Bactericidal properties of the Vibrio cholerae non O1 bacteriolysin

The influence of the preparation of hemocytolysin, obtained from V. cholerae non O1 (strain P-11702), on the growth of V. cholerae cells was studied. The study revealed that hemocytolysin is capable of inducing partial or complete bacterial lysis on the place of its application, depending on the protein load of the substance and the inoculation dose of microbes. Two electrophoretic fractions with molecular weights of 14 and 14.5 kD induced the cytolysis of sheep, rabbit, guinea-pig red blood cells and showed a bactericidal effect. The different sensitivity of Vct+ and Vct- strains of V. cholerae to different doses of hemocytolysin was studied.     

Characterization of enteropathogenic Vibrio cholerae non O1/O139 isolated in Uzbekistan

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.     

Histopathological changes in experimental cholera with a non toxigenic non- O1 non-O139 Vibrio cholerae strain isolated from Kolkata, India

This study was conducted to understand the pathophysiological changes in experimental rabbit ileal loop model using the Vibrio cholerae strain non-O1non-O139, isolated as sole pathogen from clinically diagnosed cholera patients in Kolkata. Significant amount of haemorrhagic fluid accumulation was observed in all the test loops of rabbit model where the strain of V.cholerae was inoculated as compared to control loops. Microscopic examination of the accumulated fluid showed the presence of erythrocytes and pus cells. Histology revealed structural alteration of the villous epithelium with inflammatory cells infiltration in all the layers of the gut mucosa including the nerve plexus region. Preliminary observation with a haemagglutinin protease extracted from the non-O1 non-O139 strain, was also studied in different concentrations in the same animal model which showed similar type of macroscopic and microscopic response in the ileal loops as seen with the original strain. The results highlight that along with other pathways, inflammatory cells and the enteric neurons have an important role in the pathophysiology of diarrhoea and the isolated protease may be the probable virulence factor in initiating the disease process in this non-O1non-O139 strain induced cholera.     

Complete genome sequence of a sucrose-nonfermenting epidemic strain of Vibrio cholerae O1 from Brazil

We report the genome sequence of Vibrio cholerae strain IEC224, which fails to ferment sucrose. It was isolated from a cholera outbreak in the Amazon. The defective sucrose phenotype was determined to be due to a frameshift mutation, and a molecular marker of the Latin American main epidemic lineage was identified.     

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

Immunosensor for the detection of Vibrio cholerae O1 using surface plasmon resonance

An immunosensor for the detection of Vibrio cholerae O1 was developed on the basis of surface plasmon resonance (SPR). A protein G layer was fabricated by means of the chemical coupling between the free amine (-NH₂) groups of protein G and the activated carboxyl groups present on a self-assembled monolayer (SAM) consisting of a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2). A monoclonal antibody, which was confirmed to be specific to V. cholera O1 by the Western blotting technique, was immobilized on the protein G layer. The formation of the SAM, the protein G layer and the sequential binding of the antibody against V. cholera O1 were investigated with SPR spectroscopy. As the number of fabricated layers increased, the minimum angle of plasmon resonance was increased accordingly. The target bacteria, V. cholera O1, was measured with the fabricated immunosensor, whose detection range was between 10⁵ and 10⁹ cells/mL.     

Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1.

DNA colony hybridization with a polynucleotide clonal DNA probe for heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was used to screen 197 isolates of V. cholerae O1. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae O1 GP156, heated at 100 degrees C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely neutralized by an anti-NAG-ST monoclonal antibody (mAb2F). The preparation from V. cholerae O1 GP156 also inhibited the binding of mAb2F to NAG-ST in a competitive ELISA. V. cholerae O1 GP156 was confirmed to possess a gene encoding cholera toxin (CT). These results indicate that a heat-stable enterotoxin is produced by certain strains of CT-producing V. cholerae O1.     

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1

BT23, a Vibrio cholerae O1 El Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Cholera toxin gene polymerase chain reaction for detection of non-culturable Vibrio cholerae O1

Cholera enterotoxin is a major antigenic determinant for virulence of Vibrio cholerae O1 which can enter into a viable but non-culturable (N-C) state, not detectable by conventional culture methods, yet remain capable of producing enterotoxin and potentially pathogenic. PCR was applied in the current study to detect the chilera toxin (ctx) gene of N-C cells, thus eliminating the necessity of culture. Sets of oligonucleotide primers were designed, based on the ctxAB operon of V. cholerae O1, to detect the presence of the ctx gene. DNA from both culturable and N-C cells of V. cholerae O1 was amplified by PCR using sets of primers flanking 302-, 564- and 777-bp fragments of the ctx gene. The PCR method employed was capable of detecting the ctx gene in N-C V. cholerae in aquatic microcosms and in diarrheal stool samples from three patients who had distinct clinical symptoms of cholera but were culture-negative for V. cholerae O1 and non-O1 and enterotoxigenic Escherichia coli. Forty cycles of a two-step reaction (30 s each at 94 and 60°C) were optimal and more time efficient than a three-step PCR described previously. The procedure, from the point of heating microcosms or broth culture samples to observation on gels, requires < 4 h to complete.J.A.K. Hasan, A. Huq, M.A.R. Chowdhury, and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD, USA. M. Shahabuddin is with the National Institute of Health. Bethesda, MD, USA. L. Loomis is with New Horizons Diagnostics Corporation, Columbia, MD, USA.     

Virulence properties of rough and smooth strains of Vibrio cholerae O1

A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains. A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme-linked immunosorbent assay (ELISA) in Richardson's and AKI media. All the smooth and rough strains, except one, showed a titre of 1: 10 and 1: 100 in Richardson's and AKI media, respectively. Both types of strains produced enterotoxin in rabbit ileal loop (RIL). The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P <0.05). However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P >0.05) between these properties. This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae , tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

Vibrio cholerae O22 might be a putative source of exogenous DNA resulting in the emergence of the new strain of Vibrio cholerae O139

The new epidemic strain O139 of Vibrio cholerae, the etiologic agent of cholera, has probably emerged from the pandemic strain O1 El Tor through a genetic rearrangement involving the horizontal transfer of exogenous O-antigen- and capsule-encoding genes of unknown origin. In V. cholerae O139, these genes are associated with an insertion sequence designated IS1358_O139. In this work, we studied the distribution of seven genes flanking the IS1358_O139 element in 13 serovars of V. cholerae strains. All these O139 genes and an IS1358 element designated IS1358_O22-1 were only found in V. cholerae O22 with a similar genetic organization. Sequence analysis of a 4.5-kb fragment containing IS1358_022-1 and the adjacent genes revealed that these genes are highly homologous to those of V. cholerae O139. These results suggest that strains of V. cholerae O22 from the environment might have been the source of the exogenous DNA resulting in the emergence of the new epidemic strain O139.     

Development of a multiplex single-tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection

A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctxA gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers directed to rfbN gene were added to the reaction. The detection limit of the multiplex reaction was determined using V. cholerae O1 DNA and V. cholerae O1 grown in alkaline peptone water (APW). STNPCR was shown to be approximately 100-fold more sensitive than simple PCR and 10 times less sensitive than two-step nested PCR. This drawback is compensated by a lower risk of cross-contamination. The addition of a second target did not impair the detection limit of STNPCR (as little as 1 pg of V. cholerae O1 DNA detected). MSTNPCR could specifically detect up to three V. cholerae O1 cells or colony forming units (cfu) directly from the APW growth. A diagnostic kit consisting of a set of microtubes having the inner primers fixed onto the inside of the tube cap and a set of tubes containing the reaction mixture was evaluated for stability, and it proved to be stable for five months at -20 degrees C. Therefore, MSTNPCR would be useful in the detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.     

Expression and detection of different biotype-associated cell-bound haemagglutinins of Vibrio cholerae O1

The expression of two cell-bound haemagglutinins, one sensitive to L-fucose (FSHA) and the other to D-mannose (MSHA), on Vibrio cholerae O1 strains of both the classical and the El Tor biotypes was studied by (i) agglutination of chicken and human group O erythrocytes in the presence of L-fucose or D-mannose, (ii) binding of the bacteria to L-fucose- and D-mannose-coated agarose beads, and (iii) agglutination of the bacteria by 'biotype-specific' antisera. All of the 12 classical strains studied that were isolated before 1979 gave FSHA of human O erythrocytes whereas only 6 of 17 classical strains isolated during recent epidemics expressed FSHA; a few of the classical strains expressed MSHA in addition to FSHA. All the El Tor strains gave MSHA of chicken erythrocytes and one strain also expressed FSHA. Both the cell-bound HAs were optimally expressed during the exponential phase of growth; FSHA markedly decreased during the late exponential phase while the MSHA usually persisted into the stationary phase. The expression of FSHA and MSHA correlated very well with the direct binding of vibrios to fucose- and mannose-coated agarose beads, respectively. Antiserum 'specific' for classical vibrios agglutinated classical strains expressing FSHA and also the El Tor strain exhibiting FSHA. Similarly, the anti-El Tor serum agglutinated all El Tor strains and also classical strains expressing MSHA, suggesting that the 'biotype-specific' sera were specific for the biotype-associated cell-bound HAs.     

Molecular-epidemiological characteristic and possible origin of Vibrio cholerae non O1/non O139 with complete and limited set of virulence genes

Study of molecular-epidemiological characteristics of Vibrio cholerae non O1/non O139 serogroup with complete and limited set of virulence genes was performed. Differences of their genes composition as compared to these of O1 serogroup (classic and El Tor biovars) were revealed, which points to their origin from avirulent environmental cholera vibrios.     

Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae O1 and non O1 in Romania

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.