Molecular Epidemiology of Reemergent Vibrio cholerae O139 Bengal in India

We report the prevalence of the O139 serogroup in Calcutta, India, after its reemergence in August 1996 and the spread of the reemerged clone to other parts of the country by using previously established molecular markers. Phenotypically, the reemerged Vibrio cholerae O139 displayed a difference compared to those that appeared in late 1992 and 1993 in that the current O139 strains are sensitive to co-trimoxazole. Ribotyping with the enzyme BglI produced two rRNA restriction patterns in the O139 strains isolated after August 1996, and these patterns were identical to those exhibited by strains of O139 isolated in 1992. Three clones of V. cholerae O139 are currently prevailing in the country, with strains exhibiting three bands after HindIII digestion and hybridization with a ctxA probe being dominant. The reemergence of V. cholerae O139 in Calcutta after a 32-month quiescent period reestablishes the O139 serogroup as an entity which is likely to play a crucial role in the temporal antigenic variations among the serogroups of V. cholerae causing cholera.     

Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.

Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V. cholerae O1. After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V. cholerae O139 were measured in acute- and convalescent-phase sera from 33 V. cholerae O139-infected and 18 V. cholerae O1-infected patients and in single serum samples from 20 healthy control subjects. The responses in these individuals to V. cholerae O1 strains were also determined. Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera. These findings may explain the basis for the lack of heterologous protection between the two serogroups of V. cholerae. Healthy controls had higher background levels of vibriocidal antibody to V. cholerae O1 than to V. cholerae O139.     

Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V. cholerae O139 Bengal in Bangladesh.

The emergence of Vibrio cholerae O139 Bengal in 1993, its rapid spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 during 1992 and 1993, and the subsequent reemergence of V. cholerae O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged El Tor vibrios. We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and Africa before the emergence of V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 to determine whether the reemerged El Tor vibrios were genetically different from the El Tor vibrios which existed before the emergence of V. cholerae O139. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin (zot) or in DNA sequences flanking the genes showed that the El Tor strains isolated before the emergence of V. cholerae O139 belonged to four different ribotypes and four different ctx genotypes. Of 32 El Tor strains isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to a single new ribotype and a distinctly different ctx genotype. These results provide evidence that the reemerged El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor vibrios which were replaced by the O139 vibrios. Further analysis showed that all the strains carried the structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR). All strains of the new clone produced cholera toxin (CT) in vitro, as assayed by the GM1-dependent enzyme-linked immunosorbent assay, and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios. Further studies are required to assess the epidemic potential of the newly emerged clone of V. cholerae O1 and to understand the mechanism of emergence of new clones of toxigenic V. cholerae.     

Phage specific for Vibrio cholerae O139 Bengal.

From the stool of a Vibrio cholerae O139 Bengal-infected patient, a phage that specifically lysed capsulated V. cholerae O139 strains only was isolated. The phage is useful for the confirmatory diagnosis of V. cholerae O139 infection and for the differentiation of variants that lack the capsule.     

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.     

Escalating association of Vibrio cholerae O139 with cholera outbreaks in India

Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only V. cholerae O1 biotype ElTor or V. cholerae O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of V. cholerae O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of V. cholerae O139.     

Phenotypic and genotypic changes in Vibrio cholerae O139 Bengal.

To find reasons for the recent decline of Vibrio cholerae O139 Bengal cholera in Bangladesh, phenotypic and genotypic changes in O139 isolates obtained from patients with cholera from 1993 to 1996 were studied. The isolates were tested for the presence of ctx and tcpA genes, hemagglutinin/protease (HA/P), capsule, D-mannose-sensitive hemagglutinin (MSHA), L-fucose-sensitive hemagglutinin (FSHA), tube test (tube) and CAMP test (CAMP) hemolytic activities, resistance to 2,4-diamino-6,7-diisopropyl pteridine (O/129) and trimethoprim-sulfamethoxazole (TMP-SMX), and genotype by pulsed-field gel electrophoresis (PFGE). All isolates possessed ctx and tcpA genes, HA/P, and a capsule. Most isolates were negative for FSHA, but although the majority of the isolates were positive for MSHA, no discernible trend in the activity was found during the study period. All early isolates were CAMP hemolysin positive and resistant to the vibriostatic compound O/129 and TMP-SMX, the two properties that could be used for the presumptive diagnosis of O139 cholera. However, subsequently, isolates that were CAMP hemolysin negative and susceptible to TMP-SMX and O/129 were increasingly encountered, with all the 1996 isolates being so, which suggested that these properties can no longer be used for the presumptive diagnosis of O139 cholera. V. cholerae O139 isolates that were CAMP hemolysin positive and resistant to O/129 and TMP-SMX produced a disease of greater severity than that caused by the CAMP hemolysin-negative and susceptible isolates on the basis of the lengths of stay of the hospitalized patients. The study period witnessed the evolution of four different genotypes by PFGE. All of these data suggested that the V. cholerae O139 isolates have undergone changes in some properties. However, how these changes influenced their prevalence relative to that of V. cholerae O1 in human infection is not clear. Studies of the environmental factors will provide the key for an understanding of the relative abundance of these vibrios.     

Molecular analysis of toxigenic Vibrio cholerae O139 Bengal strains isolated in Bangladesh between 1993 and 1996: evidence for emergence of a new clone of the Bengal vibrios.

Vibrio cholerae O139 Bengal emerged in 1992 and rapidly spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 in Bangladesh during 1992 and 1993. The subsequent emergence of a new clone of V. cholerae O1 of the El Tor biotype that transiently displaced the O139 vibrios during 1994 to 1995 and the recent reemergence of V. cholerae O139 and its coexistence with the El Tor vibrios demonstrated temporal changes in the epidemiology of cholera in Bangladesh. We studied clonal diversity among V. cholerae O139 strains isolated from cholera patients and environmental surface water since their first appearance until their transient disappearance in 1994 as well as the O139 strains that reemerged during 1995 to 1996 and were isolated in the capital Dhaka and four rural districts of Bangladesh to investigate the origin of the reemerged strains. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA and cholera toxin (CT) (ctxA) or in DNA sequences flanking these genes revealed four different ribotypes and four different ctx genotypes among the 93 strains of V. cholerae O139 studied. Ribotypes I and II and ctx genotypes A through C were shared by strains isolated from the epidemic outbreak during 1992 and 1993 in Bangladesh and India, ribotype III was represented by a single CT-negative O139 strain from Argentina, and 16 of 27 (59.2%) of the reemerged strains isolated during 1995 and 1996 belonged to a new ribotype of O139 vibrios designated ribotype IV. All 16 strains belonging to ribotype IV also belonged to a new ctx genotype (genotype 4). These results provide evidence for the emergence of a new clone of toxigenic V. cholerae O139 in Bangladesh. Further analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon and the presence of an O139-specific genomic region in all O139 strains. The PCR amplicon corresponding to the rfaD gene of a CT-negative O139 strain from Argentina was smaller in length than those of the toxigenic O139 strains but was identical to those of seven non-O1 and non-O139 strains. All O139 strains except the CT-negative strain carried structural and regulatory genes for CT and toxin-coregulated pili (ctxA, tcpA, tcpI, and toxR). These results suggest that the O139 Bengal strains possibly emerged from an El Tor strain but that the CT-negative non-Bengal O139 strain might have emerged from a non-O1, non-O139 strain. Thus, strains belonging to the O139 serogroup may have emerged from similar serotype-specific genetic changes in more than one progenitor strain of V. cholerae.     

Phagocytosis of Vibrio cholerae O139 Bengal by human polymorphonuclear leukocytes

Capsulated bacteria exhibit serum (complement) resistance and resistance to phagocytosis, which result in disseminated infections. Vibrio cholerae O139 strains possess a thin capsule and have been found to be partially serum resistant in a previous study. In the present study, compared to a standard capsulated Klebsiella pneumoniae strain, which showed total resistance to killing by phagocytosis, V. cholerae O139 strains were shown to be only partially resistant, with most strains showing <40% survival. These findings may explain the relative rarity of V. cholerae O139 bacteremia in cholera caused by this organism.     

Phagocytosis of Vibrio cholerae O139 Bengal by Human Polymorphonuclear Leukocytes

Capsulated bacteria exhibit serum (complement) resistance and resistance to phagocytosis, which result in disseminated infections. Vibrio cholerae O139 strains possess a thin capsule and have been found to be partially serum resistant in a previous study. In the present study, compared to a standard capsulated Klebsiella pneumoniae strain, which showed total resistance to killing by phagocytosis, V. cholerae O139 strains were shown to be only partially resistant, with most strains showing <40% survival. These findings may explain the relative rarity of V. cholerae O139 bacteremia in cholera caused by this organism.     

Characterization of a toxigenic Vibrio cholerae O139 strain belonging to a new ribotype and isolated from a diarrheal patient

We characterized a Vibrio cholerae O139 strain isolated from a diarrheal patient admitted to Taluk Hospital, Cherthala, Alleppey, Kerala, India, on 9 June 2000. The V. cholerae O139 strain possesses the core of the CTX genetic element, colonization toxin-coregulated pilus, the adherence outer membrane protein, and the central regulatory protein encoded by toxR and produces cholera toxin (200 pg/ml). We provide molecular evidence showing that toxigenic V. cholerae O139 strain ALO95 belongs to a distinct genotype characterized by a unique ribotype designated B-VII and has a unique enterobacterial repetitive intergenic consensus sequence PCR fingerprint profile designated E-V.     

Reduction in capsular content and enhanced bacterial susceptibility to serum killing of Vibrio cholerae O139 associated with the 2002 cholera epidemic in Bangladesh

Vibrio cholerae O139 emerged in 1992 as a major cause of epidemic cholera. However, the incidence of disease due to this new serogroup subsequently decreased for almost a decade. In April 2002, there was a dramatic resurgence of V. cholerae O139 in Bangladesh. We compared the phenotypic properties of the bacterial isolates and the immunological responses in patients with disease due to V. cholerae O139 during the 2002 epidemic with those dating to the emergence of this disease in 1993 to 1995. Strains isolated from patients in the two time periods were compared with respect to capsular polysaccharide, their resistance to the bactericidal effect of serum, and their capacity to be used as target strains in complement-mediated vibriocidal assays. Phase-contrast microscopy showed that strains isolated in 2002 had less capsular material than those isolated from 1993 to 1995 (P = <0.001), a finding confirmed by electron microscopic studies. Strains isolated in 2002 were more susceptible to the bactericidal activity of serum compared to strains from 1993 to 1995 (P = 0.013). Compared to results using a standard O139 strain, a modified vibriocidal assay utilizing a 2002 strain, CIRS 134, as the target organism detected higher vibriocidal responses in both O139-infected cholera patients as well as O139 vaccine recipients. The vibriocidal assay utilizing the less encapsulated 2002 strain, CIRS 134, is a more sensitive indicator of adaptive immune responses to recent infection with V. cholerae O139. Consequently, this assay may be useful in studies of both O139-infected patients and recipients of O139 vaccines.     

Characterization of Aeromonas trota strains that cross-react with Vibrio cholerae O139 Bengal.

It has previously been shown that Vibrio cholerae O139 Bengal shares antigens with V. cholerae serogroups O22 and O155. We detected six surface water isolates of Aeromonas trota that agglutinated in polyclonal antisera to V. cholerae O139 and V. cholerae O22 but not in antiserum to V. cholerae O155. On the basis of agglutinin-absorption studies, the antigenic relationship between the cross-reacting bacteria were found to be in an a,b-a,c fashion, where a is the common antigenic epitope and b and c are unique epitopes. The antigen sharing between A. trota strains and V. cholerae O139 was confirmed in immunoblot studies. However, A. trota strains did not react with two monoclonal antibodies specific for V. cholerae O139 and, consequently, tested negative in the Bengal SMART rapid diagnostic test for V. cholerae O139 which uses one of the monoclonal antibodies. A polyclonal antiserum to a cross-reacting A. trota strain cross-protected infant mice against cholera on challenge with virulent V. cholerae O139. All A. trota strains were cytotoxic for HeLa cells, positive for adherence to HEp-2 cells, and weakly invasive for HEp-2 cells; one strain was heat-stable toxin positive in the suckling mouse assay; however, all strains were negative for cholera toxin-like enterotoxin. Studies on bacteria that share somatic antigen with V. cholerae O139 may shed further light on the genesis of V. cholerae O139.     

Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India

In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V. cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000. All of the representative V. cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of V. cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V. cholerae O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the V. cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the E. coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which V. cholerae and ETEC were concomitantly involved.     

Vibrio cholerae O139 Bengal possesses a capsular polysaccharide which may confer increased virulence

A newly described Vibrio cholerae serogroup—0139 Bengal, the causative agent of the recent large epidemics of cholera-like disease in the Indian subcontinent and neighbouring countries—possesses a high molecular weight capsular polysaccharide (CPS) that can be visualized by electron microscopy and in composition differs from the lipopolysaccharide (LPS). The CPS and LPS can be separated from each other by a two-step extraction procedure, a phenol-water extraction in order to extract all polysaccharides from the bacterial suspension followed by a phenol-chloroform-petroleum ether (PCP) extraction. The CPS is mainly composed of 3,6-dideoxyhexose (abequose or colitose), quinovosamine and glucosamine. The LPS of the O139 Bengal strain appears to possess a short polysaccharide which contains glucose, galactose, glucosamine and heptose, Both the LPS and CPS are immunogenic. They react in an enzyme immunoassay with rabbit antibodies generated against whole heat-killed bacteria. By analogy with other capsulated bacteria, the possession of a capsule may confer increased virulence of O139 Bengal.     

Production, characterization, and application of monoclonal antibodies to Vibrio cholerae O139 synonym Bengal.

Mouse monoclonal antibodies (MAbs) were derived against acetone-treated whole cells of the newly recognized Vibrio cholerae O139 serogroup which is causing epidemics of cholera-like disease in India and Bangladesh. Four MAbs specifically recognized the lipopolysaccharide antigens of V. cholerae O139. MAbs ICL9 and ICL13 were of the immunoglobulin M (IgM) isotype, ICL11 was of the IgG3 isotype, and ICL12 was of the Ig2b isotype. A fifth MAb, ICL10, of the IgG2b isotype cross-reacted with V. cholerae O91. All five MAbs recognized V. cholerae O139 in an enzyme-linked immunosorbent assay, slide agglutination test, motility inhibition test, and indirect immunofluorescence test. During a 1-month evaluation of these MAbs in our clinical laboratory, all 86 cases diagnosed as V. cholerae O139 by a rabbit polyclonal antiserum were also detected by these MAbs, establishing their utility as highly sensitive and specific diagnostic reagents. With these MAbs, it should now be possible to screen for the V. cholerae O139 serogroup in epidemic and endemic diarrhea cases and in environmental and food samples.     

Isolation of sucrose late-fermenting and nonfermenting variants of Vibrio cholerae O139 Bengal: implications for diagnosis of cholera.

The sucrose-containing selective medium thiosulfate-citrate-bile salt-sucrose agar missed a sucrose nonfermenting and four sucrose late-fermenting variant strains of Vibrio cholerae O139 Bengal from diarrheal stools. These strains were, however, correctly identified as V. cholerae O139 on a sucrose-deficient selective medium, taurocholate-tellurite-gelatin agar.