Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee's cholera phage IV and the phage produced by the hemolytic vibrio lysogenized with the infection of Mukerjee's cholera phage IV

Strains of hemolytic Vibrio cholerae O1 (El Tor vibrio) which are sensitive to Mukerjee's cholera phage group IV were isolated from cholera patients in North-East Thailand in 1986. Plaques of the phage on these hemolytic V. cholerae O1 were usually translucent but almost transparent on some strains, just like the plaques on non-hemolytic V. cholerae O1 (classical vibrio). These hemolytic V. cholerae O1 were lysogenized with the infection of cholera phage IV, and the lysogenized strains produced phage different from cholera phage IV. These hemolytic strains were classified into Cured type in prophage typing of V. cholerae O1, El Tor, because they were also lysogenized with Kappa phage and were hemolytic. When Cured-type V. cholerae O1, El Tor previously isolated in various countries were examined for the sensitivity to cholera phage IV, some of the isolates were sensitive.     

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.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Influence of water temperature, salinity, and pH on survival and growth of toxigenic Vibrio cholerae serovar 01 associated with live copepods in laboratory microcosms.

The influence of water temperature, salinity, and pH on the multiplication of toxigenic Vibrio cholerae serovar O1 cells and their attachment to live planktonic crustaceans, i.e., copepods, was investigated by using laboratory microcosms. By increasing water temperatures up to 30 degrees C, a pronounced effect on the multiplication of V. cholerae was demonstrated, as was attachment of the cells to live copepods. These were measured by culturable counts on agar plates and direct observation by scanning electron microscopy, respectively. Of the three salinities examined (5, 10, and 15%), maximum growth of V. cholerae and attachment to copepods occurred at 15%. An alkaline pH (8.5) was optimal both for attachment and multiplication of V. cholerae, as compared with pH 6.5 and 7.5. It is concluded that conditions affecting attachment of V. cholerae serovar O1 to live copepods observed under laboratory conditions may also occur in the natural estuarine environment and, thereby, are significant in the epidemiology of cholera.     

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.     

Ecological relationships between Vibrio cholerae and planktonic crustacean copepods.

Strains of Vibrio cholerae, both O1 and non-O1 serovars, were found to attach to the surfaces of live copepods maintained in natural water samples collected from the Chesapeake Bay and Bangladesh environs. The specificity of attachment of V. cholerae to live copepods was confirmed by scanning electron microscopy, which revealed that the oral region and egg sac were the most heavily colonized areas of the copepods. In addition, survival of V. cholerae in water was extended in the presence of live copepods. Attachment of viable V. cholerae cells to copepods killed by exposure to -60 degrees C was not observed. Furthermore, survival of V. cholerae was not as long in the presence of dead copepods as in the live copepod system. A strain of Vibrio parahaemolyticus was also seen to attach to copepod surfaces without effect on survival of the organism in water. The attachment of vibrios to copepods was concluded to be significant since strains of other bacteria, including Pseudomonas sp. and Escherichia coli, did not adhere to live or dead copepods. Attachment of V. cholerae to live copepods is suggested to be an important factor of the ecology of this species in the aquatic environment, as well as in the epidemiology of cholera, for which V. cholerae serovar O1 is the causative agent.     

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production

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

Adherence to human small intestines of capsulated Vibrio cholerae O139

Abstract Capsulated cells of V. cholerae O139 adhered to formalis-fixed or native mucosa of the small intestines from an adult and a child. The primary adherence target was mucus. Capsulated O139 cells adhered better to the antigen sampling cells (M cells) of ileal Peyer's patch than to the absorptive cells. O139 cells on the mucosa appeared as small aggregates. Similar organisms were found on the mucosa of duodenal biopsy samples from patients infected with V. cholerae O139. The findings indicated that capsulated cells of V. cholerae O139 tend to autoagglutinate and contribute to the effective adherence to the intestinal mucosa.     

Role of a protease in the adherence and enterotoxicity ofVibrio mimicus

The role of protease in the intestinal adherence and enterotoxicity ofVibrio mimicus strain E-33 was investigated by usingin vivo ligated rabbit ileal loop model.V. mimicus protease (VMP) was negative in fluid accumulation in the rabbit ileal loop. However, pre-treatment of the loop with purified VMP induced the elevation of fluid accumulation caused by the vibrios with the enhancement of bacterial adherence to the corresponding intestinal mucosa. The augmentation in the fluid accumulation could also be observed in the loop upon supplementation with VMP. In contrast, adherence to the mucosa was reduced by the simultaneous inoculation of VMP. The elevation in the fluid accumulation could also be observed in the loop supplemented or pre-treated with protease from V.cholerae orV. vulnificus. Furthermore, when the vibrios in the loops were accompanied by anti-VMP IgG antibody or inhibitors for VMP, such as ethyleneglycol-bis (\-aminoethylether)-N, N, N’, N’,-tetraacetic acid or tetraethylenepentamine, reductions in the adherence indices with consequent reductions in the fluid accumulations were observed. It is therefore, suggested that the protease produced by the pathogen contributes significantly toward the pathogenicity ofV. mimicus.     

The protective activity of tea against infection by Vibrio cholerae O1

Extracts of black tea exhibited bactericidal activity against Vibrio cholerae O1. The tea extract inhibited the haemolysin activity of V. cholerae O1, El Tor and the morphological changes of Chinese hamster ovary cells induced by cholera toxin. Tea extract also reduced fluid accumulation induced by cholera toxin in sealed adult mice and by V. cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea has protective activity against V. cholerae O1.     

Preliminary in vitro evaluation of the antimicrobial activity of terpenes and terpenoids towards Erwinia amylovora (Burrill) Winslow et al.

Extracts of black tea exhibited bactericidal activity against Vibrio cholerae O1. The tea extract inhibited the haemolysin activity of V. cholerae O1, El Tor and the morphological changes of Chinese hamster ovary cells induced by cholera toxin. Tea extract also reduced fluid accumulation induced by cholera toxin in sealed adult mice and by V. cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea has protective activity against V. cholerae O1.     

Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as the CTX phage [corrected]

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rstC and rstB1 (truncated) genes downstream of ORF500. att-fs2-dir and att-fs2-rev sequences homologous to that of att-CTXphi were found between orf500 and rstC of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of attRS of the CTXphi phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.     

Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as cholera toxin X

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rst C and rst B1 (truncated) genes downstream of ORF500. att -fs2-dir and att- fs2-rev sequences homologous to that of att -CTXφ were found between orf 500 and rst C of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of att RS of the CTXφ phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.     

The agglutinability of cholera O-monoclonal immunoglobulins]

A set of 10 monoclonal antibodies specific for vibrio species and of 5 monoclonal antibodies specific for serovar (Ogawa) was studied. These monoclonal antibodies were directed toward V. cholerae O1 antigen in agglutination reaction and on slide plates. Monoclonal antibodies agglutinating typical strains of cholera vibrios with titration range from 1: 6000 to 1: 25,000 were selected. MA were revealed to interact with cholera vibrio strains with reduced agglutinability. The set of agglutinating O monoclonal immunoglobulins was developed for laboratory identification of cholera O1 vibrios.     

Virulence genes in environmental strains of Vibrio cholerae

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.     

O139霍乱弧菌生物学特性研究

１９９２年以来,许多国家和地区先后暴发了Ｏ１３９霍乱大流行。本文从微生物学和分子遗传学的角度对来自不同地区的四株Ｏ１３９霍乱弧菌的生物学特性进行了研究。结果表明四株Ｏ１３９霍乱弧菌均呈典型弧形、单端单鞭毛,培养要求不高、耐碱,固体平板上菌落呈不透明。电镜下显示有菌毛、荚膜结构。有较广的抗生素敏感谱及霍乱Ｈｅｉｂｅｒｇ氏Ⅰ群的糖发酵能力。ＤＮＡＧ＋ＣＭＯＬ％测定值均在霍乱弧菌范围之内且数值接近。质粒图谱检测发现四株中有三株含有一个４．１０ＭＤａ大小的质粒,而另一株不含质粒。Ｏ１３９霍乱弧菌的生物学特性大多数与Ｏ１群菌相似,两者重大的区别在于Ｏ１３９菌具荚膜结构。     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

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

A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139.

Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.