Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site.     

Purification of El Tor cholera enterotoxins and comparisons with classical toxin.

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.     

Biotype-specific tcpA genes in Vibrio cholerae

Abstract The tcpA gene, encoding the structural subunit of the toxin-coregulated pilus, has been isolated from a variety of clinical isolates of Vibrio cholerae , and the nucleotide sequence determined. Strict biotype-specific conservation within both the coding and putative regulatory regions was observed, with important differences between the El Tor and classical biotypes. V. cholerae O139 Bengal strains appear to have El Tor-type tcpA genes. Environmental O1 and non-O1 isolates have sequences that bind an E1 Tor-specific tcpA DNA probe and that are weakly and variably amplified by tcpA -specific polymerase chain reaction primers, under conditions of reduced stringency. The data presented allow the selection of primer pairs to help distinguish between clinical and environmental isolates, and to distinguish El Tor (and Bengal) biotypes from classical biotypes from classical biotypes of V. cholerae . While the role of TcpA in cholera vaccine preparations remains unclear, the data strongly suggest that TcpA-containing vaccines directed at O1 strains need include only the two forms of TcpA, and that such vaccines directed at (O139) Bengal strains should include the TcpA of El Tor biotype.     

The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton.

The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats. The V. cholerae O1 El Tor biotype and V. cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V. cholerae O1 classical biotype strains do not. Although V. cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains. Since V. cholerae O1 El Tor and V. cholerae O139 have displaced V. cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton. We found that mutation of mshA in V. cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex. The effect of the mutation was more pronounced for V. cholerae O139, essentially eliminating adherence. Adherence of the V. cholerae O1 classical mshA mutant was unaffected. The results suggest that MSHA is a factor contributing to the ability of V. cholerae to adhere to plankton. The results also showed that both biotypes of V. cholerae O1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.     

Detection and differentiation of the gene for toxin co-regulated pili (tcpA) in Vibrio cholerae non-O1 using the polymerase chain reaction

Abstract The polymerase chain reaction has been used to differentiate the gene which encodes the toxin co-regulated pili ( tcpA ) of the El Tor and classical biotypes of Vibrio cholerae O1. The same PCR primers were applied to strains belonging to non-O1 serogroups that produced cholera toxin. The size of fragment amplified was either identical to the tcpA of biotype El Tor (471 bp) or to the tcpA of biotype classical (617 bp). All strains belonging to the novel epidemic serogroup O139 generated a 471-bp fragment identical to El Tor tcpA . The present study suggests that there may be an association between non-O1 serogroup and tcpA type.     

Facile identification of protein sequences by mass spectrometry. B subunit of Vibrio cholerae classical biotype Inaba 569B toxin

A mass spectrometric method was applied to the B subunit of Vibrio cholerae classical biotype Inaba 569B toxin to determine its amino acid sequence and to confirm the differences in the amino acid sequences predicted from the nucleotide sequences of the genes of El Tor biotype strains 62746 and 2125 toxins. In this method, the Staphylococcus aureus protease V8 digest of the CNBr-treated B subunit of the classical biotype toxin was examined directly by fast-atom-bombardment mass spectrometry without separation of individual peptides. The values of molecular ion signals observed in the mass spectra were compared with the amino acid sequences of the classical biotype and El Tor biotype toxins. All the observed mass values coincided with those calculated from the published sequences of the B subunit except those of the sequences at positions 12-29 and 69-79. Peptides with these sequences were isolated by high-performance liquid chromatography and analyzed by Edman degradation or by combination of mass spectrometry and enzymatic degradation. The results revealed that the amino acid residues at positions 22 and 70 were Asp instead of Asn in the published sequences of classical biotype toxin. It was also found that Asn at position 44 was partially deaminated to Asp. The amino acid sequence of the classical biotype toxin was found to be different only at positions 18 (His----Tyr), 47 (Thr----Ile) and 54 (Gly----Ser) from that of El Tor biotype toxins.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

Polylysogeny and prophage induction by secondary infection in Vibrio cholerae

Strains of Vibrio cholerae O1, biotypes El Tor and classical, were infected with a known temperate phage (PhiP15) and monitored over a 15-day period for prophage induction. Over the course of the experiment two morphologically and three genomically distinct virus-like particles were observed from the phage-infected El Tor strain by transmission electron microscopy and field inversion gel electrophoresis, respectively, whereas only one phage, PhiP15, was observed from the infected classical strain. In the uninfected El Tor culture one prophage was spontaneously induced after 6 days. No induction in either strain was observed after treatment with mitomycin C. Data indicate that El Tor biotypes of V. cholerae may be polylysogenic and that secondary infection can promote multiple prophage induction. These traits may be important in the transfer of genetic material among V. cholerae by providing an environmentally relevant route for multiple prophage propagation and transmission.     

Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B

The Hly region from the chromosome of Vibrio cholerae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.     

Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor

A mismatch amplification mutation PCR assay was developed and validated for rapid detection of the biotype specific cholera toxin B subunit of V. cholerae O1. This assay will enable easy monitoring of the spread of a new emerging variant of the El Tor biotype of V. cholerae O1.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Genome sequence of the human pathogen Vibrio cholerae Amazonia

Vibrio cholerae O1 Amazonia is a pathogen that was isolated from cholera-like diarrhea cases in at least two countries, Brazil and Ghana. Based on multilocus sequence analysis, this lineage belongs to a distinct profile compared to strains from El Tor and classical biotypes. The genomic analysis revealed that it contains Vibrio pathogenicity island 2 and a set of genes related to pathogenesis and fitness, such as the type VI secretion system, present in choleragenic V. cholerae strains.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Detection and quantitative assessment of a Vibrio cholerae O1 species in several foods by a novel enzyme immunoassay.

A selected antibody enzyme immunoassay (SAEIA) for the general detection of Vibrio cholerae O1 species has been developed using the immunological reagents of a rabbit antiserum specific for V. cholerae O1 classical Inaba 569B and immobilized cell fragments of V. cholerae O1 El Tor 85P6, and beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G as tracer. The SAEIA was specific for V. cholerae O1 species and showed low cross-reaction values to other microorganism species tested including Vibrio parahaemolyticus. The detection limit of the SAEIA was 4,500 cells per assay for all the 13 strains of V. cholerae O1 examined. Quantitative comparison on the growth of the El Tor 85P4 in several foods cultured for 24 hr were studied using the SAEIA. Preceding the experiments, little inhibition of every food homogenate for the measurement of the SAEIA was first demonstrated and then the homogenate was directly used for an assay sample. The interaction of the growth of Escherichia coli to that of V. cholerae O1 in a food was also found to be little under the mixed culturing of both bacteria using the SAEIA.     

TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae

The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non-O1 serotypes.