New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh

The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.     

Analysis of expression of toxin-coregulated pili in classical and El Tor Vibrio cholerae O1 in vitro and in vivo.

The expression of toxin-coregulated pili (TCP) and their structural subunit TcpA was compared in 20 strains of Vibrio cholerae of the classical and El Tor biotypes. Bacteria were isolated from the intestines of rabbits with experimental cholera and compared with the same strains grown under optimal TCP expression conditions in vitro. Immunoblotting revealed that TcpA production was induced in both biotypes after vibrios entered the intestinal milieu; TcpA-negative inocula gave rise to TcpA-positive vibrios after multiplication in the gut. The levels of TcpA expressed during growth in the intestine were, for most strains, comparable to those attained under optimal growth conditions in vitro. Of 11 classical strains tested, 10 expressed TCP antigen on the bacterial surface at levels comparable to or exceeding those seen after growth in vitro as determined by an inhibition enzyme-linked immunosorbent assay. In contrast, only one of the nine El Tor strains studied produced detectable amounts of TCP surface antigen in vivo and no fimbriae or surface antigen reacting with anti-TCP serum was found on El Tor vibrios from human cholera stools. Distinct TCP fimbriae were observed by immunoelectron microscopy on classical-biotype vibrios grown either in rabbit intestines or in vitro but were not detected on El Tor vibrios. The results show that TCP is expressed on V. cholerae O1 of the classical biotype but not on V. cholerae O1 of the El Tor biotype in the intestines of rabbits with experimental cholera infection.     

2,3-butanediol synthesis and the emergence of the Vibrio cholerae El Tor biotype

Vibrio cholerae is an aquatic bacterium that causes the severe diarrheal disease cholera. V. cholerae strains of the O1 serogroup exist as two biotypes, classical and El Tor. Toxigenic strains of the El Tor biotype emerged to cause the seventh pandemic of cholera in 1961 and subsequently displaced strains of the classical biotype both in the environment and as a cause of cholera within a decade. The factors that drove emergence of the El Tor biotype and the displacement of the classical biotype are unknown. Here, we show a unique difference in carbohydrate metabolism between these two biotypes. When grown with added carbohydrates, classical biotype strains generated a sharp decrease in medium pH, resulting in loss of viability. However, growth of El Tor biotype strain N16961 was enhanced due to its ability to produce 2,3-butanediol, a neutral fermentation end product, and suppress the accumulation of organic acids. An N16961 mutant (SSY01) defective in 2,3-butanediol synthesis showed the same defect in growth that classical biotype strains show in media rich in carbohydrates. Importantly, the SSY01 mutant was attenuated in its ability to colonize the intestines of infant mice, suggesting that host carbohydrates may be available to V. cholerae within the intestinal environment. Similarly, the SSY01 mutant failed to develop biofilms when utilizing N-acetyl-D-glucosamine as a carbon source. Because growth on N-acetyl-D-glucosamine likely reflects the ability of a strain to grow on chitin in certain aquatic environments, we conclude that the strains of classical biotype are likely defective compared to those of El Tor in growth in any environmental niche that is rich in chitin and/or other metabolizable carbohydrates. We propose that the ability to metabolize sugars without production of acid by-products might account for the improved evolutionary fitness of the V. cholerae El Tor biotype compared to that of the classical biotype both as a global cause of cholera and as an environmental organism.     

Genome variability in the altered variants of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> biovar El Tor isolated in Russia

Molecular-genetic analysis of 56 clinical strains of the Vibrio cholerae biovar El Tor isolated during the period 1965–2010 in Russia and neighboring countries was carried out. Thirty-one isolates (55.3%) bearing the gene ctxB in the genome of the prophage CTXφ in cholera vibrios of the classical biovar were found. These strains are altered variants of the cholera El Tor pathogen. The nucleotide sequence of their genes ctxB and rstR included in the prophage CTXϕ, which are related with the production of the cholera toxin, was determined. It was found that altered variants bear different alleles of these genes (ctxB1 or ctxB7; rstR^El and/or rstR^Class) and belong to four gene types: ctxB1, rstR^Class; ctxB1, rstR^El; ctxB1, rstR^Class/rstR^El; and ctxB7, rstR^Class/rstR^El. It was shown that these gene variants produce 42 to 48 times more cholera toxin than typical strains. Isolates in which the pathogenicity island VSP-2 contains a long deletion were found among altered variants. The study data are evidence of the genomic variability of altered pathogen variants of the seventh cholera pandemic and require the development of new gene-diagnostic testing systems to improve the efficiency of epidemiological control of cholera distribution.     

The toxin-coregulated pilus is a colonization factor and protective antigen ofVibrio choleraeEl Tor

We have previously shown that insertional inactivation oftcpA, the gene encoding the major pilin subunit of the toxin-coregulated pilus (TCP), rendersVibrio choleraeO1 strains of El Tor biotype virtually avirulent in the infant mouse cholera model (IMCM). We now report that more refined mutants, bearing an in-frame deletion intcpA, show a similar dramatic attenuationin vivo. In mixed-infection competition experiments the ratio of wild-type:mutant vibrios increasedc. 10³–10⁵fold during a period ofin vivogrowth. An attempt to complement the ΔtcpAmutants by providing a functional El TortcpAgenein transwas only partially successful. Sera raised against El Tor TcpA were able to passively protect infant mice against challenge with TCP-positive strains of homologous biotype and were also protective against isolates of the novel O139 serovar. These sera failed to protect against challenge with a strain of classical biotype, nor could antibodies to classical TCP confer immunity to El Tor challenge. We conclude that TCP is a critical colonization factor ofV. choleraeO1 El Tor and that antibodies to TCP are sufficient to confer protection against such strains in the IMCM. Our data suggest that the biotype-specific epitopes carried by TcpA are of greater vaccine significance than those epitopes common to both proteins.     

Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.     

Comparison of Vibrio cholerae pathogenicity islands in sixth and seventh pandemic strains

Epidemic Vibrio cholerae strains possess a large cluster of essential virulence genes on the chromosome called the Vibrio pathogenicity island (VPI). The VPI contains the tcp gene cluster encoding the type IV pilus toxin-coregulated pilus colonization factor which can act as the cholera toxin bacteriophage (CTXPhi) receptor. The VPI also contains genes that regulate virulence factor expression. We have fully sequenced and compared the VPI of the seventh-pandemic (El Tor biotype) strain N16961 and the sixth-pandemic (classical biotype) strain 395 and found that the N16961 VPI is 41,272 bp and encodes 29 predicted proteins, whereas the 395 VPI is 41,290 bp. In addition to various nucleotide and amino acid polymorphisms, there were several proteins whose predicted size differed greatly between the strains as a result of frameshift mutations. We hypothesize that these VPI sequence differences provide preliminary evidence to help explain the differences in virulence factor expression between epidemic strains (i.e., the biotypes) of V. cholerae.     

Emergence and progression of Vibrio cholerae O1 El Tor variants and progenitor strains of Mozambique variants in Kolkata, India

Analysis of 75 Vibrio cholerae O1 strains isolated from hospitalized patients in Kolkata from 1989 to 1994 revealed the existence of true El Tor along with El Tor variants that possessed the classical allele of ctxB (ctxB(cl)) in strains having an El Tor backbone. Based on the existence of different combinations of ctxB and rstR alleles and their localization sites in the genome, these strains were classified into multiple genetic groups. Of 75 clinical strains, 11 were identified as non-toxigenic. These 8 strains were also devoid of pTLC, which is uncommon among the O1 strains. However, Mozambique variants isolated in 2004 were typically negative for pTLC, but these strains possessed tandemly arranged CTX prophages with ctxB(cl) in the small chromosome. Genetic manipulation studies with laboratory-generated kanamycin-tagged pCTX-Kan (derived from tandemly arranged small chromosome-localized ctxB(cl) bearing CTX prophages of 1992 VC53, a progenitor strain of the Mozambique variant) demonstrated that integration specificity of the pCTX-Kan was somewhat towards small chromosome. Such integration could be the prime step towards generation of the Mozambique variant. Based on the existence of multiple alleles of CTX? and their infections with non-toxigenic strains, we propose that the El Tor variant strains could have emerged following these genetic events. This study demonstrated existence of different 'intermediate strains' in a time frame that overlapped with a period of V. cholerae O139 emergence. Identification of these intermediate strains gave impetus to believe stepwise generation of the El Tor variants, and all these events profoundly influenced V. cholerae epidemiology in the following years.     

Vibrio cholerae classical biotype strains reveal distinct signatures in Mexico

Vibrio cholerae O1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, before the El Tor (ET) biotype initiated the seventh pandemic in Asia in the 1970s by completely displacing the CL biotype. Although the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. cholerae CL and ET biotypes, including a hybrid ET, were found associated with areas of cholera endemicity in Mexico between 1991 and 1997. In this study, CL biotype strains isolated from areas of cholera endemicity in Mexico between 1983 and 1997 were characterized in terms of major phenotypic and genetic traits and compared with CL biotype strains isolated in Bangladesh between 1962 and 1989. According to sero- and biotyping data, all V. cholerae strains tested had the major phenotypic and genotypic characteristics specific for the CL biotype. Antibiograms revealed the majority of the Bangladeshi strains to be resistant to trimethoprim-sulfamethoxazole, furazolidone, ampicillin, and gentamicin, while the Mexican strains were sensitive to all of these drugs, as well as to ciprofloxacin, erythromycin, and tetracycline. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA revealed characteristic banding patterns for all of the CL biotype strains although the Mexican strains differed from the Bangladeshi strains in 1 to 2 DNA bands. The difference was subtle but consistent, as confirmed by the subclustering patterns in the PFGE-based dendrogram, and can serve as a regional signature, suggesting the pre-1991 existence and evolution of the CL biotype strains in the Americas, independent from Asia.     

Genomic relatedness of the new Matlab variants of Vibrio cholerae O1 to the classical and El Tor biotypes as determined by pulsed-field gel electrophoresis

The genomes of the recently described Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes were compared with those of El Tor and classical biotypes by the use of pulsed-field gel electrophoresis. Dendrograms constructed using the unweighted-pair group method using average linkages generated from NotI restriction patterns of whole-chromosomal DNA grouped these strains into two major clusters that were found to be similar but not identical to those of either of the biotypes. Strains that clustered with the classical biotype appear to have been derived from the classical strains, which are thought to be extinct.     

Cholera toxin expression by El Tor Vibrio cholerae in shallow culture growth conditions

Vibrio cholerae O1 classical, El Tor and O139 are the primary biotypes that cause epidemic cholera, and they also express cholera toxin (CT). Although classical V. cholerae produces CT in various settings, the El Tor and O139 strains require specific growth conditions for CT induction, such as the so-called AKI conditions, which consist of growth in static conditions followed by growth under aerobic shaking conditions. However, our group has demonstrated that CT production may also take place in shallow static cultures. How these type of cultures induce CT production has been unclear, but we now report that in shallow culture growth conditions, there is virtual depletion of dissolved oxygen after 2.5 h of growth. Concurrently, during the first three to 4 h, endogenous CO₂ accumulates in the media and the pH decreases. These findings may explain CT expression at the molecular level because CT production relies on a regulatory cascade, in which the key regulator AphB may be activated by anaerobiosis and by low pH. AphB activation stimulates TcpP synthesis, which induces ToxT production, and ToxT directly stimulates ctxAB expression, which encodes CT. Importantly, ToxT activity is enhanced by bicarbonate. Therefore, we suggest that in shallow cultures, AphB is activated by initial decreases in oxygen and pH, and subsequently, ToxT is activated by intracellular bicarbonate that has been generated from endogenous CO₂. This working model would explain CT production in shallow cultures and, possibly, also in other growth conditions.     

Immune response genes modulate serologic responses to Vibrio cholerae TcpA pilin peptides.

Cholera is an enteric disease caused by Vibrio cholerae. Toxin-coregulated pilus (TCP), a type 4 pilus expressed by V. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at the H-2 locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.     

Genovariants of Vibrio cholerae biovar El Tor: Construction,molecular-genetic,and proteomic analyses

Experimental modeling of the emergence of virulent Vibrio cholerae El Tor genovariants is presented. It has been shown that the obtained genovariants differed neither in phenotypic or genotypic traits from natural genetically altered strains that emerged in populations of wild-type strains. It has been established, using the PCR and sequencing methods, that the genovariants formed in the process of conjugation carried in their genome a fragment of the CTX^Classφ prophage genome with the ctxB1 gene of classical-type cholera vibrios. It has been shown that changes in the prophage’s structure led to higher levels of toxigenicity and virulence in the genovariants compared to a typical recipient strain. A proteomic analysis has also revealed changes in the expression of 26 proteins performing various functions in the cell, such as metabolism, energy exchange, transport of amino acids, etc.). These data are indicative of the effect produced by the new DNA region in the genome of the genovariants on the expression level of some house-keeping genes. The obtained results confirm the idea that horizontal gene transfer is one of the mechanisms leading to the emergence of genovariants in the populations of wild-type strains.     

Molecular characterization of a new variant of toxin-coregulated pilus protein (TcpA) in a toxigenic non-O1/Non-O139 strain of Vibrio cholerae

A toxigenic non-O1/non-O139 strain of Vibrio cholerae (10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new tcpA gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the tcpA sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classical tcpA. Further, tcpA of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpA gene in V. cholerae which probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.