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SUMMARYA total of 45 strains ofVibrio choleraeO1 isolated from 10 different places in India where they were associated with cases of cholera between the years 2007 and 2008 were examined by molecular methods. With the help of phenotypic and genotypic tests the strains were confirmed to be O1 El Tor biotype strains with classicalctxBgene. Polymerase chain reaction (PCR) analysis by double – mismatch amplification mutation assay PCR showed 16 of these strains carried thectxB-7allele reported in Haitian strains. Sequencing of thectxBgene in all the 45 strains revealed that in 16 strains the histidine at the 20th amino acid position had been replaced by asparagine and this single nucleotide polymorphism did not affect cholera toxin production as revealed by beads enzyme-linked immunosorbent assay. This study shows that the newctxBgene sequence was circulating in different places in India. Seven representatives of these 45 strains analysed by pulsed – field gel electrophoresis showed four distinctNot Idigested profiles showing that multiple clones were causing cholera in 2007 and 2008.

Cholera is a public health threat in all developing countries. Kolkata, a city in eastern India, is an endemic zone for cholera. During the course of a comprehensive investigation on the distribution of phages of Vibrio cholerae O1 and O139 in freshwater bodies in Kolkata, we were able to isolate the phages of V. cholerae O1 and O139. Vibrio cholerae O1 phages were found at all the sites and exhibited a distinct seasonal cycle, with a primary peak (13.6–17.2 PFU mL−1) during monsoon (June to August) in both 2006 and 2007. Vibrio cholerae O139 phages were present in the environment and were predominant during monsoon in the year 2006, except for late winter and early summer from February to April. In contrast, in the year 2007, the O139 phages could be isolated only during July to December, with the highest counts of 12.0 PFU mL−1 determined in August. The multiplex PCR results showed that 90 samples were positive for wbe of V. cholerae O1, 32 samples for O139 ( wbf ) and 18 samples for both. This study shows that surveillance of vibriophages indicates the presence of V. cholerae O1 and O139 in water bodies in and around Kolkata and could therefore serve as a powerful biomonitoring agent.

The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32.1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139.

To the Editor: The epidemic cholera caused by Vibrio cholerae O1 appeared in Latin America in 1991 after a 100-year absence. Following its explosive appearance in Peru, travelers on the Amazon River brought cholera to Brazil by April 1991. It spread southward along the Atlantic Coast of Brazil, reaching Rio de Janeiro in February 1993. Phage typing is a useful tool for studying the source or origin of V. cholerae for epidemiologic importance. Because of limitations of the Basu and Mukerjee scheme, a new phage-typing scheme for V. cholerae O1 was developed at the National Institute of Cholera and Enteric Diseases, India (1–3). During the course of a comprehensive study on the phage typing of V. cholerae O1, most strains isolated in Brazil were found to be sensitive with a set of 10 El Tor phages (ATCC 51352-B1–B10) (4). This finding prompted us to explore or ascertain the natural habitat of V. cholerae and cholera phages, if any, in an environmental reservoir in Brazil, particularly in Rio de Janeiro. Samples were collected from selected points where sewage water connected with the main canal receives domestic and industrial effluents from the city on its way to the Atlantic Ocean. Two major environmental parameters, temperature and pH, were recorded at each station during collection. Samples were processed for phage isolation according to the procedure adopted for V. cholerae phages (5,6). Portions of the samples were also analyzed for enteric pathogens. A total of 32 sewage samples were collected in 8 months; 12 were isolated from Rio de Janeiro, 10 from the Amazon region in the North, and 10 from northeastern Brazil. Each sample was divided into two parts; one section was used to isolate O1 phages using MAK 757 (ATCC 51352) as a propagating strain. The other portion was used for non-O1 phages; the non-O1 V. cholerae strain was used for propagation starting from O2 to O139. The procedure was repeated on nutrient agar for the appearance of plaques. The phages were purified from a single discrete plaque by the soft agar (0.8%) overlay method with the propagating strain of V. cholerae non-O1 (O2 to O139) until homogenous plaques were obtained. Phage lysates were prepared in nutrient broth (Difco Laboratories, Detroit, MI) with the propagating strain V. cholerae (both O1 and non-O1). High-titer phages were obtained by the plate lysis procedure by using the agar overlay method (5) with multiplicity of infection 0.01 at an incubation temperature of 37°C. Concentration, purification, and electron microscopy study of these phages were performed as described by Ghosh et al. (1989) (7), using a FEI Tecnai 12 Bio Twin transmission electron microscope (FEI Europe BV, Eindhoven, Holland, the Netherlands). Measurements were made with Analysis (SIS GmbH, Munster, Germany) software. The homogeneity of each phage was studied by plaque morphology on nutrient agar following 10-fold serial dilution. Each high-titer phage (108–109 pfu/mL) was then used to determine routine test dilution (RTD) by the soft agar overlay method. In this experiment, the RTD used was the highest dilution that failed to give confluent or complete lysis. A variety of enteropathogens were included for susceptibility against these phages. A single colony from nutrient agar plate was injected into 5 mL of nutrient broth and incubated 2–3 h under stationary conditions at 37°C. A bacterial lawn was made on nutrient agar with this broth culture mixed with 3.5 mL of molten soft agar. The phage at RTD was spotted onto the plate and incubated at 37°C for 18 h. The next morning, the appearance of the zone of lysis was recorded. V. cholerae MAK 757 (ATCC 51352) was included as a positive control. Of the 32 samples examined, two non-O1 phages (O6 and O34) sensitive to V. cholerae O6 and O34 were isolated from the same site, the Sarapui River in Rio. The plaques of both the phages were observed as clear and round, with a diameter of 3 to 4 mm. A total of 107 strains of Vibrio and Enterobacteriaceae were tested against these two phages. All strains were untypeable, except for the O6 and O34 serotypes, which were lysed by the phages specific for O6 (DR1) and O34 (DR2). During the study period, the recorded temperature was 25°C–38oC, and pH ranged from 8 to 10. No correlation was observed with these two parameters and the isolation of phages. The morphology of the phages was studied by negative staining electron microscopy. The O34 phages have a hexagonal head with a long tail. The diameter (distance between opposite apices) of the head is 83.0 ± 0.3 nm, while the length and width of the tail are 111.0 ± 0.8 nm and 17.0 ± 0.5 nm, respectively (Figure 1). The O6 phage has a similar form, with a head diameter of 77.5 ± 0.3 nm and a long tail 100.0 ± 0.6 nm in length and 19.0 ± 0.4 nm in width (Figure 2). The other enteropathogens isolated from the sewage samples were enteropathogenic Escherichia coli, Salmonella spp., and Shigella spp. (data not shown). Figure 1 Electron micrograph of O34 vibriophage. Bar represents 100 nm. Figure 2 Electron micrograph of O6 vibriophage. Bar represents 100 nm. The use of vibriophages as tools for studying the source or origin of V. cholerae has contributed greatly to the understanding of the epidemiology of this disease. The emergence of V. cholerae O1 in South America in 1991 provoked major social and economic damage. A total of 60,000 clinical cases were reported from 1990 to 1996 in the coastal city of Rio de Janeiro. In 1997, approximately 2,600 confirmed cases of cholera were reported. In 1998, the number of cholera cases was 376. In 1999, the number of cases increased to 4,142; in 2000, the number decreased to 821 cases. This extreme variation in cholera cases continued during 2001, 2002, and 2003 (until February): the number of cholera cases reported were 665, 174, and 10, respectively. However, since 1993, no cholera cases caused by O1 have been reported. Only cases of non-O1 have been encountered, with O6 and O34 the predominant serotypes. A nationwide survey conducted by the National Reference Center for Cholera under Instituto Oswaldo Cruz is ongoing to isolate more phages in Brazil and neighboring countries. To date, serotyping is the only identification tool for the characterization of non-O1 strains of V. cholerae (8). However, serotyping is only performed at a limited number of laboratories. For this study, all isolates from Brazil were sent to laboratories outside the country for serotyping. This step was expensive and time-consuming and posed risks during transit. An alternative method is the use of phages for identifying non-O1 strains. This method offers an affordable monitoring system in less-developed countries such as Brazil. Phage O6 and O34 should at the least be useful for confirming the diagnosis of V. cholerae O6 and O34 infection and for differentiating V. cholerae O1 and non-O1 strains.

Background There is not much information on the differences in clinical, epidemiological and spatial characteristics of diarrhea due to V. cholerae and V. parahaemolyticus from non-coastal areas. We investigated the differences in clinical, epidemiological and spatial characteristics of the two Vibrio species in the urban slums of Kolkata, India. Methods The data of a cluster randomized cholera vaccine trial were used. We restricted the analysis to clusters assigned to placebo. Survival analysis of the time to the first episode was used to analyze risk factors for V. parahaemolyticus diarrhea or cholera. A spatial scan test was used to identify high risk areas for cholera and for V. parahaemolyticus diarrhea. Results In total, 54,519 people from the placebo clusters were assembled. The incidence of cholera (1.30/1000/year) was significantly higher than that of V. parahaemolyticus diarrhea (0.63/1000/year). Cholera incidence was inversely related to age, whereas the risk of V. parahaemolyticus diarrhea was age-independent. The seasonality of diarrhea due to the two Vibrio species was similar. Cholera was distinguished by a higher frequency of severe dehydration, and V. parahaemolyticus diarrhea was by abdominal pain. Hindus and those who live in household not using boiled or treated water were more likely to have V. parahaemolyticus diarrhea. Young age, low socioeconomic status, and living closer to a project healthcare facility were associated with an increased risk for cholera. The high risk area for cholera differed from the high risk area for V. parahaemolyticus diarrhea. Conclusion We report coexistence of the two vibrios in the slums of Kolkata. The two etiologies of diarrhea had a similar seasonality but had distinguishing clinical features. The risk factors and the high risk areas for the two diseases differ from one another suggesting different modes of transmission of these two pathogens.

The conventional phage typing scheme proposed by S. Basu and S. Mukerjee (Experientia 24:299-300, 1968) has been used routinely for identification of the strains at the Vibrio Phage Reference Laboratory since 1968. However, because of limitations of this scheme, a new phage typing scheme using five newly isolated phages was incorporated into the conventional scheme. A different definition of routine test dilution (almost confluent lysis) was found to be more useful than the one previously used (confluent lysis). The 1,000 strains tested could be clustered into 27 types with the five new phages. With the new scheme of 10 phages (5 new phages and 5 phages of Basu and Mukerjee), the 1,000 strains could be grouped into 146 types. The new phages were different from each other and also from those of Basu and Mukerjee, as revealed by lytic pattern, electron microscopy, restriction endonuclease digestion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antiphage antiserum studies. With the new typing scheme, 99.6% of the strains were typeable. Phage type 115 was the most common and includes 119 (11.9%) of the 1,000 strains tested. Next most common were phage types 142 (9.4%), 143 (7.0%), 104 and 116 (both 5.4%), 3 (5.3%), 5 (4.1%), 4 (3.9%), 24 (2.1%), and 100 (1.7%). The larger number of types would be useful for further classification of the strains for epidemiological purposes. This newly developed scheme is highly applicable to, and could be widely adopted for, phage typing of Vibrio cholerae O1 biotype El Tor strains.

AtypicalVibrio choleraeO1 strains – hybrid strains (strains that cannot be classified either as El Tor or classical biotype) and altered strains (El Tor biotype strains that produce classical cholera toxin) – are currently prevalent in Asia and Africa. A total of 74 hybrid and altered strains that harboured classical cholera toxin were investigated by multilocus variable-number tandem repeat analysis (MLVA). The results showed that the hybrid/altered strains could be categorized into three groups and that they were distant from the El Tor strain responsible for the seventh cholera pandemic. Hybrid/altered strains with a tandem repeat of the classical CTX prophage on the small chromosome were divided into two MLVA groups (group I: Mozambique/Bangladesh group; group III: Vietnam group), and altered strains with the RS1–CTX prophage containing the El Tor typerstRand classicalctxBon the large chromosome were placed in two MLVA groups (group II: India/Bangladesh group; group III: India/Vietnam group).

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.

Retrospective analysis led to the detection of twoVibrio choleraevariant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml−1) resistance, positivity in Voges–Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to KolkataV. choleraeO1 strains isolated on or before 1992.NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.

Cholera is an acute diarrhoeal disease caused by Vibrio cholerae, of the 206 serogroups of Vibrio cholerae only O1 and O139 which produce CTX are known to cause epidemics. New variants of Eltor biotype exhibiting characters of classical biotype has been reported from different counter parts of the world since 2002. The present study was conducted to determine biotypes, serotypes and phage types of Vibrio cholerae prevalent in Hyderabad and to determine the antibiotic susceptibility pattern of the isolates.

Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of EcoR1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.

We isolated Vibrio parahaemolyticus from plankton samples collected from different freshwater areas in the vicinity of Calcutta, India. Levels of this organism in association with freshwater plankton were low (less than 90 organisms per g). All of the 16 strains of V. parahaemolyticus isolated in this study were Kanagawa negative, and all except 1 were untypable. The occurrence of V. parahaemolyticus in association with freshwater plankton suggests that adsorption onto plankton may aid the survival of this moderately halophilic organism in freshwater milieus.

Repeated subculturing of Kanagawa-negative strains of Vibrio parahaemolyticus on Wagatsuma agar induced the production of a hemolysin which was not the thermostable direct hemolysin. Crude hemolysin exhibited a 30 to 40% lethal toxicity in mice after intraperitoneal injection. A 21-kilodalton protein band was observed with all the environmental isolates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results suggested that a certain percentage of environmental strains of V. parahaemolyticus is responsible for pathogenesis.

The seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes was studied in 1982 and 1983. The occurrence of this organism in water and sediments at the three sites studied was very infrequent and was restricted to the summer months, although it was not always isolated during these months. The association of V. parahaemolyticus with plankton was chiefly confined to the summer months and progressively declined with the onset of monsoons, remaining below detectable levels during the postmonsoon and winter months. The incidence and counts of V. parahaemolyticus were consistently higher in association with plankton than with water and sediment samples. V. parahaemolyticus could be recovered throughout the period of investigation from freshly caught and market samples of freshwater fishes. The highest recovery rate of this halophile from fishes was invariably from fecal samples. Most of the strains isolated in this study were untypable, and those which could be typed were predominantly serotypes encountered in the environment. All the isolates were Kanagawa negative. From this study, it could be concluded that the survival of V. parahaemolyticus in freshwater ecosystems is transient and dependent on a biological host.

Serological analysis of the O and K antigens was performed on 324 isolates of Vibrio parahaemolyticus obtained from three hydrobiologically dissimilar aquatic environments. Only 50.9% of the strains could be serotyped. The largest number of untypable strains and the lowest serological diversity were observed from the freshwater collection. Three serotypes, O2:K28, O5:K17, and O2:K3, dominated among all biotopes. There appears to be some distinction between serotypes of environmental and clinical origins.

The modified Elek test and Wagatsuma agar were compared for their ability to detect the Kanagawa activity of 142 strains of Vibrio parahaemolyticus. The performance of the modified Elek test was on a par with that of the Wagatsuma agar as far as positivity was concerned, and the test was far superior to Wagatsuma agar in eliminating doubtful results. The results of the modified Elek test were not unduly influenced by the different types of agar used.

Crude hemolysin from four KP+ strains of Vibrio parahaemolyticus belonging to serotype 02:K3 exhibited a major protein band (molecular weight, 65 kilodaltons (kDa)) in addition to a previously known thermostable direct hemolysin band (molecular weight, 21 kDa) in SDS – polyacrylamide slab gel electrophoresis. These strains showed maximum virulence leading to 100% mouse lethality within 2-6 h. It is hypothesized that this 65-kDa protein may play a vital role in the pathogenesis of the disease caused by V. parahaemolyticus.