Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. The disease is characterized by a devastating watery diarrhea which leads to rapid dehydration, and death occurs in 50 to 70% of untreated patients. Cholera is a waterborne disease, and the importance of water ecology is suggested by the close association of V. cholerae with surface water and the population interacting with the water. Cholera toxin (CT), which is responsible for the profuse diarrhea, is encoded by a lysogenic bacteriophage designated CTXΦ. Although the mechanism by which CT causes diarrhea is known, it is not clear why V. cholerae should infect and elaborate the lethal toxin in the host. Molecular epidemiological surveillance has revealed clonal diversity among toxigenic V. cholerae strains and a continual emergence of new epidemic clones. In view of lysogenic conversion by CTXΦ as a possible mechanism of origination of new toxigenic clones of V. cholerae, it appears that the continual emergence of new toxigenic strains and their selective enrichment during cholera outbreaks constitute an essential component of the natural ecosystem for the evolution of epidemic V. cholerae strains and genetic elements that mediate the transfer of virulence genes. The ecosystem comprising V. cholerae, CTXΦ, the aquatic environment, and the mammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen.

Epidemiology, Genetics, and Ecology of Toxigenic Vibrio cholerae

Vibrio parahaemolyticus is recognized as a cause of food-borne gastroenteritis, particularly in the Far East, where raw seafood consumption is high. An unusual increase in admissions of V. parahaemolyticus cases was observed at the Infectious Diseases Hospital in Calcutta, a city in the northeastern part of India, beginning February 1996. Analysis of the strains revealed that a unique serotype, O3:K6, not previously isolated during the surveillance in Calcutta accounted for 50 to 80% of the infections in the following months. After this report, O3:K6 isolates identical to those isolated in Calcutta were reported from food-borne outbreaks and from sporadic cases in Bangladesh, Chile, France, Japan, Korea, Laos, Mozambique, Peru, Russia, Spain, Taiwan, Thailand, and the United States. Other serotypes, such as O4:K68, O1:K25, and O1:KUT (untypeable), that had molecular characteristics identical to that of the O3:K6 serotype were subsequently documented. These serotypes appeared to have diverged from the O3:K6 serotype by alteration of the O:K antigens and were defined as "serovariants" of the O3:K6 isolate. O3:K6 and its serovariants have now spread into Asia, America, Africa, and Europe. This review traces the genesis, virulence features, molecular characteristics, serotype variants, environmental occurrence, and global spread of this unique clone of V. parahaemolyticus.

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Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants

Some strains of Vibrio parahaemolyticus, a marine bacterium, can cause gastroenteritis in humans through consumption of seafood. It was reported in the late 1960s that almost all clinical strains, but very few environmental strains, manifest Kanagawa phenomenon (KP), β-type hemolysis on Wagatsuma agar (8, 19). KP is caused by high-level production of thermostable direct hemolysin. Thermostable direct hemolysin is encoded by the tdh gene (13, 17), which was detected almost exclusively in clinical strains in an early study (11). The role of thermostable direct hemolysin in enterotoxigenicity was demonstrated by construction and examination of the tdh-deficient mutant of a KP-positive strain (10). Investigation of an outbreak in the Maldives in 1985 revealed that some clinical strains do not possess the tdh gene but carry the tdh-related hemolysin (trh) gene (14). The trh sequence was approximately 70% identical to the tdh sequence. There is much greater strain-to-strain divergence among trh sequences than among tdh sequences. The trh sequences in different strains, however, can be clustered into two groups represented by the trh1 and trh2 genes, which have 84% sequence identity (5). Strains possessing either the tdh gene, the trh gene, or both were shown to be strongly associated with gastroenteritis (5, 20).

Surveillance for V. parahaemolyticus infection was initiated in January 1994 in Calcutta, India. A group of strains belonging to serovar O3:K6 and possessing the tdh gene but not the trh gene appeared suddenly in February 1996 and was shown to be responsible for the high incidence of V. parahaemolyticus infection since then in Calcutta (16). Serovar O3:K6 was not isolated before February 1996 in Calcutta. In addition, the O3:K6 strains isolated in Calcutta were shown to exhibit unique profiles in an arbitrarily primed PCR (AP-PCR) analysis (16). Strains belonging to the same group, i.e., O3:K6 strains possessing the tdh gene but not the trh gene and showing the unique AP-PCR profiles, were also detected among those isolated from travelers arriving in Japan from Southeast Asian countries from 1995 on (16). Thus, the Calcutta O3:K6 strains and the above strains from the travelers were considered to belong to a single clone (16). These results suggested that this unique clone, referred to below as a new O3:K6 clone, might have emerged recently and become prevalent not only in Calcutta, India, but also in other parts of the world.

We examined this hypothesis, and we present evidence in this study for the first pandemicity in the history of V. parahaemolyticus. Clinical strains isolated over 22 years, starting from 1977, in a hospital in Bangladesh were available. The emergence of the new O3:K6 clone in 1996 but not earlier was demonstrated by examination of these strains. Next, we showed by AP-PCR analysis that the clinical strains of serovar O3:K6 isolated in six other countries, including the United States, from 1997 on belong to the same clone. We then developed a novel PCR method to identify the strains belonging to the new O3:K6 clone. We utilized the toxRS operon sequence to develop this PCR method. The toxR and toxS genes in the toxRS operon encode transmembrane proteins involved in the regulation of virulence-associated genes and are well conserved in the genus Vibrio (3, 7, 18; J. H. Rhee, S. E. Lee, S. Y. Kim, S. H. Shin, C. M. Kim, P. Y. Ryu, K. C. Leong, S. H. Choi, and S. S. Chung, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. B-171, p. 84, 1998; V. Vuddhakul, T. Nakai, C. Matsumoto, T. Oh, T. Nishino, M. Nishibuchi, and J. Okuda, submitted for publication). We used the intraspecies variation of the toxRS sequence to develop a cluster-specific PCR method that allowed for confirmation of the clonality of the new O3:K6 strains. By using this PCR method in our investigation, we found emerging strains that were almost indistinguishable from the new O3:K6 clone, although the strains belonged to different serovars.

Pandemic Spread of an O3:K6 Clone of Vibrio parahaemolyticus and Emergence of Related Strains Evidenced by Arbitrarily Primed PCR and toxRS Sequence Analyses

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.

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

Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXTMO10, these genes are clustered within a composite transposon-like structure found near the element's 5′ end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXTET, does not contain the same resistance genes as SXTMO10. In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

Molecular analysis of antibiotic resistance gene clusters in vibrio cholerae O139 and O1 SXT constins.

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

Clonal diversity among the recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread

Distribution of genes encoding cholera toxin, zonula occludens toxin, accessory cholera toxin, and El Tor hemolysin Vibrio cholerae of diverse origins

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.

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Molecular Epidemiology of Reemergent Vibrio cholerae O139 Bengal in India

Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country. The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V. cholerae O1 which emerged in Calcutta in 1994. Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1. The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V. cholerae 0139 in Calcutta. However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of 0139 in Calcutta. The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V. cholerae O1. None of the strains in this study carried plasmids. Molecular studies clearly show that the new expanded drug resistant clone of V. cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India

Stool specimens obtained from 123 hospitalized patients with acute secretory diarrhea admitted to the Infectious Diseases Hospital, Calcutta, India, were examined for isolation of Vibrio cholerae O1 by direct or enrichment plating on selective media for cholera toxin (CT) by bead enzyme-linked immunosorbent assay (bead-ELISA) and for the CT gene by polymerase chain reaction (PCR). V. cholerae O1 was isolated either by direct culture or by enrichment culture from 70 stool specimens, all of which gave positive results by PCR. Eleven specimens which were culture negative and bead-ELISA positive also gave positive results by PCR. In addition, 13 more specimens which were negative by both the culture method and bead-ELISA, were positive by PCR. With the combined results of both the culture method and the CT bead-ELISA, a confirmed laboratory diagnosis of cholera could be made from 81 stool specimens, while the combined results of the three methods, including PCR, yielded a positive result for 94 specimens examined. From these data, we conclude that PCR provides a more sensitive and specific assay for rapid diagnosis of cholera than currently available methods.

Detection of cholera toxin gene in stool specimens by polymerase chain reaction: comparison with bead enzyme-linked immunosorbent assay and culture method for laboratory diagnosis of cholera.

P. K. Bag

Papers

Clonal diversity among the recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread

Distribution of genes encoding cholera toxin, zonula occludens toxin, accessory cholera toxin, and El Tor hemolysin Vibrio cholerae of diverse origins

Molecular Epidemiology of Reemergent Vibrio cholerae O139 Bengal in India

Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India

Detection of cholera toxin gene in stool specimens by polymerase chain reaction: comparison with bead enzyme-linked immunosorbent assay and culture method for laboratory diagnosis of cholera.