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.     

Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine

Vibrio cholerae O1 can enter a state in which they remain viable but are non-culturable. Presumably, such bacteria can be pathogenic if they retain the capacity to proliferate in the human intestine following ingestion. Two groups of volunteeers were given inocula containing viable but non-culturable V. cholerae O1 of the attenuated vaccine strain CVD 101 (viable CVD 101 organisms readily colonize the human intestine). Volunteers in one of the two groups excreted viable CVD 101, demonstrating that, in the environment of the human intestine, previously non-culturable vibrios can regain the capacity to multiply. These observations support the proposition that viable but non-culturable bacterial enteropathogens may pose a potential threat to health.     

Cryptic Appearance of a New Clone of Vibrio cholerae Serogroup O1 Biotype El Tor in Calcutta,India

In this study, pulsed-field gel electrophoresis (PFGE) was applied to determine if the Vibrio cholerae O1 strains which reappeared after being temporarily displaced in Calcutta by the O139 serogroup were different from those isolated before the advent of the O139 serogroup. NotI digestion generated a total of 11 different patterns among the 24 strains of V. cholerae randomly selected to represent different time frames. Among the V. cholerae O1 strains isolated after July 1993, 4 PFGE banding patterns designated as H through K were observed with pattern H dominating. Pattern H was distinctly different from all other patterns encountered in this study including patterns A, B and C of V. cholerae O1 El Tor, which dominated before November 1992, and pattern F, which was the dominant V. cholerae O139 pattern. Further, pattern H was also different from the NotI banding patterns of the representative strains of the 4 toxigenic clonal groups of V. cholerae O1 El Tor currently prevailing in different parts of the world. NotI fragments of the new clone of V. cholerae O1 did not hybridize with an O139 specific DNA probe, indicating that there was no O139 genetic material in the new clone of V. cholerae O1. Hybridization data with an O1-specific DNA probe again differentiated between the clones of V. cholerae O1 existing before the genesis of the O139 serogroup and the O1 strains currently prevalent.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Biological Activities of Lipopolysaccharide Isolated from Vibrio cholerae O139, a New Epidemic Strain for Recent Cholera in Indian Subcontinent

Biological activities of lipopolysaccharide (LPS) isolated from Vibrio cholerae O139, a new causative agent for recent cholera epidemic in Indian subcontinent, were investigated in comparison with those of LPS from O1 V. cholerae. V. cholerae O139 LPS exerted mitogenic activity, lethal toxicity and Shwartzman reaction to the same extent as those observed for O1 V. cholerae LPS, although these activities except for lethal toxicity were obviously lower than those of Salmonella typhimurium LT-2 LPS used as a reference. It was, therefore, suggested that O139 LPS does not contribute to the high infective and pathogenic potentials of the V. cholerae O139 strain as in the case of O1 V. cholerae.     

Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin

Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera. There is no vaccine available against cholera caused by this serotype. V. cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen.This study evaluated several synthetic schemes for preparation of conjugates of V. cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents. Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS_AH) or of CSA (CSA_AH), and 2) binding of CPS_AH to CSA or of CPS to CSA_AH. Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA_AH (EDC:CPS-CSA_AH) was statistically significantly less immunogenic than the other three conjugates. Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V. cholerae O139 inoculum.Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V. cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.     

Interaction of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1/non-O139 with Copepods,Cladocerans and Competing Bacteria in the Large Alkaline Lake Neusiedler See,Austria

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community.     

Serological Reactivity of Humans to a Vibrio cholerae Common Antigen

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. Received: 11 August 1997 / Accepted: 22 September 1997     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Morphological and physical characterization of the capsular layer of Vibrio cholerae O139

The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae. Received: 23 March 1998 / Accepted: 28 July 1998     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaD_vco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbD_vco139. The DNA region between rfaD_vco139 and rfbQRS_vco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.     

Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co‐culture with eukaryotic cells

VBNC Vibrio cholerae O139 VC‐280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co‐cultured with CHO cells. Other eukaryotic cell lines, including HT‐29, Caco‐2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC‐280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC‐280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co‐culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.     

Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor

Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double-stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site-specific manner. DNA sequences that cross-hybridize with K139 phage DNA are present in all strains of V. cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139-related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as ‘kappa’. In order to test whether K139 phage is involved in lysogenic conversion of V. cholerae, we constructed a novel mini-transposon, Tn10d-bla, which was designed to produce β-lactamase fusions to phage-encoded, exported proteins. All Tn 10d-bla insertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N-terminal hydrophobic signal sequence. ORF1 was designated the glo gene (G protein-like O RF) because its amino acid sequence shows similarity to eukaryotic Gs(α) protein (34.5% identity over an 81-amino-acid overlap) and its C-terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP-binding proteins. LD₅₀ assays with isogenic Glo⁺ and Glo^? K139 lysogens suggest that glo encodes a secreted virulence determinant of V. cholerae     

Two strains ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae serogroup O139 synonym “Bengal”

Two strains (O22 reference strain, 169–68, and strain 490–93 isolated from a patient with diarrhea in Thailand) ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae O139 synonym Bengal are described. The O antigens of these two strains were closely related to that ofV. cholerae O139 in an a,b-a,c type of relationship, but were not completely identical with serogroup O139. Therefore, both these strains are not classified into the O139 serogroup ofV. cholerae, because they have their own major antigens. As the strain 490–93 could not be placed into any of the 154 established O serogroups ofV. cholerae, this strain was assigned to a new serogroup, O155. For practical use, the diagnostic antiserum prepared against the O139 reference strain (MO45, ATCC 51394) ofV. cholerae must be absorbed with reference strains 169–68 and 490–93 representing serogroups O22 and O155 ofV. cholerae to remove cross-reacting agglutinins of the O22 and O155 strains, respectively.     

Isolation,identification, and characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from the Danube River in Slovakia

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.     

Seasonal Cholera Caused by Vibrio cholerae Serogroups O1 and O139 in the Coastal Aquatic Environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.     

Analysis of Vibrio cholerae O139 Bengal Isolated from Different Geographical Areas Using Macrorestriction DNA Analysis

Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains. NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns. Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity. These results further reiterate the spread of an identical clone of V. cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.     

Molecular phylogenetic analysis ofVibrio cholerae O1 El Tor strains isolated before, during and after the O139 outbreak based on the intergenomic heterogeneity of the 16S-23S rRNA intergenic spacer regions

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eightrrn operons (rrna-rrnh) ofVibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O139 outbreak. ISR classes ‘a’ and ‘g’ were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O139 serogroup and post-O139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.