) ml-1 and 10(3) larva-1 . Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 x 10(2) bacteria larva-1 on day 3 post-hatching to approximately 10(5) bacteria fish-1 16 days post-hatching . Sixteen days after hatching, Vibrio spp . accounted for approximately 3 x 10(4) cfu larva-1 exposed to V . pelagius on days 2, 5 and 8 post-hatching . However, only 10(3) of the Vibrio spp . belonged to V . pelagius . When larvae were exposed to Aer . caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp . (10(4) larva-1), of which 9 x 10(3) belonged to Aer . caviae . Later in the experiment, at the time when high mortality occurred, 9 x 10(5) Aer . caviae were detected . Introduction of V . pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer . caviae and with the control group . It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria (V . pelagius) to the rearing water. Mol Microbiol, 1998 May, 28(3), 501 - 20 Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle; Klose KE et al.; Vibrio cholerae, the bacterium that causes cholera, has a pathogenic cycle consisting of a free-swimming phase outside its host, and a sessile virulent phase when colonizing the human small intestine . We have cloned the V . cholerae homologue of the rpoN gene (encoding sigma54) and determined its role in the cholera pathogenic cycle by constructing an rpoN null mutant . The V . cholerae rpoN mutant is non-motile; examination of this mutant by electron microscopy revealed that it lacks a flagellum . In addition to flagellar synthesis, sigma54 is involved in glutamine synthetase expression . Moreover, the rpoN mutant is defective for colonization in an infant mouse model of cholera . We present evidence that the colonization defect is distinct from the non-motile and Gln phenotypes of the rpoN mutant, implicating multiple and distinct roles of sigma54 during the V . cholerae pathogenic cycle . RNA polymerase containing sigma54 (sigma54-holoenzyme) has an absolute requirement for an activator protein to initiate transcription . We have identified three regulatory genes, flrABC (flagellar regulatory proteins ABC) that are additionally required for flagellar synthesis . The flrA and flrC gene products are sigma54-activators and form a flagellar transcription cascade . flrA and flrC mutants are also defective for colonization; this phenotype is probably independent of non-motility . An flrC constitutive mutation (M114-->I) was isolated that is independent of its cognate kinase FlrB . Expression of the constitutive FlrCM114-->I from the cholera toxin promoter resulted in a change in cell morphology, implicating involvement of FlrC in cell division . Thus, sigma54 holoenzyme, FlrA and FlrC transcribe genes for flagellar synthesis and possibly cell division during the free-swimming phase of the V . cholerae life cycle, and some as yet unidentified gene(s) that aid colonization within the host. Int J Hematol, 1998 Feb, 67(2), 175 - 8 Vibrio vulnificus septicemia in a patient with severe aplastic anemia; Tsuzuki M et al.; A 53-year-old man with severe aplastic anemia developed sporadic Vibrio vulnificus septicemia 1 day after eating raw fish and shellfish . Although V . vulnificus infection is potentially fatal, he was saved by immediate and sensitive antibiotic administration . Patients with chronic hematologic disease are susceptible to infection by this organism and are prone to developing septicemia when they eat raw seafood . It is necessary for a patient with this infection to be given effective antibiotics as quickly as possible. Mutat Res, 1998 Feb 26, 398(1-2), 131 - 41 Construction of a umuC'-luxAB plasmid for the detection of mutagenic DNA repair via luminescence; Justus T et al.; This paper describes a novel system for the detection of mutagenic DNA repair in Escherichia coli . The DNA damage inducible umuC gene of Escherichia coli has been fused to the luxAB genes from Vibrio harvleyi that encode the enzyme luciferase . Mutagenicity has been assessed semi-quantitatively by the induction of bioluminescence . This system is simple, rapid and equivalent in sensitivity to other currently available test procedures . Its use in the detection of known SOS mutagens MMS, MNNG and UV is described. Ecotoxicol Environ Saf, 1998 May-Jun, 40(1-2), 173 - 6 Assessment of the ecotoxic potential of soil contaminants by using a soil-algae test; Hammel W et al.; To assess the ecotoxic potential of soil contaminants, a test with the soil alga Chlorococcum infusionum has been developed, enabling investigations of soil pollutions with soluble and fairly soluble chemicals . Three soil types artificially contaminated with Sb compounds and five soils from a historical mining area, which were highly polluted with Sb, As, Hg, and Cu, were used as test soils . For antimony, EC50 values from 125 mg/kg up to > 1000 mg/kg, depending on soil type, were determined . Two of five soils from the mining area caused toxic effects . Additionally, aqueous extracts of all soils were exposed in established tests (daphnid, alga, bacterium) . In contrast with the soil-algae test, no toxic effects were found . Aquatic tests with SbO/K tartrate were performed to point out the toxicity of antimony . The following EC50 values in milligrams of Sb per liter were determined: Scenedesmus subspicatus, 59 mg/liter; Chlorococcum infusionum, 43 mg/liter; Daphnia magna, 8 mg/liter; and Vibrio fisheri, 7 mg/liter. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7035 - 9 CTXphi immunity: application in the development of cholera vaccines; Kimsey HH et al.; CTXphi is a filamentous bacteriophage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae . CTXphi is unusual among filamentous phages because it encodes a repressor and forms lysogens . CTXphi can infect the existing live-attenuated V . cholerae vaccine strains derived from either the El Tor or classical V . cholerae biotypes and result in vaccine reversion to toxinogenicity . Intraintestinal CTXphi transduction assays were used to demonstrate that El Tor biotype strains of V . cholerae are immune to infection with the El Tor-derived CTXphi, whereas classical strains are not . The El Tor CTXphi repressor, RstR, was sufficient to render classical strains immune to infection with the El Tor CTXphi . The DNA sequences of the classical and El Tor CTXphi repressors and their presumed cognate operators are highly diverged, whereas the sequences that surround this "immunity" region are nearly identical . Transcriptional fusion studies revealed that the El Tor RstR mediated repression of an El Tor rstA-lacZ fusion but did not repress a classical rstA-lacZ fusion . Likewise, the classical RstR only repressed a classical rstA-lacZ fusion . Thus, similar to the mechanistic basis for heteroimmunity among lambdoid phages, the specificity of CTXphi immunity is based on the divergence of the sequences of repressors and their operators . Expression of the El Tor rstR in either El Tor or classical live-attenuated V . cholerae vaccine strains effectively protected these vaccines from CTXphi infection . Introduction of rstR into V . cholerae vaccine strains should enhance their biosafety. Antimicrob Agents Chemother, 1998 Jun, 42(6), 1319 - 22 Minocycline and cefotaxime in the treatment of experimental murine Vibrio vulnificus infection; Chuang YC et al.; We conducted an in vivo study with the mouse model of Vibrio vulnificus infection to evaluate the efficacies of therapy with minocycline or cefotaxime alone and in combination . V . vulnificus was introduced subcutaneously into the area over the right thigh . The inoculum size ranged from 1.0 x 10(3) to 1.2 x 10(8) CFU from experiment to experiment but was constant for all animals in the same experiment . Antibiotics were given intraperitoneally 2 h after the bacteria were inoculated . In experiments 1 to 4, the standard dose for humans was used to treat the infection, while in experiment 5, five times the standard dose for humans was used to treat the infection . In experiment 1, with a small inoculum of 5 x 10(3) CFU, all mice in the saline-treated control group and the cefotaxime-, minocycline-, and combined antibiotic-treated groups survived . In experiment 2, with a moderate inoculum of 1.2 x 10(5) CFU, all the mice in the three antibiotic-treated groups survived, while only two of nine mice in the control group survived . In experiment 3, with a large inoculum of 8.0 x 10(7) CFU, six of nine mice in the combined antibiotic-treated group survived, while only one of nine mice in the cefotaxime-treated group and none of the mice in the control and minocycline-treated groups survived . In experiment 4, with a large inoculum of 1.2 x 10(8) CFU, 8 of 20 mice in the combined antibiotic-treated group survived, while none of the 20 mice in the control group, the group treated with cefotaxime alone, and the group treated with minocycline alone survived . In experiment 5, in which mice were infected with a large inoculum of 6.6 x 10(7) CFU and treated with five times the standard human dose of antibiotics, 10 of 12 mice in the combined antibiotic-treated group survived, while only 4 of 12 mice in the minocycline-treated group, 1 of 12 mice in the cefotaxime-treated group, and none of the mice in the control group survived . In experiments 3 to 5, the difference in the survival rates between the combined antibiotic-treated and minocycline-treated groups was statistically significant (P < 0.05) . These results indicate that combination therapy with cefotaxime and minocycline is distinctly more advantageous than therapy with the single antibiotic regimen for the treatment of severe experimental V . vulnificus infections. Microb Pathog, 1998 May, 24(5), 321 - 6 Endogenous nitric oxide in MDCK-I cells modulates the Vibrio cholerae haemagglutinin/protease (HA/P)-mediated cytotoxicity; Wu Z et al.; Previously, we have shown that the Vibrio cholerae haemagglutinin/protease (HA/P) accounts for significant remaining toxicity of CVD110, an attenuated V . cholerae 01 El Tor live oral vaccine-strain . The present report demonstrates that endogenous nitric oxide (NO) production modulates HA/P-mediated cytotoxicity in Madin-Darby canine kidney cell strain I (MDCK-I) epithelial cells . The basal levels of endogenous NO suppressed the cytotoxicity of HA/P, whereas inhibition of NO production with nitro-L-arginine methyl-ester (L-NAME) made the MDCK-I cells susceptible even to low concentrations of the cytotoxin . The inhibition of NO production caused a reinforcement of the HA/P- mediated distortion of a tight junction-associated protein ZO-1 and increment of filamentous actin at the apical and the lateral membrane domains . The mechanism by which NO exerts its modulatory action is not likely to be from its direct interaction with the zinc-containing catalytic domain of HA/P, since two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D, L-penicillamine (SNAP), did not affect the proteolytic activity of HA/P . In conclusion, the endogenous NO in the MDCK-I cells has a modulating effect on the cytotoxicity of HA/P and thus protects the cells against the cytotoxin . J Bacteriol, 1998 Jun, 180(12), 3166 - 73 OpaR, a homolog of Vibrio harveyi LuxR, controls opacity of Vibrio parahaemolyticus; McCarter LL; Vibrio parahaemolyticus is an organism well adapted to communal life on surfaces . When grown on a surface or in a viscous layer, the bacterium induces a large gene system and differentiates to swarmer cells capable of movement over and colonization of surfaces . V . parahaemolyticus displays additional phenotypic versatility manifested as variable colony morphology, switching between translucent and opaque colony types . Although not itself luminescent, V . parahaemolyticus produces autoinducer molecules capable of inducing luminescence in Vibrio harveyi . To examine the role of quorum signaling in the lifestyles of V . parahaemolyticus, the functional homolog of the gene encoding the V . harveyi autoinducer-controlled transcriptional regulatory protein LuxR was cloned . Sequence analysis of the clone predicted an open reading frame with a deduced product 96% identical to LuxR . Introduction of the clone carrying the luxR-like locus into V . parahaemolyticus dramatically affected colony morphology, converting a translucent strain to an opaque one . When the coding sequence for the luxR homolog was placed under the control of the Ptac promoter, conversion to the opaque phenotype became inducible by isopropyl-beta-D-thiogalactopyranoside . Allelic disruption of the luxR-like gene on the chromosome of an opaque strain produced a translucent strain proficient in swarming ability . Primer extension mapping demonstrated opaR transcription in opaque but not translucent cell types . It is postulated that this gene, which has been named opaR, encodes a transcription factor controlling cell type . The underlying genetic basis for opaque-translucent variation may be the consequence of a genomic alteration detected in the opaR locus of opaque and translucent strains. Dev Comp Immunol, 1998 Jan-Feb, 22(1), 27 - 37 Lysozyme and antiprotease activity in the lesser octopus Eledone cirrhosa (Lam.) (Cephalopoda); Malham SK et al.; Antiprotease and lysozyme activities were detected in various tissue samples including the haemocytes and haemolymph of Eledone cirrhosa . Injection of live Vibrio anguillarum caused an increase in lysozyme activity in the branchial heart over 48 hours and a decrease in the lysozyme activity of haemocytes over 24 hours . Haemocytes from control PBS injected animals demonstrated increased lysozyme levels 4 hours after injection whereas it decreased after the injection of live bacteria in PBS . The lysozyme activity of the haemolymph was not affected by these procedures . Bacteria injections had no effect on the antiprotease activity of the organ samples but increased the antiprotease activity of the haemocytes compared to controls in the 4 h samples . Haemolymph antiprotease activity decreased at a greater rate following bacteria injection than in control PBS injected animals . Haemocyte numbers/ml increased for both the control and bacteria injected animals with a greater increase demonstrated for the bacteria injected animals in the 4 h sample . Concomittant with the increase in the numbers of circulating haemocytes live V . anguillarum were cleared from the circulation of E . cirrhosa in less than 4 hours. Bull World Health Organ, 1998, 76(1), 63 - 71 A single dose of live oral cholera vaccine CVD 103-HgR is safe and immunogenic in HIV-infected and HIV-noninfected adults in Mali; Perry RT et al.; Despite considerable experience with single-dose, live, oral cholera vaccine CVD 103-HgR in Asia, Europe, and the Americas, the vaccine had not been evaluated in sub-Saharan Africa or on individuals infected with human immunodeficiency virus (HIV) . We therefore conducted a randomized, placebo-controlled, double-blind, cross-over clinical trial in 38 HIV-seropositive (without clinical acquired immunodeficiency syndrome (AIDS)) and 387 HIV-seronegative adults in Mali to assess its safety and immunogenicity . Adverse reactions (fever, diarrhoea and vomiting) were observed with similar frequency among vaccine and placebo recipients . The vaccine strain was not isolated from the coprocultures of any subject . The baseline geometric mean titre (GMT) of serum vibriocidal antibody was significantly lower in HIV-seropositives (1:23) than in HIV-seronegatives (1:65) (P = 0.002) . Significant rises in vibriocidal antibody were observed in 71% of HIV-seronegatives and 58% of HIV-seropositives, and in 40% of HIV-seropositives with CD4+ counts below 500 per microliter . Following immunization, the peak vibriocidal GMT in HIV-seronegatives was 1:584 versus 1:124 in HIV-seropositives (P = 0.0006); in HIV-seropositives with CD4+ counts < 500 per microliter, the peak vibriocidal GMT was 1:40 (P = 0.03 versus other HIV-seropositives) . CVD 103-HgR was safe in HIV-infected Malian adults, although serological responses were significantly attenuated among HIV-seropositives (particularly in those with CD4+ counts < 500 per microliter) relative to HIV-seronegatives . These results encourage further evaluations of this single-dose, oral cholera vaccine in high-risk populations such as refugees in sub-Saharan AfricaPIP: In response to the 1994 cholera outbreak that swept through Rwandan refugee camps near Goma, Zaire, in 1994, the World Health Organization explored the immunogenicity of a new generation of single-dose, live oral cholera vaccines . One such vaccine, CVD 103-HgR, has been evaluated in Asia, Europe, and the Americas, but not in sub-Saharan Africa or in individuals infected with HIV . Therefore, the present study evaluated the safety and immunogenicity of this new vaccine in a randomized, placebo-controlled, double-blind, crossover clinical trial in Mali . Enrolled were 38 HIV-positive individuals without full-blown AIDS and 387 HIV-negative adults . Adverse reactions (fever, diarrhea, and vomiting) occurred with equal frequency in vaccine and placebo recipients . The vaccine strain was not isolated from the coprocultures of any subject . The baseline geometric mean titre (GMT) of serum vibriocidal antibody was significantly lower in HIV-positive subjects (1:23) than HIV-negatives (1:65) . Significant rises in vibriocidal antibody were observed in 71% of HIV-seronegatives and 58% of HIV-positives and in 40% of HIV-positives with CD4 counts below 500/mcl . After immunization, the peak vibriocidal GMT in HIV-negative subjects was 1:584 compared with 1:124 in HIV-positive subjects . In HIV-positives with a CD4 count below 500/mcl, the peak vibriocidal GMT was 1:40 . Although serologic responses were significantly attenuated among HIV-positive subjects, especially those with CD4 counts below 500/mcl, CVD 103-HgR was safe in HIV-infected Malian adults . Further evaluations of this single-dose oral cholera vaccine are recommended in high-risk populations such as refugees in sub-Saharan Africa . Microbiology, 1998 May, 144 ( Pt 5), 1299 - 308 A chitin-binding domain in a marine bacterial chitinase and other microbial chitinases: implications for the ecology and evolution of 1,4-beta-glycanases; Svitil AL et al.; To examine the ecology and evolution of microbial chitinases, especially the chitin-binding domain, one of the chitinase genes (chiA) from the marine bacterium Vibrio harveyi was analysed . The deduced amino acid sequence of ChiA is not very similar overall to other proteins, except for two regions, the putative catalytic and chitin-binding domains . Among all bacterial chitinases sequenced to date, there is no relationship between percentage similarity of catalytic domains and chitin-binding domains in pairwise comparisons, suggesting that these two domains have evolved separately . The chitin-binding domain appears to be evolutionarily conserved among many bacterial chitinases and is also somewhat similar to cellulose-binding domains found in microbial cellulases and xylanases . To investigate the role of the chitin-binding domain, clones producing versions of ChiA with or without this domain were examined . One version with the domain (ChiA1) bound to and hydrolysed chitin, whereas a truncated ChiA without the putative chitin-binding domain (ChiA2) did not bind to chitin, but it could hydrolyse chitin, although not as well . ChiA1 diffused more slowly in agarose containing colloidal chitin than ChiA2, but diffusion of the two proteins in agarose without colloidal chitin was similar . These results indicate that the chitin-binding domain helps determine the movement of chitinase along N-acetylglucosamine strands and within environments containing chitin. Microbiology, 1998 May, 144 ( Pt 5), 1213 - 21 Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae; Lan R et al.; Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae . The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons . The site for BglI, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate . This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation . Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon . Spacers of 431, 509, 607 and 711 bp were found . A total of at least eight rrn recombination events were detected . Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G . In addition there was variation due to deletion of tandem operons . The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study . This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study . Recombination between rrn operons may be a factor in ribotype variation in all systems . The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved. Appl Environ Microbiol, 1998 Jun, 64(6), 2318 - 22 Bioencapsulation of two different vibrio species in nauplii of the brine shrimp (Artemia franciscana) Gomez-Gil B, Herrera-Vega MA, Abreu-Grobois FA, Roque A. Two groups of nauplii from the brine shrimp (Artemia franciscana) were enriched with different bacteria, and the dynamics of bacterial uptake by the nauplii were observed . This study showed that the efficiency of Artemia nauplii in bioencapsulating bacteria strongly depends on the type of bacteria used, time of exposure, and status (live or dead) of the bacteria. Rev Soc Bras Med Trop, 1998 Mar-Apr, 31(2), 187 - 92 {Vibriocidal and agglutinating antibodies in the urban population in the municipality of Manacapuru/AM (1992-1993)}; Goncalves Eda G et al.; A serological study was carried out involving 1,196 individuals residents in the urban area of Manacapuru--Amazonas, to evaluate the behavior of vibriocidal and agglutinating antibodies . A systematic random sampling procedure was employed to obtain the sample . A year later a 2nd sample of serum was obtained from 120 individuals selected among the participants of the survey . Vibriocidal antibodies microtitulation and seroagglutination in tubes were employed . The correspondence between the studied antibodies was determined by the correlation coefficient calculated according to the frequency of the titles detected . The analysis of the results revealed positive correlation between the antibodies (r = 1.0) and a decrease in titles in a large proportion of the positive samples one year later. J Infect Dis, 1998 Jun, 177(6), 1594 - 9 Enteric infections in an endemic area induce a circulating antibody-secreting cell response with homing potentials to both mucosal and systemic tissues; Qadri F et al.; Enteric infections induce a response of circulating pathogen-specific antibody-secreting cells (ASC) . The expression of homing receptors (HRs) on these cells was studied in patients with diarrhea caused by Vibrio cholerae in Bangladesh, an area in which cholera is endemic . The gut HR, alpha4beta7, was expressed by approximately 80% of the ASC, indicating mucosal homing of these cells . However, the peripheral lymph node HR, L-selectin, was also expressed by approximately 80% of the ASC specific to either cholera toxin or O antigen . In earlier findings after oral immunization in nonendemic areas, alpha4beta7 has been expressed by approximately 100% and L-selectin by approximately 50% of the ASC . In comparison, the present data speak for a more systemic targeting of the immune response associated with long-lasting immunity in an endemic area . The results thus provide insight for the continued development and evaluation of vaccines. Vaccine, 1998 Jan-Feb, 16(2-3), 201 - 7 Immunogenicity and protective role of three formulations of oral cholera vaccine; Kalambaheti T et al.; Three formulations of oral cholera vaccine were compared with respect to their immunogenicity and protective ability in a rat ileal loop model . Eight-week-old Wistar rats were divided into five groups . The first group received orally vaccine A consisting of liposome-associated V . cholera lipopolysaccharide, fimbriae and procholeragenoid, whereas the rats of groups 2 and 3 received orally vaccines B and C consisting of heatkilled fimbriated and non-fimbriated whole cell V . cholerae, respectively . Rats of groups 4 and 5 were controls that received orally liposomes alone and normal saline solution, respectively . It was found that vaccine A elicited stronger immune responses to all three V . cholerae antigens . The antibody responses were detected in both serum and intestinal lavage samples . Vaccine B elicited only modest serum and intestinal responses to V . cholerae fimbriae (anti-F) . No detectable immune response was found in rats of group 3 immunized with vaccine C . Rats immunized with vaccines A and B had a similar order of magnitude of numbers of vibrios adhered to their intestinal mucosa . These numbers were less than those associated with the intestinal tissues of control rats of groups 4 and 5 by about two orders of magnitude . Although without any detectable immune response, rats of group 3 that were immunized with vaccine C showed some reduction in numbers of vibrios associated with their intestinal mucosa . The numbers of vibrios recovered from the intestinal segments of rats of all treatment groups were in the order group 1 = 2 < 4 = 5 . Electron micrography also revealed patches of vibrio colonization on the mucosa of rats of groups 3, 4 and 5 . These features were not found in the groups vaccinated with vaccines A and B . The inhibition of vibrio colonization afforded by the vaccines was biotype- and serotype-non-specific . The results suggest that the heat-killed whole cell fimbriated V . cholerae may be an alternative vaccine preparation to the liposome-associated refined antigen vaccine at a lower cost. Antibiot Khimioter, 1998, 43(3), 14 - 8 {Antibiotic sensitivity of Vibrio cholerae 01 isolated from humans}; Khaitovich AB; Antibiotic susceptibility of 1479 Vibrio cholerae 01 strains isolated from humans between 1991 and 1995 was studied . The antibiotics used belonged to different chemical groups . The assay method was that of serial dilutions in solid media . The isolates showed high susceptibility to tetracyclines, gentamicin, erythromycin, rifampicin, ampicillin, carbenicillin and cefazolin . The susceptibility to kanamycin and monomycin was moderate and that to chloramphenicol, streptomycin and polymixin B was low . The time and area peculiarities of the circulating cultures were observed . The susceptibility of the isolates was either changing or unchanging . The constant susceptibility levels were detected with respect to gentamicin, erythromycin, monomycin, kanamycin, streptomycin and polymyxin B . The susceptibility changing in time was shown with respect to tetracyclines, rifampicin and chloramphenicol . A gradual increase in the resistance to betalactams with greater numbers of the plasmid resistance pattern strains was recorded . Regular surveillance of the changes in the V.cholerae 01 antibiotic susceptibility is needed to timely correct the treatment and prophylaxis regimens. Indian J Med Res, 1998 Apr, 107, 151 - 4 Haemolysin production by environmental isolates of Vibrio parahaemolyticus from Andamans; Ghosh AR et al.; Sixty marine water samples were collected from various coastal sites around Port Blair at different times during August 1996 to July 1997 . The specimens were subjected to standard procedure for isolation and identification of V . parahaemolyticus . Forty four V . parahaemolyticus isolates were detected from these specimens and all showed clear haemolysis on Wagatsuma agar plates . The haemolytic activity was abolished by heating the culture supernatants at 60 degrees C for 10 min and enhanced when plates were kept at 4 degrees C . When isolates were subjected to PCR assay for tdh gene, only one showed the presence of the gene . The results indicate the existence of pathogenic V . parahaemolyticus in the marine environment of these islands. J Biochem (Tokyo), 1998 Jun, 123(6), 1169 - 73 Flagellin-containing membrane vesicles excreted from Vibrio alginolyticus mutants lacking a polar-flagellar filament; Nishioka N et al.; Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-) . Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy . Their hooks were covered with a sheath structure which is contiguous to the outer membrane . The filament proteins, flagellins, were shed into the culture medium of these mutants . These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments . After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded . We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation . These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella. Microb Pathog, 1998 Mar, 24(3), 175 - 83 Heterogeneity in the organization of the CTX genetic element in strains of Vibrio cholerae O139 Bengal isolated from Calcutta, India and Dhaka, Bangladesh and its possible link to the dissimilar incidence of O139 cholera in the two locales; Basu A et al.; After a lapse of 33 months, Vibrio cholerae O139, the new serogroup associated with cholera, has re-emerged in Calcutta, India and has become the dominant serogroup causing cholera from September 1996 . In neighbouring Bangladesh, V . cholerae O1 biotype El Tor continues to be the dominant cause of cholera with the O139 serogroup accounting for only a small proportion of cases . Comparison of the phenotypic traits of representative O139 strains from Calcutta and Dhaka isolated between December 1996 and April 1997 showed similar phenotypic traits with the exception that Dhaka O139 strains were susceptible to streptomycin whilst Calcutta O139 strains were resistant . The Dhaka and Calcutta O139 strains displayed identical ribotypes but showed remarkable differences in the structure and organization of the CTX genetic element . In the Dhaka O139 strains, two copies of the CTX element were arranged in tandem and this resembled the pattern displayed by the 1992 epidemic strains of O139 . The Calcutta O139 strains, in contrast, carried three copies of the CTX genetic element arranged in tandem with the loss of a conserved BglII restriction site in the RS1 element and the appearance of a new HindIII site in the same region . While there may be other factors, it appears that the reorganization of the CTX genetic element in the Calcutta O139 strains may have contributed to the resurgence of this serogroup in Calcutta . Biochim Biophys Acta, 1998 Apr 23, 1384(1), 1 - 6 Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus; Lee JH et al.; The vmc gene encoding a metalloprotease of Vibrio mimicus (ATCC 33653) was cloned in Escherichia coli and sequenced . The vmc gene contained 1884 nt sequence which codes a polypeptide of 628 amino acids with a predicted molecular mass of 71,275 Da . The deduced amino acid sequence had the similarity of 68.5% with V . parahaemolyticus metalloprotease . The consensus sequence of a zinc binding motif (HEXXH) was identified to be HEYTH . The zymography analysis showed a gelatinolytic protein band around molecular mass of 61 kDa, and this result suggested that the cloned metalloprotease may undergo processing during secretion. Infect Immun, 1998 Jun, 66(6), 2601 - 6 An epimerase gene essential for capsule synthesis in Vibrio vulnificus; Zuppardo AB et al.; The extracellular capsule polysaccharide (CPS) of Vibrio vulnificus is a primary virulence factor which allows survival of the bacteria in the human host . To study the genes involved in expression of the capsule, we generated mutants that lost the ability to produce CPS following the insertion of a minitransposon into the genome of an encapsulated, clinical strain of V . vulnificus . A genomic region, from one nonencapsulated mutant, containing the transposon and flanking V . vulnificus DNA was cloned, and a probe complementary to the chromosomal DNA immediately adjacent to the transposon was used to locate this fragment in the genome of the encapsulated parent strain . The fragment, which contained a putative capsule gene, was cloned and, when supplied in trans, complemented the mutation in the nonencapsulated mutant to restore capsule production . In addition, virulence studies, using the 50% lethal dose assay, showed that the restoration of capsule production also restored the virulence of the organism . Sequence analysis of the gene disrupted by the transposon revealed that it matched a nucleotide-sugar epimerase of Vibrio cholerae O139, with 75 and 85% identities at the nucleotide and amino acid levels, respectively . In addition, computer analysis recognized epimerases of various organisms as highly similar to the putative epimerase of V . vulnificus . Finally, a combination of PCR amplification and Southern blotting showed that this epimerase is common to at least 10 strains of V . vulnificus that each express a serologically distinct CPS . Our results indicate that the epimerase gene is essential for capsule expression in V . vulnificus. Infect Immun, 1998 Jun, 66(6), 2535 - 9 The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V . cholerae O139; Jouravleva EA et al.; We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V . cholerae strains in plaque assays . We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection . Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection . Susceptibility is restored by mshA complementation of deletion mutants . Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain . Monoclonal antibody against MSHA inhibits plaque formation . We conclude that MSHA is the receptor for phage 493 . The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493 . However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA . Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays . Nevertheless, they express the mshA pilin gene . They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains . Resistance to 493 in plaque assays is thus not equivalent to resistance to infection . The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139. Environ Health Perspect, 1998 Apr, 106 Suppl 2, 583 - 91 Correlations of Vibrio fischeri bacteria test data with bioassay data for other organisms; Kaiser KL; Linear relationships of the median lethal concentrations of several hundreds of chemicals for a variety of organisms with Vibrio fischeri median effective concentrations are investigated . Significant correlations can be developed for many aquatic species including the fishes fathead minnow, bluegill, catfish, goldfish, goldorfe, guppy, killifish, rainbow trout, sheepshead minnow, and zebrafish; the water flea Daphnia sp.; such crustaceans as Artemia sp . and Crangon sp.; the ciliate Tetrahymena pyriformis; and algae, such as Chlorella sp . These interspecies relationships can be used to estimate order-of-magnitude type toxic effects of many substances for these aquatic organisms . Highly significant relationships can be obtained when selecting compounds on a chemical basis, such as alcohols, ketones, aromatics, etc., which allow the calculation of the compounds' toxicities to the corresponding aquatic species with increased accuracy and confidence . Analogous correlations with mammalian (rat and mouse) oral, intraperitoneal, and intravenous median lethal dose (LD50) data are much weaker than those for most aquatic species . However, there are significant differences between these three routes of administration and the intravenous LD50 data show the best relationship with the Vibrio data. J Clin Microbiol, 1998 Apr, 36(4), 1103 - 4 Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem; Abbott SL et al.; Two unusual cases of Aeromonas infection are described, one associated with bacteremia (Aeromonas schubertii) and another in which the organism was recovered from an infected gall bladder (Aeromonas veronii biotype veronii) . These strains were initially identified as Vibrio damsela and Vibrio cholerae by the Vitek and API 20E systems, respectively . Use of appropriate screening tests and familiarity with the newer Aeromonas species could prevent initial misidentifications and potential public health consequences. Microbios, 1997, 92(371), 83 - 9 Estimation of the viability of Vibrio cholerae 0139 by assessing cell membrane integrity; Decamp O et al.; Changes in the viability of Vibrio cholerae 0139 Bengal, estimated by cellular membrane integrity, in batch culture over 35 days, were investigated . Data indicated an initial period of rapid growth with up to 30% of bacterial mortality, followed by a period of slower growth, lower culturability but higher viability, from day 7 onwards . The size of viable bacteria significantly decreased during the incubation time, whilst the size of dead bacteria showed a less pronounced decrease . V . cholerae 0139 changed from a straight or curved rod shape to a spherical shape . This study shows that BacLight dyes are a fast and useful tool to examine health risk-associated bacteria, providing useful information about their viability and concentration. Kansenshogaku Zasshi, 1998 Mar, 72(3), 218 - 22 {Survival of Vibrio cholerae non-O1 in river sediment during cold season}; Uchiyama H; To assess the existence of Vibrio cholerae non-O1 in the environment water system during the cold season, the organism was incubated in both river sediment or terrestrial soil as particle matter-water (1:1) suspension . The low temperature condition was set to 5 degrees and 10 degrees C . At 5 degrees C, V . cholerae non-O1 did not grow in any medium, sediment and soil . At 10 degrees C, the decrease of the number of organisms was delayed in river sediment as compared with peptone medium . At 30 degrees C, the organism grew in river sediment and its extract solution . In soil, the organism was decreased immediately under low temperature . Though the number of organisms was decreased temporarily in soil, its number was maintained within 14 days at 30 degrees C . But in the extract solution from soil, the organism was decreased immediately under 5 degrees, 10 degrees, 20 degrees, 30 degrees C . It showed that survival of V . cholerae non-O1 was maintained in particle substances contained water . These results suggested that V . cholerae non-O1 would be able to exist in river sediment during the cold season. Plasmid, 1998, 39(3), 235 - 44 Plasmid-mediated histamine biosynthesis in the bacterial fish pathogen Vibrio anguillarum; Barancin CE et al.; Histamine production in bacteria-contaminated fish is the result of the presence of bacterial histidine decarboxylase activity, which converts histidine present in muscle proteins to histamine . The fish pathogen Vibrio anguillarum harbors a plasmid-encoded histidine decarboxylase gene (angH) that is essential for biosynthesis of the siderophore anguibactin . However, the role of angH in histamine biosynthesis by this pathogen has not been fully determined . Thus, the objectives of this study were to monitor the production and release of histamine by the wild-type as well as by a plasmidless strain and angH isogenic mutants generated by allelic exchange . Reverse transcription polymerase chain reaction showed that only the wild-type strain expressed angH, while no angH message was detected in the mutants and the plasmidless derivative . The iron uptake-deficient phenotype of one of the angH mutants confirmed the location of the mutation and the unique role of this gene in iron acquisition . Thin-layer chromatography, gas chromatography, and mass spectrometry showed that histamine was released by the strain harboring a wild-type angH gene when grown in excess histidine . This biogenic amine was not detected in the culture supernatants of the plasmidless derivative and the angH mutant when cultured under the same experimental conditions . These results indicate that angH is essential for histamine biosynthesis in V . anguillarum, a compound responsible for food poisoning and potentially involved in bacterial virulence . Thin-layer chromatography of wild-type culture supernatants and beta-galactosidase assays using the isogenic angH mutant demonstrated that the expression of this gene is independent of the histidine concentration of the medium under both iron-rich and iron-limiting conditions . Gene, 1998 Mar 16, 209(1-2), 65 - 70 The interaction of the Vibrio cholerae transcription factors, Fur and IrgB, with the overlapping promoters of two virulence genes, irgA and irgB; Watnick PI et al.; irgA, a virulence gene in Vibrio cholerae, encodes a 77kDa outer membrane protein . irgA expression is activated by irgB, which encodes a LysR-type transcription factor and is divergently transcribed from a promoter overlapping that of irgA . Expression of irgA and irgB is repressed by iron and Fur . A 200bp DNA fragment containing the irgA-irgB intergenic region was inserted between the Escherichia coli phoA and lacZ genes, respectively, to generate operon fusions to the two promoters, and this construct was crossed into the chromosomal lacZ gene of V . cholerae . This DNA fragment was sufficient to produce regulation of irgA-phoA and irgB-lacZ transcription by iron, Fur and IrgB . Purified V . cholerae Fur and IrgB overexpressed in E . coli bound simultaneously to this DNA fragment in gel shift experiments, and footprints of both proteins on the irgA-irgB intergenic region were observed using DNaseI footprinting . The Fur footprint overlapped a Fur box, previously identified by homology with the E . coli Fur box . The position of the IrgB footprint was consistent with activation of irgA transcription and repression of irgB transcription by IrgB . We present a model for the interaction of Fur and IrgB in transcriptional regulation of irgA. Eur J Biochem, 1998 Apr 1, 253(1), 319 - 27 Structural characterization of the lipopolysaccharide O-antigen and capsular polysaccharide of Vibrio ordalii serotype O:2; Sadovskaya I et al.; Structures of the capsular and O-chain polysaccharides of Vibrio ordalii serotype O:2, the causative agent of vibriosis in salmonid fish, were determined by high-field NMR techniques, mass spectrometric methods and partial hydrolysis . Both polymers were shown to be composed of linear tetrasaccharide repeating units, having the structure: carbohydrate sequence {see text}. Appl Environ Microbiol, 1998 Apr, 64(4), 1573 - 5 Chemotaxis of pathogenic Vibrio strains towards mucus surfaces of gilt-head sea bream (Sparus aurata L.); Bordas MA et al.; Vibrio anguillarum and Vibrio alginolyticus exhibited significant adhesion to and chemotactic abilities towards mucus collected from the skin, gills, and intestine of gilt-head sea bream . Quadratic polynomial models for chemotaxis designed to estimate the influence of temperature demonstrated a differential bacterial chemotaxis depending of the source of the mucus, with the chemotaxis towards intestinal mucus being the least influenced. J Bacteriol, 1998 May, 180(9), 2298 - 305 Nucleotide sequence and spatiotemporal expression of the Vibrio cholerae vieSAB genes during infection; Lee SH et al.; The iviVII gene of Vibrio cholerae was previously identified by a screen for genes induced during intestinal infection . In the present study, nucleotide sequence analysis revealed that iviVII is a 1,659-bp open reading frame, herein designated vieB, that is predicted to be last in a tricistronic operon (vieSAB) . The deduced amino acid sequence of VieS exhibited similarity to the sensor kinase component, and those of VieA and VieB were similar to the response regulator components, respectively, of the two-component signal transduction family . Analysis of transcriptional fusions to a site-specific DNA recombinase reporter, tnpR, revealed that vieS and vieA are transcribed during in vitro growth in a vieAB-independent and vieA-dependent manner, respectively . In contrast, transcription of vieB occurred exclusively during infection and was not dependent upon VieB . We conclude that the vieSAB genes are differentially regulated, at least during laboratory growth . Use of a V . cholerae strain harboring a vieB::tnpR transcriptional fusion allowed the kinetics and location of vieB expression within the intestine to be determined . We found that vieB transcription is induced shortly after infection of the proximal and mid-small intestine. Infect Immun, 1998 May, 66(5), 1968 - 72 Validation of a volunteer model of cholera with frozen bacteria as the challenge; Sack DA et al.; To evaluate a standardized inoculum of Vibrio cholerae for volunteer challenge studies, 40 healthy adult volunteers were challenged at three different institutions with a standard inoculum prepared directly from vials of frozen, virulent, El Tor Inaba V . cholerae N16961, with no further incubation . Groups of 5 volunteers, with each group including 2 volunteers with blood group O, were given a dose of 10(5) CFU, and 34 of the 40 volunteers developed diarrhea (mean incubation time, 28 h) . Transient fevers occurred in 15 (37.5%) of the volunteers . V . cholerae was excreted by 36 of 40 volunteers . Five additional volunteers received 10(4) CFU, and four developed diarrhea but with a lower average purging rate than required for the model . Of the 40 volunteers, 37 developed rises in their vibriocidal and antitoxin titers similar to those in previous groups challenged with freshly harvested bacteria . We conclude that challenge with frozen bacteria results in a reproducible illness similar to that induced by freshly harvested bacteria . Use of this model should minimize differences in attack rates or severity when groups are challenged at different times and in different institutions. Appl Environ Microbiol, 1998 May 1, 64(5), 1910 - 8 Grazing Pressure by a Bacterivorous Flagellate Reverses the Relative Abundance of Comamonas acidovorans PX54 and Vibrio Strain CB5 in Chemostat Cocultures Hahn MW, Hofle MG. The response of the bacterial strains Comamonas acidovorans PX54 (beta subclass of the class Proteobacteria) and Vibrio strain CB5 (gamma subclass of the class Proteobacteria) to grazing by the bacterivorous flagellate Ochromonas sp . was examined in one-stage chemostat experiments under conditions of low growth rates with a complex carbon source . The two bacterial strains were cultured together; they were cultured without flagellates in the first phase of the experiments and in the presence of the flagellates in the second phase . Monoclonal and polyclonal antibodies were used to determine the numbers and sizes of C . acidovorans PX54 and Vibrio strain CB5 cells . The flagellates caused strong changes in total bacterial cell numbers, in the relative abundances of the individual bacterial strains, and in bacterial cell size distribution . Vibrio strain CB5 dominated the total bacterial cell numbers during the flagellate-free phase of the experiments with a relative abundance of 93%, but this declined to 33% after inoculation with the flagellate . In contrast to Vibrio strain CB5, C . acidovorans PX54 responded to grazing with a strong expansion of cell length distribution toward large, filamentous cells . These changes in cell morphology resulted in a high percentage of inedible cells in the C . acidovorans PX54 population but not in the Vibrio strain CB5 population, which caused the observed change in the relative abundances of the strains . Batch culture experiments without the flagellate demonstrated that the elongation of C . acidovorans PX54 cells was dependent on their growth rate . This indicates that the occurrence of filamentous C . acidovorans PX54 cells is not a direct response to chemical stimuli released by the flagellates but rather a response to increased growth rates due to flagellate grazing. Appl Environ Microbiol, 1998 May, 64(5), 1721 - 4 Improved isolation of Vibrio vulnificus from seawater and sediment with cellobiose-colistin agar; Hoi L et al.; An improved selective medium, cellobiose-colistin (CC) agar, gave a significantly higher (P < 0.05) isolation rate of Vibrio vulnificus from water and sediment samples than did modified cellobiose-polymyxin B-colistin (mCPC) agar . In a total of 446 alkaline peptone water preenrichments amended with polymyxin B, V . vulnificus was isolated from 154 preenrichments (35%) with mCPC agar and from 179 preenrichments (40%) with CC agar . CC agar gave a higher plating efficiency of V . vulnificus cells than did cellobiose-polymyxin B-colistin (CPC) agar, mCPC agar, or thiosulfate-citrate-bile salts-sucrose (TCBS) agar; the only significant difference was observed with TCBS agar, which gave much lower plating efficiencies than the other selective media . Determination of MICs demonstrated that the concentrations of colistin and polymyxin B in CPC agar inhibit growth of a proportion of V . vulnificus strains. Science, 1998 Apr 24, 280(5363), 605 - 8 A distinctive class of integron in the Vibrio cholerae genome; Mazel D et al.; The ability of bacteria to acquire and disseminate heterologous genes has been a major factor in the development of multiple drug resistance . A gene, intI4, was identified that encodes a previously unknown integrase that is associated with a "gene-VCR" organization (VCRs are Vibrio cholerae repeated sequences), similar to that of the well-characterized antibiotic resistance integrons . The similarity was confirmed by IntI1-mediated recombination of a gene-VCR cassette into a class 1 integron . VCR cassettes are found in a number of Vibrio species including a strain of V . metschnikovii isolated in 1888, suggesting that this mechanism of heterologous gene acquisition predated the antibiotic era. Diagn Microbiol Infect Dis, 1998 Mar, 30(3), 187 - 91 Acalculous cholecystitis and septicemia caused by non-O1 Vibrio cholerae: first reported case and review of biliary infections with Vibrio cholerae; West BC et al.; The first case of septicemic acute acalculous cholecystitis caused by non-O1 Vibrio cholerae is described in a healthy traveler, and biliary tract infections from V . cholerae are reviewed . Immediately after a vacation in Cancun, Mexico, a 55-year-old man developed acute cholecystitis . Blood and bile cultures grew non-O1 V . cholerae . At surgery, the gallbladder was acalculous, inflamed, distended, and nearly ruptured . Pathogenetic factors may have included diarrhea prophylaxis with bismuth subsalicylate, distension of the gallbladder from illness-induced fasting, and bacterial toxins in the gallbladder . The patient received i.v . cephapirin, followed by oral cephradine for a total of 10 days, and he made a quick and complete recovery . V . cholerae should be considered in the differential diagnosis of persons from endemic areas who present with cholecystitis or acute jaundice. Ecotoxicol Environ Saf, 1998 Mar, 39(3), 185 - 94 Development of a soil extraction procedure for ecotoxicity characterization of energetic compounds; Sunahara GI et al.; The acetonitrile-sonication extraction method (US EPA Method 8330) associated with aquatic-based toxicity tests was examined to study the ecotoxicity of energetic substances in soil . Three studies were carried out: (1) toxicological characterization of different energetic substances to select a representative toxicant and to validate the choice of bioassays; (2) choice of an appropriate solvent to transfer acetonitrile extracts to the bioassay incubation media; and (3) optimization of Method 8330 using soil samples spiked with the toxicant . Initial studies indicated that pure 2,4,6-trinitrotoluene (TNT) was toxic to Vibrio fischeri {Microtox; IC50 (15 min) of 4.2 microM}, whereas RDX was less toxic (IC20 = 181 microM) and HMX was not toxic up to its limit of water solubility (< 22 microM) . Selected pure TNT metabolites were less toxic than TNT . Similar results were found using the 96-h Selenastrum capricornutum growth inhibition test . The toxicity of pure TNT in different solvents (acetonitrile, acetone, and DMSO) and that from Method 8330-extracted TNT-spiked soil samples were compared to TNT dissolved in water . Data indicated that DMSO was the most appropriate solvent to transfer the acetonitrile extracts . A modified Method 8330 may be used in conjunction with bioassays and chemical analyses to examine the ecotoxicity of soils contaminated with energetic substances. Microbiol Immunol, 1998, 42(3), 237 - 9 Filamentous phage fs1 of Vibrio cholerae O139; Nakasone N et al.; Filamentous phage, fs1, was obtained from Vibrio cholerae O139 . The lysogenized strains produced a large amount of fs1 phage in the culture supernatant . This phage was previously reported as novel fimbriae of that organism . The genome of the phage was a 6.5 kb single-stranded DNA . The capsid of fsl consists of a small molecule peptide (about 2.5 kDa). Lett Appl Microbiol, 1998 Feb, 26(2), 110 - 2 Occurrence and distribution of Vibrio vulnificus in tropical fish and shellfish from Cochin (India); Thampuran N et al.; The incidence and distribution of Vibrio vulnificus in marine and brackish-water fish and shellfish from coastal areas of Cochin on the west coast of India were studied . For marine fish collected in very fresh condition from vessels, a level of incidence of 16.6% was noted . Frequency of isolation was greater from the intestine than from the muscle . The greater Most Probable Number (MPN) count determined by the three tube method ranged from 15 to 910 g-1 in the positive samples. Indian J Med Sci, 1997 Nov, 51(11), 417 - 9 Gastroenteritis due to Vibrio cholerae El-Tor Ogawa in Dhule; Nitsure S et al.; For V . Cholerae isolation, stool sample is better than rectal swab . Direct oxidase test on stool is easy and reliable . V . cholerae El-Tor Ogawa is predominant type in Dhule area . New phage type T27 was reported . Tetracyclin resistance needs further studies. FEMS Immunol Med Microbiol, 1998 Mar, 20(3), 201 - 7 Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139; Hoshino K et al.; A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences . The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V . cholerae O1 and O139, respectively . Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard . In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup . All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V . cholerae strains have the genetic potential of producing cholera toxin. Chemosphere, 1998 Apr, 36(10), 2345 - 57 Toxicity and anaerobic biodegradability of pyridine and its derivatives under sulfidogenic conditions; Liu SM et al.; Attempts were made to correlate the chemical structure of pyridine and 15 pyridine derivatives with both their biodegradability by estuarine sediment microorganisms under anaerobic conditions and also with their toxicity to the marine bacterium Vibrio fischeri Beijerinck 1889 by using the Microtox bacterial assay . Among monosubstituted pyridines, comparisons of different substituents at positions C-2, C-3, or C-4 atom of the pyridine ring showed that isomers of carboxylpyridine (COOHPYR), hydroxypyridine (OHPYR), and cyanopyridine (CNPYR) were more susceptible to biotransformation than isomers of chloropyridine (ClPYR) and methylpyridine (CH3PYR) in anoxic estuarine sediment slurries under sulfidogenic conditions . Isomers with the functional group at the C-2 or C-3 atom of the pyridine ring were biotransformed faster than those with the same functional group at C-4 . The only exception was 4-ClPYR, which was biotransformed within 130 days, while 2- and 3-ClPYR continued to persist in the anoxic sediment slurries . Median effect concentrations (EC50) of pyridine and pyridine derivatives were in the range of 0.027 to 49.1 mmol/L . Pyridine derivatives with -CN and -OH functional groups tended to be less toxic, while pyridine derivatives with -CH3, -Cl, and -COOH functional groups tended to be more toxic . Isomers with the substituent at C-2 were less toxic than the C-3 or C-4 isomers . There was no clear correlation between the pseudo-first-order rate constants for the microbial transformation of pyridine and its derivatives and their toxicity to the marine bacterium. Biochim Biophys Acta, 1998 Mar 6, 1370(1), 77 - 86 Topological study of Vibrio alginolyticus NhaB Na+/H+ antiporter using gene fusions in Escherichia coli cells; Enomoto H et al.; NhaB, an Na+/H+ antiporter, of Vibrio alginolyticus is a 528-amino-acid protein . Hydropathy profile-based computer analysis predicted that the NhaB might contain up to 13 membrane-spanning domains . To examine this hypothesis, we applied the phoA fusion method to the cloned nhaB gene . Eighteen plasmid-borne nhaB-phoA fusion genes were constructed in Escherichia coli cells and the alkaline phosphatase activity and expression level of the fusion proteins analyzed . These results and the results obtained with additional constructs indicated that V . alginolyticus NhaB has a unique topology consisting of nine transmembrane segments with the N-terminus in the cytoplasm and the C-terminus in the periplasm . Vaccine, 1998 Apr, 16(7), 678 - 84 Immunogenicity of liposome-associated and refined antigen oral cholera vaccines in Thai volunteers; Chaicumpa W et al.; A mixture of Vibrio cholerae antigens made up of crude fimbrial extract, lipopolysaccharide and procholeragenoid was administered orally to Thai volunteers either as free antigen or associated with liposomes . All vaccines and controls were administered in three doses given at 14 day intervals . Nine volunteers received liposome-associated vaccine and seven received free vaccine . Liposomes without antigens were given to eight volunteers and seven volunteers received 5% NaHCO3 solution alone . Both vaccines had 100% immunogenicity as determined by serum vibriocidal antibody responses . Liposomes were shown by indirect ELISA to localize the immune response against lipopolysaccharide and fimbriae to the intestinal mucosa . Vaccines given liposome-associated antigens had a higher rate of antigen-specific antibody response than did individuals who had received free antigens . The vaccines induced intestinal antibodies of IgM and/or IgA isotypes, but not IgG antibody. Chemotherapy, 1998 Mar-Apr, 44(2), 108 - 11 Evaluation of different antibiotics in inhibiting colonization of Vibrio cholerae O1 and O139 in the rabbit intestine; Chakrabarti MK et al.; The effects of furazolidone, erythromycin and azithromycin in inhibiting colonisation of Vibrio cholerae O1 and O139 in the rabbit intestine were tested . Both V . cholerae O1 and O139 highly colonised the gut in control rabbits . The colonisation of furazolidone-resistant strains in the rabbit intestine was prevented effectively by both erythromycin and azithromycin . In furazolidone-sensitive strains, the efficacies of erythromycin and azithromycin were very much comparable to furazolidone . These results suggested that azithromycin may be subjected to clinical trial in comparison to furazolidone and erythromycin for the treatment of cholera due to O1 and O139 infection in children. Jpn J Med Sci Biol, 1997 Jun, 50(3), 113 - 21 Molecular typing of Vibrio vulnificus isolates by random amplified polymorphic DNA (RAPD) analysis; Ryang DW et al.; This study was undertaken to determine molecular types and genetic similarity among V . vulnificus isolates by RAPD analysis . We compared these results with serotypes of V . vulnificus . Ninety-seven V . vulnificus strains including 69 strains from Chonnam University Hospital (CUH; Kwangju, Korea), 13 from Wonkwang University Hospital (WUH; Iksan, Korea), 13 from the Japanese National Institute of Health (JNIH) and two reference strains (ATCC 33815 and ATCC 27562) were analyzed . Four molecular types comprising all the strains were obtained by RAPD analysis . Type I was the most common (60/95) and included 58 strains from CUH . Type I showed a further subdivision into seven subtypes . Type II (23/95) composed of 11 strains from CUH, nine from WUH, three from JNIH and two reference strains . Six type III strains comprised four WUH strains and two JNIH strains . All six strains of type IV were from JNIH . The range of genetic similarity values among V . vulnificus isolates was 0.24 to 1.00 . The serotypes of 95 strains were 04 (84.2%), 014 (3.2%), 01 (2.1%), 013 (2.1%), and R (2.1%) . The most common 04 serotype strains were distributed among types I (60 strains), II (23 strains), III and IV (six strains) . Although the V . vulnificus isolates showed a wide range of genetic similarity values, RAPD analysis could separate V . vulnificus strains into four molecular types, and the isolates from the same hospitals tended to belong to the same molecular type . There was no specific correlation between molecular types and serotypes of V . vulnificus. Appl Environ Microbiol, 1998 Apr, 64(4), 1459 - 65 Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica); Motes ML et al.; This study investigated the temperature and salinity parameters associated with waters and oysters linked to food-borne Vibrio vulnificus infections . V . vulnificus was enumerated in oysters collected at three northern Gulf Coast sites and two Atlantic Coast sites from July 1994 through September 1995 . Two of these sites, Black Bay, La., and Apalachicola Bay, Fla., are the source of the majority of the oysters implicated in V . vulnificus cases . Oysters in all Gulf Coast sites exhibited a similar seasonal distribution of V . vulnificus: a consistently large number (median concentration, 2,300 organisms {most probable number} per g of oyster meat) from May through October followed by a gradual reduction during November and December to < or = 10 per g, where it remained from January through mid-March, and a sharp increase in late March and April to summer levels . V . vulnificus was undetectable (< 3 per g) in oysters from the North and South Carolina sites for most of the year . An exception occurred when a late-summer flood caused a drop in salinity in the North Carolina estuary, apparently causing V . vulnificus numbers to increase briefly to Gulf Coast levels . At Gulf Coast sites, V . vulnificus numbers increased with water temperatures up to 26 degrees C and were constant at higher temperatures . High V . vulnificus levels (> 10(3) per g) were typically found in oysters from intermediate salinities (5 to 25 ppt) . Smaller V . vulnificus numbers (< 10(2) per g) were found at salinities above 28 ppt, typical of Atlantic Coast sites . On 11 occasions oysters were sampled at times and locations near the source of oysters implicated in 13 V . vulnificus cases; the V . vulnificus levels and environmental parameters associated with these samples were consistent with those of other study samples collected from the Gulf Coast from April through November . These findings suggest that the hazard of V . vulnificus infection is not limited to brief periods of unusual abundance of V . vulnificus in Gulf Coast oysters or to environmental conditions that are unusual to Gulf Coast estuaries. Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 3134 - 9 A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains; Karaolis DK et al.; The bacterial species Vibrio cholerae includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera . While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains . Initially, two ToxR-regulated genes (aldA and tagA) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains . The region containing aldA and tagA comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP) . The PAI is 39.5 kb in size, has low %G+C (35%), contains putative integrase and transposase genes, is flanked by att sites, and inserts near a 10Sa RNA gene (ssrA), suggesting it may be of bacteriophage origin . We found this PAI in two clinical non-O1/non-O139 cholera toxin-positive strains, suggesting that it can be transferred within V . cholerae . The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic Escherichia coli, a transposase from Vibrio anguillarum, and several ORFs with no known homology . As the PAI contains the CTXPhi receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera . We propose to call this island VPI (V . cholerae pathogenicity island). Curr Microbiol, 1998 Apr, 36(4), 196 - 201 Serological reactivity of humans to a Vibrio cholerae common antigen; Sciortino CV Jr; Over the course of seven pandemics, Vibrio cholerae serotypes have varied . In 1992 the appearance of a new serotype, O139 Bengal, began the eighth cholera pandemic . Several new O139 antigens have been identified, yet a common V . cholerae antigen has not been described . In this study, a monoclonal antibody specific against an 18.7-kDa outer membrane antigen reacted in dotblot analysis with 292 epidemiologically diverse V . cholerae isolates including O1, non-O1, and O139 serotypes . Serum collected from volunteers experimentally challenged with V . cholerae O139, and rabbit antisera to V . cholerae O1, were reactive with the 18.7-kDa antigen by Western immunoblot . This is the first report that the 18.7-kDa antigen is present in V . cholerae O139. Biochim Biophys Acta, 1997 Dec 31, 1362(2-3), 109 - 15 Molecular analysis of a filamentous phage (fsl) of Vibrio cholerae O139; Honma Y et al.; A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl) . The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell . The DNA sequence of fsl RF was determined . It consisted of 6340 bp and had a G + C content of 43.5% . Fifteen possible ORFs were found in fsl . One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V . cholerae and gene I of the coliphage group . ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage . Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage. FEMS Immunol Med Microbiol, 1997 Dec, 19(4), 323 - 9 Evaluation of different subcellular fractions of Vibrio cholerae O139 in protection to challenge in experimental cholera; Bondre VP et al.; Various cellular fractions of Vibrio cholerae O139 were prepared and evaluated in the rabbit ileal loop model of experimental cholera for identification of the protective antigen(s) relevant for vaccine development . Lipopolysaccharides (LPS) and capsular polysaccharides (CPS) of O139 strains and its cell surface, membrane and cytosolic fractions were assayed for antibacterial immunity, whereas the cholera toxin was examined for antitoxic immunity . The lipopolysaccharides, membrane fraction and cholera toxin induced moderate protection, however there was a significant synergistic effect when cholera toxin was combined with membrane proteins or lipopolysaccharides . The O139 strains strongly resembled O1 strains in the profile of proteins and immunological cross reactivity, yet there was no cross protection . The results warrant further investigation of the pathogenesis of O139 strains and identify the critical somatic antigens relevant to protection. Biochemistry, 1998 Mar 10, 37(10), 3237 - 42 Menaquinone-7 in the reaction center complex of the green sulfur bacterium Chlorobium vibrioforme functions as the electron acceptor A1; Kjaer B et al.; Photosynthetically active reaction center complexes were prepared from the green sulfur bacterium Chlorobium vibrioforme NCIMB 8327, and the content of quinones was determined by extraction and high-performance liquid chromatography . The analysis showed a stoichiometry of 1.7 molecules of menaquinone-7/reaction center . No other quinones were detected in the isolated reaction centers, whereas membrane preparations also contained chlorobiumquinone . The possible involvement of quinones in electron transport was investigated by electron paramagnetic resonance (EPR) spectroscopy . A highly anisotropic radical was detected by Q-band EPR spectroscopy in both membranes and isolated reaction centers following dark reduction with sodium dithionite and photoaccumulation at 205 K . At 34 GHz, the EPR spectrum is characterized by a g tensor with gxx = 2.0063, gyy = 2.0052, gzz = 2.0020 and delta B of 0.7 mT, consistent with its identification as a quinone . This spectrum is highly similar in terms of g values and line widths to photoaccumulated A1- in photosystem I of Synechococcus sp . PCC 7002 . The results indicate that menaquinone-7 in the green sulfur bacterial reaction center is analogous to phylloquinone in photosystem I. Indian J Exp Biol, 1998 Jan, 36(1), 76 - 85 Activity of bacterial phosphomonoesterases in batch culture; Sharma A et al.; The phosphomonoesterases catalyse the hydrolysis of primary esters of phosphoric acid which help the bacteria to survive in phosphate stressed environment . Ninety-five bacterial isolates were obtained from domestic sewage and industrial effluents of gelatine and soap factories at Jabalpur on a medium enriched with phosphate and were screened for phosphatase production . The phosphatase producers were tentatively identified as Escherichia coli, Vibrio vulnificus, Aeromonas hydrophila, Staphylococcus aureus, Pseudomonas maltophilia and Micrococcus varians . The in vitro studies on the production of phosphomonoesterases by bacteria was conducted . The maximum alkaline phosphatase production was recorded on 8th day of incubation by E.coli and P.maltophilia, on 10th day of incubation by V.vulnificus while M.varians and P.maltophilia produced higher acid phosphatase on 4th and 10th day of incubation respectively . The detailed investigations were done to find out the effect of various physical and chemical factors on phosphomonoesterases activity and the optimum conditions required for enzyme activity. Tijdschr Diergeneeskd, 1997 Nov 1, 122(21), 600 - 3 {Severe watery diarrhea caused by Vibrio cholerae in lambs}; Visser IJ et al.; An outbreak of watery diarrhoea in lambs is described . Seventeen lambs died within 24 hours after the start of the diarrhoea . At necropsy Vibrio cholerae was isolated from the organs and intestines of three lambs . The strains did not react with O1 or O139 antisera, the strains responsible for cholera epidemics among humans . It is concluded that the diarrhoea in the lambs was caused by V . cholerae non-01/non-139 . This microorganism had not been described before in lambs in the Netherlands. World Health Forum, 1997, 18(3-4), 262 - 5 Sanitation for rural communities: first win the people's support; Rao S et al.; A latrine project in an Indian village which failed because the inhabitants were scarcely brought into its planning and execution is contrasted with a moderately successful scheme in another village where a concerted effort was made to educate the community about the value of latrines and to obtain the people's participationPIP: In 1981, at the beginning of the International Drinking Water Supply and Sanitation Decade, a range of rural sanitation programs was launched in India in response to the spread of Vibrio cholerae, diarrhea outbreaks, and other subjects of public health concern . However, despite these initiatives, only 14% of the country's rural population had access to adequate sanitation by 1994 . The authors focus upon two village latrine construction projects in southern India in an attempt to explain why so little progress has been made . The latrine project in one Indian village failed because community members were not adequately involved in its planning and execution . The second project in another village, in which a concerted effort was made to teach the community about the value of latrines and to obtain community participation in project planning and implementation, was moderately successful . All of the households which accepted latrines in this latter village are still using them today . The introduction of sanitation on a large scale in rural areas is unlikely to succeed without community education and participation . FEMS Microbiol Lett, 1998 Mar 15, 160(2), 183 - 9 Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae; Sengupta TK et al.; A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10,325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein . Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V . cholerae . On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V . cholerae . Expression of 10,325 pili was favored in AKI rather than in NB medium and at 30 degrees C rather than at 37 degrees C . Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10,325 . An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10,325, but not against V . cholerae O1 strains . Such protection was shown to be mediated by inhibition of intestinal colonization in vivo. Zh Mikrobiol Epidemiol Immunobiol, 1998 Jan-Feb, (1), 21 - 4 {The etiology of El Tor cholera}; Savel'ev VN et al.; Vibrio cholerae eltor has been shown to exist in two variants: epidemic (V . cholerae eltor Hly-, tox+) and nonepidemic, or endemic (V . cholerae eltor Hly+, tox-); each of these variants determines the corresponding form of manifestation of the infection among the population and requires a differentiated complex of antiepidemic measures, as well as different tactics for the treatment of patients. Infect Immun, 1998 Apr, 66(4), 1648 - 53 A recombinant live attenuated strain of Vibrio cholerae induces immunity against tetanus toxin and Bordetella pertussis tracheal colonization factor; Chen I et al.; An attenuated strain of Vibrio cholerae was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor from Bordetella pertussis (Tcf) . In vitro, high levels of protein were obtained when the Escherichia coli nirB promoter was used and the bacteria were grown with low aeration . Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin . Bacterial viability was essential for the induction of anti-TetC antibodies . Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type B . pertussis . These data are in agreement with the putative role of Tcf in Bordetella tracheal colonization . In conclusion, we have demonstrated that V . cholerae may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved. Epidemiol Infect, 1998 Feb, 120(1), 1 - 5 Association of a disease approximating cholera caused by Vibrio cholerae of serogroups other than O1 and O139; Bhattacharya MK et al.; One hundred and six patients suffering from severe dehydrating diarrhoea were studied of whom 36 patients were positive for Vibrio cholerae . Out of 36, 15 were positive for V . cholerae O1, 10 for V . cholerae O139 and 11 for V . cholerae non-O1 non-O139 . O1 and O139 were positive for the 301-bp ctxA amplicon and 471-bp tcpA amplicon indicating that the strains possessed toxigenic capability whereas no non-O1 non-O139 strain possessed ctxA or tcpA genes . Post-admission severity of purging and amount of ORS required were less in the V . cholerae non-O1 non-O139 group (P < 0.05) compared to the V . cholerae O1 and O139 groups . It appears from this study that a cholera-like clinical condition can be caused in the absence of CT as exemplified by strains of non-O1 non-O139. Microbiol Immunol, 1998, 42(1), 41 - 5 Adhesive property of toxin-coregulated pilus of Vibrio cholerae O1; Tamamoto T et al.; The adhesive property of toxin-coregulated pilus (TCP) to the human intestine jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods . The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium . V . cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti-TCP IgG . The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine . These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium. Biol Chem, 1998 Feb, 379(2), 193 - 200 Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals; Maeda H et al.; We found that human matrix metalloproteases (MMPs) may be processed from their proenzyme forms (proMMP) to their active forms by two new and unique mechanisms: Firstly, by bacterial proteases such as Pseudomonas elastase and Vibrio cholerae protease, which cleave off the N-terminal autoinhibitory domain (so-called cysteine switch) from proMMPs . The second mechanism depends on free radical generation by activated polymorphonuclear leukocytes (PMNs) . In this case, peroxynitrite (ONOO-) or nitrogen dioxide radical (.NO2), the reaction products of either superoxide (O2.-) or molecular oxygen (O2) and nitric oxide (.NO), are the key reactants . Both O2.- and .NO are generated by activated macrophages and PMNs as a result of immunologic responses involving various proinflammatory cytokines . .NO2 or ONOO- seems to interact with a single cysteine residue in the propeptide autoinhibitory domain, or so-called cysteine switch of proMMPs, thus transforming proMMPs into their active conformation . Furthermore, reactive oxygen species are known to inactivate the alpha1-protease inhibitor (alpha1-PI), a potent neutrophil elastase inhibitor in plasma . In addition, we found that such radicals activate MMPs which degrade and inactivate alpha1-PI by proteolysis . Thus, the activation of MMPs, accompanied by the inactivation of alpha1-PI, will bring about enhanced proteolytic damage to the matrix tissues of the infected sites by both MMPs and elastase. Microbios, 1997, 91(368-369), 175 - 80 Susceptibility of different isolates of Vibrio harveyi to antibiotics; Liu PC et al.; The susceptibility of six Vibrio harveyi strains to antibiotics was studied . Four strains originally isolated from diseased penaeids and two reference strains originally isolated from either sea water (ATCC 25919) or diseased Talorchestia sp . (ATCC 14126) were used in the present study . Results revealed that all three strains isolated in Taiwan exhibited resistance against nitrofurantoin, novobiocin and sulphonamide . The two reference strains and the strain isolated in Indonesia were susceptible to these three antibiotics. Hybridoma, 1998 Feb, 17(1), 63 - 7 Production and characterization of a monoclonal antibody against mannose-sensitive hemagglutinin of Vibrio cholerae; Falero G et al.; We have generated murine monoclonal antibodies (MAb) against Vibrio cholerae mannose-sensitive hemagglutinin (MSHA) using conventional hybridoma procedures . Seven hybridomas were obtained and one characterized . Hybridoma 2F12/F1 secreted an antibody of the IgG3 type that reacted with a 17-kDa antigen corresponding to the product of the mshA gene . This MAb inhibited mannose-sensitive agglutination of chicken erythrocytes by EL tor and O139 vibrios . Vibrios expressing MSHA activity inhibited binding of the antibody secreted by 2F12/F1 to MSHA-coated microtiter plates. FEBS Lett, 1998 Feb 27, 423(3), 371 - 5 Changes in the periplasmic linker and in the expression level affect the activity of ToxR and lambda-ToxR fusion proteins in Escherichia coli; Jappelli R et al.; In order to assess the potentiality of Vibrio cholerae ToxR protein and of bacteriophage lambda repressor as indicators of the dimerization of periplasmic proteins in Escherichia coli, we have constructed a series of plasmids encoding transmembrane fusion proteins . The amino-terminal part, containing the DNA binding domain of either ToxR or lambda repressor, is located in the cytoplasm and acts as reporter for dimerization . As models of periplasmic proteins we have used alkaline phosphatase (a dimer) and beta-lactamase (a monomer) . Both the expression level and the distance between the transmembrane segment and the periplasmic protein substantially affect the activity of the reporter domains. Mol Microbiol, 1998 Feb, 27(4), 797 - 805 Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization; Chiang SL et al.; The pathogenesis of cholera begins with colonization of the host intestine by Vibrio cholerae . The toxin co-regulated pilus (TCP), a fimbrial structure produced by V . cholerae, is absolutely required for colonization (i.e . the persistence, survival and growth of V . cholerae in the upper intestinal milieu), but many other aspects of the colonization process are not well understood . In this study, we use signature-tagged transposon mutagenesis (STM) to conduct a screen for random insertion mutations that affect colonization in the suckling mouse model for cholera . Of approximately 1100 mutants screened, five mutants (approximately 0.5%) with transposon insertions in TCP biogenesis genes were isolated, validating the use of STM to identify attenuated mutants . Insertions in lipopolysaccharide, biotin and purine biosynthetic genes were also found to cause colonization defects . Similar results were observed for mutations in homologues of pta and ptfA, two genes involved in phosphate transfer . Finally, our screen identified several novel genes, disruption of which also caused colonization defects in the mouse model . These results demonstrate that STM is a powerful method for isolating colonization-defective mutants of V . cholerae. Ecotoxicol Environ Saf, 1998 Jan, 39(1), 65 - 9 Structure-toxicity relationships for three mechanisms of action of toxicity to Vibrio fischeri; Cronin MT et al.; Quantitative structure-activity relationships (QSARs) have been developed with the logarithm of the inverse of 15-min toxicity (pT15) to Vibrio fischeri (the acute Microtox test) . Statistically, robust QSARs were found for alkanones acting by the nonreactive, baseline narcosis mechanism of action {pT15 = 0.99 (log Kow)-2.08, n = 6, s = 0.24, r2 = 0.988, F = 405}; aldehydes acting by the Schiff base-forming mechanism of electrophilicity {pT15 = 0.55(log Kow)-0.58, n = 6, s = 0.07, r2 = 0.994, F = 782}; and alkenals acting by the Michael-type acceptor mechanism of electrophilicity {pT15 = 0.52(log Kow) + 0.35, n = 6, s = 0.19, r2 = 0.914, F = 54.5} . Efforts to model toxicity across mechanism of action resulted in the development of a response surface {pT15 = 0.79(log Kow)-1.17 (ELUMO)-0.41, n = 19, s = 0.46, r2 = 0.892, F = 75.3} . In addition, an excellent correlation was found between Tetrahymena pyriformis {log(1/IGC50)} and V . fischeri . {log(1/IGC50) = 0.86(pT15)-0.25, n = 19, s = 0.25, r2 = 0.957, F = 405} toxicity. Virology, 1998 Mar 15, 242(2), 314 - 8 A vibriophage, KVP40, with major capsid protein homologous to gp23* of coliphage T4; Matsuzaki S et al.; The mcp gene encoding the major capsid protein (Mcp) of vibriophage KVP40, a large-tailed DNA phage, was cloned and sequenced . The nucleotide sequence of the mcp gene was 64.4% similar to that of gene 23 of coliphage T4 . Analysis of the N-terminal amino acid sequence of purified native Mcp revealed that the mcp gene actually coded for a precursor, pro-Mcp, whose 62 N-terminal amino acids must be removed upon maturation to Mcp . Thus, mature Mcp would consist of 452 amino acid residues and have a calculated molecular mass of 47,561 Da . Comparison of amino acid sequences of Mcp and gp23*, the major capsid protein of T4, demonstrated 61.8% identity and 89.7% similarity between them . In addition, a sequence, TATAAATA, identical to a typical T4 late promoter sequence was seen in the region upstream of the mcp gene . These findings, together with their morphological similarity, suggest that KVP40 and T4 are phylogenetically related. FEMS Immunol Med Microbiol, 1998 Jan, 20(1), 45 - 54 Lipopolysaccharide O-antigen expression and the effect of its absence on virulence in rfb mutants of Vibrio cholerae O1; Iredell JR et al.; Using defined rfb mutants, defective in the biosynthesis of the O-antigen of the lipopolysaccharide (LPS), and monoclonal antibodies (MAbs) to the A, B and C LPS antigens, we have examined the distribution of the antigens and the effects of their loss . By immunogold electron microscopy, it has been possible to determine the relative amounts of the A, B and C antigens on Inaba and Ogawa cells, confirming previous studies based upon bacterial agglutination and hemagglutination inhibitions . These antigens are absent from rfb::Tn mutants selected as resistant to phages which have been shown to use the O-antigen as their receptor . These mutants were severely attenuated as measured by both LD50 and their ability to compete with the wild-type parents when analyzed in the infant mouse cholera model . These mutants were unchanged in the export of cholera toxin or other secreted proteins but revealed an altered outer membrane protein profile . The competition defect suggested an effect on TCP (toxin-coregulated pilus) . An analysis of the rfb::Tn mutants revealed that they were unable to assemble TCP on their surface, but the major subunit, TcpA, could be found as an intracellular pool . These mutants could be complemented back to wild-type using the cloned rfb region, implying that functional TCP assembly is dependent upon an intact LPS. FEMS Microbiol Lett, 1997 Nov 15, 156(2), 187 - 91 Isolation of a hemin and hemoglobin binding outer membrane protein of Vibrio vulnificus biotype 2 (serogroup E); Fouz B et al.; The eel pathogen Vibrio vulnificus biotype 2 (serogroup E) is able to use hemin (Hm) or hemoglobin (Hb) as the sole iron source for growth in vitro and in vivo . The mechanism of heme-iron acquisition in this bacterium requires a direct interaction through binding sites on the bacterial surface (constitutive outer membrane proteins) . Using affinity chromatography techniques, a unique protein of around 36.5 kDa was isolated from cell envelopes of E86 strain regardless of the affinity ligand used, hemoglobin or hemin . This protein was purified from both iron-enriched and iron-restricted grown cells . These results support the hypothesis that in this pathogen Hm- and Hb-iron acquisition is mediated by a common protein receptor which recognizes the heme prosthetic group of Hb. Med Trop (Mars), 1997, 57(3), 249 - 52 {Inc J plasmids identified for the first time in Vibrio cholerae El Tor}; Korichi MN et al.; Two epidemic outbreaks of cholera occurred in eastern Algeria in 1994 . Sixteen strains of Vibrio cholerae El Tor were isolated from stools and contaminated water . Studies to determine antibiotic sensitivity documented multiresistance in these strains . Minimal inhibiting concentrations ranged from 6 to 32 micrograms/ml for chloramphenicol, from 8 to 24 micrograms/ml for tetracycline except minocycline, and from 15 to 32 micrograms/ml for furanes . Higher values were found for other antibiotics such as trimethoprime (1,500 micrograms/ml), streptomycine (128 micrograms/ml) and sulfamides (128 micrograms/ml) . High-grade resistance of Vibrio cholerae El Tor to streptomycine and trimethoprime in association with resistance to 0:129 suggests that transposon is the underlying genetic factor . All resistance markers were located on a single structure that can be transferred to a Escherichia coli receptor and belongs to an Inc J incompatibility group . The fact that plasmid DNA could not be visualized on agarose gel after extraction is also evidence for a transferable transposon. J Med Microbiol, 1997 Aug, 46(8), 639 - 45 Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus; Iida T et al.; Although V . parahaemolyticus does not generally produce urease, several studies have reported urease-positive V . parahaemolyticus isolates from clinical sources . Recently, studies have shown a complete coincidence between the urease-producing phenotype of V . parahaemolyticus strains and the possession of the thermostable direct haemolysin (TDH)-related haemolysin (TRH) gene (trh) . TRH, like TDH, is considered to be an important virulence factor in the pathogenesis of V . parahaemolyticus gastroenteritis . The present study attempted to identify the gene ure encoding urease in V . parahaemolyticus to clarify the relationship between urease production and possession of trh . The polymerase chain reaction with mixed oligonucleotide primers targeted for conserved sequences of reported ure genes from other species was used to prepare a DNA probe to detect the V . parahaemolyticus ure gene . Colony hybridisation with this ure probe demonstrated that all the ure-positive strains produced urease . Considering the coincidence between production of urease and possession of trh in V . parahaemolyticus, it was concluded that the presence or absence of the ure gene is completely coincident with that of the trh gene in V . parahaemolyticus strains . Furthermore, the relative location of ure and trh on V . parahaemolyticus chromosomal DNA was analysed by pulsed-field gel electrophoresis . The results showed that, in all the strains examined, ure and trh were detected on the same NotI fragment, showing that the two genes localise within a relatively small portion of the chromosome DNA . These results suggest that the ure and trh genes are genetically linked in V . parahaemolyticus strains. J Clin Microbiol, 1998 Mar, 36(3), 843 - 4 Molecular evidence that a distinct Vibrio cholerae O1 biotype El Tor strain in Calcutta may have spread to the African continent; Sharma C et al.; We present molecular evidence that a distinct genotype of Vibrio cholerae O1 which appeared in Calcutta, India, in September 1993 and which is characterized by a unique ribotype that is not found in the standardized ribotyping scheme of V . cholerae and that shows a specific pulsed-field gel electrophoresis profile may have spread to the west African country of Guinea-Bissau where it was responsible for an epidemic of cholera which began in October 1994 and continued into 1996. J Clin Microbiol, 1998 Mar, 36(3), 756 - 63 Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India; Sharma C et al.; There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996 . Of the 18 strains of V . cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup . Cell-free culture supernatants of 13 representative non-O1, non-O139 V . cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin . By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes . Studies on the clonality of non-O1, non-O139 V . cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones . Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles . It is clear from this study that some serogroups of V . cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V . cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V . cholerae to include these serogroups. J Pharm Pharmacol, 1998 Jan, 50(1), 1 - 10 Recent advances in vaccine adjuvants for systemic and mucosal administration; O'Hagan DT; Although vaccines produced by recombinant DNA technology are safer than traditional vaccines, which are based on attenuated or inactivated bacteria or viruses, they are often poorly immunogenic . Therefore, adjuvants are often required to enhance the immunogenicity of these vaccines . A number of adjuvants which are particulates of defined dimensions (<5 microm) have been shown to be effective in enhancing the immunogenicity of weak antigens in animal models . Two novel adjuvants which possess significant potential for the development of new vaccines include an oil-in-water microemulsion (MF59) and polymeric microparticles . MF59 has been shown to be a potent and safe adjuvant in human subjects with several vaccines (for example HSV-2, HIV-1 and influenza virus) . An MF59 adjuvanted influenza has been recommended for approval in Italy . Microparticles prepared from the biodegradable polymers the poly(lactide-co-glycolides) (PLG) are currently undergoing extensive pre-clinical evaluation as vaccine adjuvants . Because of their controlled release characteristics, microparticles also possess considerable potential for the development of single dose vaccines . The development of single dose vaccines would offer significant advantages and would improve vaccination uptake rates in at risk populations, particularly in the developing world . In addition to systemic administration, microparticles have also also been shown to enhance the immunogenicity of vaccines when administered by mucosal routes . Therefore microparticles may allow the development of novel vaccines which can be administered by non-parenteral routes . Mucosal administration of vaccines would significantly improve patient compliance by allowing immunization to be achieved without the use of needles . An alternative approach to the development of mucosally administered vaccines involves the production of genetically detoxified toxins . Heat labile enterotoxin (LT) from Escherichia coli and cholera toxin from Vibrio cholerae are two closely related bacterially produced toxins, which are the most potent adjuvants available . However, these molecules are too toxic to be used in the development of human vaccines . Nevertheless, these toxins have been modified by site-directed mutagenesis to produce molecules which are adjuvant active, but non-toxic . The most advanced of these molecules (LTK63), which has a single amino acid substitution in the enzymatically active subunit of LT, is active as an adjuvant, but non-toxic in pre-clinical models . The approach of genetically detoxifying bacterial toxins to produce novel adjuvants offers significant potential for the future development of mucosally administered vaccines. Acta Vet Scand, 1997, 38(4), 315 - 22 Viability during storage and stability of plasmids during storage and subculturing in strains of Vibrio anguillarum; Pedersen K; The stability of plasmids, 3.3 kb - approximately 200 kb, in 8 strains of Vibrio anguillarum displaying different plasmid profiles and reactions with O-antisera was investigated over an 18 months period . All plasmid profiles proved to be resistant to storage at different temperatures but strains stored at 37 degrees C were only viable for a short period of maximum 2 months . Strains stored at 5 degrees C and 20 degrees C were viable for a longer period . Viable strains maintained their plasmid profile throughout the experiment, except in 2 cases where a 67 kb and a 200 kb plasmid were lost . Strains stored at -80 degrees C all remained viable and maintained their plasmid profile throughout the study . By subcultivating daily for up to 100 successive days, most strains maintained their plasmid profiles . Only 2 strains lost their plasmid . When picking 100 single colonies from agar plates, none of the colonies showed plasmid profiles deviating from the expected . The results suggest that plasmid profiles among V . anguillarium are very stable during subculturing, storage and laboratory handling using standard laboratory procedures, and thus, reliable for epidemiological investigations . In a second experiment, 2 pairs of 2 strains were grown together in mixed cultures . They were identical in all traits, except that one strain in each pair harboured the 67 kb pJM1-like virulence plasmid, whereas the other had lost this plasmid . The result showed that the growth rate was the same for strains with and without the plasmid, indicating that under laboratory conditions, this plasmid is neither a benefit nor the opposite for bacterial growth. FEMS Microbiol Lett, 1998 Feb 15, 159(2), 173 - 8 Distribution of the antiseptic-resistance genes qacE and qacE delta 1 in gram-negative bacteria; Kazama H et al.; The distribution of the antiseptic-resistance genes qacE and qacE delta 1 was studied in a large number of Gram-negative bacteria by a method that included the polymerase chain reaction (PCR) . A total of 117 strains of Gram-negative bacteria, isolated from clinical or environmental sources, was used in this analysis . We demonstrated the presence of these genes in 48 of 78 strains of Pseudomonas, in 20 of 26 strains of Vibrio, and in four of 13 strains of other species . These results indicate that the antiseptic-resistance genes are present in a broad range of species of Gram-negative bacteria. Am J Trop Med Hyg, 1998 Feb, 58(2), 163 - 7 Phenotypic and genotypic characterization of Vibrio cholerae isolates from a recent cholera outbreak in Senegal: comparison with isolates from Guinea-Bissau; Aidara A et al.; A total of 127 strains of Vibrio cholerae (117 V . cholerae O1 and 10 nonagglutinating strains) isolated from a recent cholera outbreak in Senegal and four strains isolated in Guinea-Bissau (during the survey of a cholera epidemic that occurred 10 months before the Senegalese one) were analyzed . Strains were characterized by conventional methods (biochemical and serologic identification, susceptibility to antimicrobial agents), polymerase chain reaction for genes encoding cholera toxin (CtxA), zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), and by ribotyping . Conventional methods showed that all strains of V . cholerae O1 belonged to serotype Ogawa, biotype El Tor and were resistant to the vibriostatic agent O129 (2,4-diamino 6,7-diisopropylpteridine phosphate), cotrimoxazole, and chloramphenicol; all strains were sensitive to tetracycline, a drug that has been extensively used in cholera therapy . Most of these V . cholerae O1 (112 strains from Senegal and four strains from Guinea-Bissau) had an intact core region (virulence cassette) and amplified a 564-basepair (bp) fragment of ctxA, a 1083-bp fragment of zot, and a 314-bp fragment of ace . Ribotyping of V . cholerae O1 strains after Bgl I restriction of total DNA revealed that ribotype B5a, which is the predominant ribotype of this seventh pandemic of cholera, was not isolated . Instead, a new ribotype was identified and designated B27 in our data bank . Since O1 isolates from Guinea-Bissau and Senegal have the same biotype, serotype, and ribotype and as the Guinea-Bissau outbreak that preceded the one in Senegal, this emerging ribotype probably came from Guinea-Bissau . Nonagglutinating strains exhibited no resistance to the O129 agent and to the tested antibiotics, they were all negative for virulence cassette, except for one strain with the ctxA and zot genes isolated from a patient with diarrhea, and there was a great variability of ribotypes among these strains . There was no difference between environmental O1 strains isolated from water and strains isolated from patients with cholera, suggesting that fecally contaminated water is an important reservoir for infection. Rev Sci Tech, 1997 Aug, 16(2), 661 - 72 Seafood-associated diseases and control in Canada; Todd EC; Paralytic shellfish poisoning (PSP) has been documented for two centuries and control programmes have been operated for fifty years . Although some illnesses are reported almost every year, the last known death from PSP occurred in 1981 . In more recent years, amnesic shellfish poisoning, ciguatera poisoning, diarrhoetic shellfish poisoning, scombroid (histamine) poisoning and tetramine poisoning have been documented . The most frequently observed of these diseases is scombroid poisoning from improperly stored fish, but PSP and ciguatera poisoning have the most serious consequences . Vibrio infections arising from naturally-contaminated shellfish are virtually unknown, and viral illnesses from polluted harvested waters are rare . Control is achieved through monitoring of waters for indicators of human pathogens . Inspection systems based on the hazard analysis and critical control point principles are being introduced into all areas of fish and shellfish harvesting . The Inspection Directorate of the Department of Fisheries and Oceans joined the new Canadian Food Inspection Agency in 1997, which co-ordinates all Federal control measures for food in Canada. Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1818 - 22 Host-derived amino acids support the proliferation of symbiotic bacteria; Graf J et al.; Animals are typically colonized by diverse bacterial symbionts, many of which are commensal and, in numerous cases, even essential for their host's proper development and growth . In exchange, the host must supply a sufficient array and quantity of nutrients to support the proliferation and persistence of its microbial community . In this investigation, we have examined such a nutritional environment by determining the symbiotic competence of auxotrophic mutants of the bioluminescent bacterium Vibrio fischeri, and have demonstrated that the host squid Euprymna scolopes provides at least 9 aa to the growing culture of symbiotic V . fischeri present in its light-emitting organ . We also collected and analyzed the extracellular fluid from this organ, in which the symbionts reside, and confirmed that it contained significant amounts of amino acids . The combined results suggested that host-derived free amino acids, as well as peptides or proteins, are a source of the amino acids that support the growth of the symbionts . This work describes a technique to sample the symbionts and their surrounding environment without contamination by host tissue components and, in combination with molecular genetic studies, allows the characterization of the nutritional conditions that support a cooperative animal-bacterial symbiosis. Int J Food Microbiol, 1997 Sep 16, 38(2-3), 133 - 42 Development and evaluation of a predictive model for the effect of temperature and water activity on the growth rate of Vibrio parahaemolyticus; Miles DW et al.; The growth rates of four strains of Vibrio parahaemolyticus were measured and compared in a model broth system . The results for the fastest growing strain, based on 77 combinations of temperature and water activity (aw) using NaCl as the humectant, were summarised in the form of a predictive mathematical model . The model, of the square-root type includes a novel term to describe the effects of super-optimal water activity, and can be used to predict generation times for the temperature range (8-45 degrees C) and water activity range (0.936-0.995) which permit growth of Vibrio parahaemolyticus . Predicted generation times from the model were compared to literature data, using bias and accuracy factors, for both laboratory media and foods . The model was shown to give realistic growth estimates, with a bias value of 1.01, and an accuracy factor of 1.38. Gut, 1998 Jan, 42(1), 11 - 2 Mid-life crisis for M cells; Owen RL; The epithelium that lines the gut is impermeable to macromolecules and microorganisms, except in Peyer's patches (PP), where the lymphoid follicle-associated epithelium (FAE) contains M cells that transport antigens and microorganisms . A cultured system that reproduces the main characteristics of FAE and M cells was established by cultivation of PP lymphocytes with the differentiated human intestinal cell line Caco-2 . Lymphocytes settled into the epithelial monolayer, inducing reorganization of the brush border and a temperature-dependent transport of particles and Vibrio cholerae . This model system could prove useful for intestinal physiology, vaccine research, and drug delivery studies. FEMS Microbiol Lett, 1998 Mar 1, 160(1), 125 - 9 Rise of cytosolic Ca2+ and activation of membrane-bound guanylyl cyclase activity in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-01; Chaudhuri AG et al.; The cytosolic calcium level ({Ca2+}i) and the membrane-bound guanylyl cyclase activity in the isolated rat intestinal epithelial cells were investigated . Heat-stable enterotoxin of Vibrio cholerae non-01 (NAG-ST) was found to increase both the {Ca2+}i and the enzyme activity . These changes occur similarly until 5 min of incubation with NAG-ST, indicating that these changes might be involved in NAG-ST induced signal transduction in rat enterocytes. FEMS Microbiol Lett, 1998 Mar 1, 160(1), 75 - 9 Purification and characterization of a monomeric isocitrate dehydrogenase from the sulfate-reducing bacterium Desulfobacter vibrioformis and demonstration of the presence of a monomeric enzyme in other bacteria; Steen IH et al.; NADP(+)-specific isocitrate dehydrogenase (EC 1.1.1.42) was p