Ogawa Serotype Research Articles

Phenotypic and genetic characterization of Vibrio cholerae O1 clinical isolates collected through national antimicrobial resistance surveillance network in Nepal

Cholera occurs in sporadic cases and outbreaks in Nepal each year. Vibrio cholerae O1 (n = 522) isolated during 2007-2010 from diarrheal patients at 10 different hospital laboratories in Nepal were characterized. Biochemical and serologic identifications showed that all the isolates belonged to serogroup O1, El Tor biotype. Except 72 isolates of Inaba serotype isolated in the year 2007, all the remaining isolates were of Ogawa serotype. All isolates were resistant to nalidixic acid and furazolidone. Resistance to tetracycline, ciprofloxacin, erythromycin and co-trimoxazole were 21, 4, 16 and 90 % respectively. Seventy-seven of these isolates were selected for further characterization for ctxB gene and MLVA typing. Two different variants of classical type cholera toxin were observed. Ogawa strains from 2007 and 2010-Western Nepal outbreak harbored CTX-3 type cholera toxin, whereas Inaba serotypes in 2007 and the remaining Ogawa serotypes in 2008-2010 harbored CTX 3b-type toxin. MLVA analysis showed circulation of four different groups of altered V. cholerae O1 El Tor strains. Two different profiles were seen among 2007 Inaba (9, 3, 6, x, x) and Ogawa (10, 7, 6, x, x) isolates. The MLVA profile of 2008 and 2009 Ogawa isolates were similar to those of Inaba strains of 2007. Isolates from 2010 also showed three different MLVA profiles; profile 9, 3, 6, x, x in 3 isolates, 11, 7, 6, x, x among 2010 Western Nepal outbreak strains and profile 8, 3, 6, x, x among isolates from Butwal and Kathmandu.

Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity

A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 109cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.

Cholera: Small outbreak in winter season of Eastern Nepal

Dear Editor, The global resurgence of cholera is becoming an increasingly important public-health challenge as the number of countries affected by this infection continues to increase.[1] The current cause of the global cholera pandemic, Vibrio cholerae O1 El Tor (including hybrid strains), has replaced classical biotype organisms around the globe, is associated with prolonged cholera outbreaks, is able to persist in aquatic reservoirs and cause endemic disease, is increasingly resistant to antimicrobials, and is associated with high case-fatality rates.[2] The first report of cholera was officially published in the years 1958-1960 in Kathmandu by a medical doctor visiting Nepal.[3] Other reports of cholera outbreaks in Nepal have been reported in literatures.[3–5] In year 2007, more than 50,000 people of the 54 Village Development Committee (VDC) s in the Saptari district of Eastern Nepal had been in effect of the diarrhea with death toll to 25.[6] After 4 years (November, 2011) in the same district, two people from Tilathi village lost their lives due to diarrheal illness. Upon the request of Ministry of Health, Nepal, a rapid response team comprising of microbiologists and epidemiologists from B.P Koirala Institute of Health Sciences was sent to elucidate the cause of the recent small outbreak of diarrheal illness. After a focus group discussion with the locals of affected village in presence of officials of District Public Health Office, Saptari, clinical and water samples were collected systematically from three wards with active cases of diarrheal illness. Five clinical samples from symptomatic individuals without any antibiotic administration and four water samples (pond and underground) were collected, transported, and investigated microbiologically following Centers for Disease Control Guidelines.[7,8] The causative agent of diarrheal illness was found to be V. cholerae, [O1: El Tor] Ogawa serotype, in three clinical and water samples each. Phenotypically, the isolates from the water samples (pond) were identical to the samples isolated from the patients. This finding suggests that the clinical isolates probably disseminated from pond water. The absence of organism in underground water keeps it safe from pathogenic organisms. In contrast to cotrimoxazole, nalidixic acid, and furazolidone; chloramphenicol and ceftriaxone are sensitive in all the isolates as depicted in Table 1. Ofloxacin, ampicillin, tetracycline, and ciprofloxacin are in decreasing order of susceptibilities. Similarity in the antibiogram among isolates A from clinical sample and E from water sample suggests the presence of same strain in both the samples, reflecting that the same strain of organism can persist in both water and humans. Dissimilar antibiogram among isolates from clinical sample suggests that the different circulating strains were responsible in causing this small outbreak. Dissimilar antibiotic sensitivity pattern among clinical isolates and environmental isolates may have been influenced by host factor, microorganism factor, and environmental factor. Extensive characterization of these strains would definitely bring some conclusion on factors responsible for genotypic variation of isolates within the same epidemic and its probable role in bacterial virulence. Table 1 Antimicrobial susceptibility pattern of Vibrio cholera isolates The villagers had recently celebrated one of their important festivals, chhath; the rituals of which are performed in ponds. Open defecation habit and use of pond water as a major source of water for drinking, cooking, and bathing had a foremost role in the cholera outbreak. The people seemed to be aware of the spread of diarrheal illnesses but were not motivated to use their toilets indeed. Numerous studies have shown that V. cholerae belong to a group of organisms whose major habitats are aquatic ecosystems, and the role of water sources in the spread of cholera has been well documented with a suggestion that environmental concentration of Vibrio species might increase in response to zooplankton or phytoplankton blooms driven by global or local aquatic conditions.[9] Three-dimensional biofilm on surfaces of V. cholerae O1 El Tor and O139 facilitates its persistence within natural aquatic habitats during interepidemic periods,[10] which might be the reason in current case too. This report of a small cholera outbreak occurred in non-summer season. From an applied perspective, clarifying the mechanisms that link seasonal environmental changes to diseases’ dynamics will aid in developing strategies for controlling diseases and improving disease forecast across a range of human and natural systems. We would like to recommend that exploration of presence of these organisms along the year and their molecular epidemiological typing would definitely benefit in tracing the method of spread of cholera in the area. This would also review the role of environmental factors responsible for cholera spread. We would like to acknowledge the support of Prof. SK Bhattacharya, Dr. Rajendra Gurung, Dr. Nimesh Poudyal, Dr. Ratna Baral, Dr. Narayan Raj Bhattarai, and Mr. Tejndra Pandit for their immense support and encouragement. We are greatful to officers of District Public Health Office, Rajbiraj, Nepal.

Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007–2009 outbreak

Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007-2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V.cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V.cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V.cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V.cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V.cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007-2009).

Safety and immunogenicity study of a killed bivalent (O1 and O139) whole-cell oral cholera vaccine Shanchol, in Bangladeshi adults and children as young as 1 year of age

Safety and immunogenicity study of an oral, killed, bivalent whole-cell, cholera vaccine, Shanchol was carried out in Bangladeshi participants. This study was conducted prior to initiating a feasibility study in Bangladesh. The double-blind, randomized placebo controlled study was carried out in adults (18-45 years), toddlers (2-5 years) and younger children (12-23 months). Two doses of the vaccine/placebo were given 14 days apart. Shanchol did not elicit major adverse events in any age group. Vibriocidal antibody responses in adults were 60% against Vibrio cholerae O1 Inaba, 72% against V. cholerae O1 Ogawa and 21% against V. cholerae O139. In toddlers, responses were 84%, 75% and 64% and in younger children it was 74%, 78% and 54% against Inaba, Ogawa and O139 serotypes. The responses in all ages were higher in vaccinees compared to pre-immune titers or to responses in placebo recipients (P<0.001). Plasma IgA antibody response to O1 Inaba LPS was seen in 61%, 73% and 45% of adults, toddlers and younger children, respectively. The safety and immunogenicity data for Shanchol is promising and warrants future use in large scale trial in cholera endemic areas, high risk Bangladeshi population and in other countries in the region.

Construction of novel vaccine strains of Vibrio cholerae co-expressing the Inaba and Ogawa serotype antigens

The approach of inducing protective immunity against cholera by oral vaccination with killed whole Vibrio cholerae cells is effective, but the complexity of current cholera vaccines makes them difficult and relatively expensive to manufacture, especially if recombinant cholera toxin B subunit is included in the formulation. In an effort to simplify the composition of a new generation of oral cholera vaccines we have generated a novel non-toxigenic candidate vaccine strain of V. cholerae O1 that stably expresses both the Ogawa and Inaba serotype antigens on its surface. This was done by introducing a functional wbeT gene without a functional promoter into the chromosome of an O1 Inaba strain. The resulting low levels of expression of the wbeT gene product allowed for the desired partial serotype switching. This strain (MS1342) can potentially replace the three virulent strains used in currently manufactured cholera vaccines. Oral immunization of mice with formalin-killed MS1342 bacteria gave rise to Ogawa-specific, Inaba-specific and cross-reactive serum antibodies that were detectable both by lipopolysaccharide (LPS)-specific ELISAs and as vibriocidal antibodies that are considered to predict protective efficacy. These responses as well as intestinal mucosal IgA anti-LPS antibody responses were fully comparable with those obtained by immunization with the internationally licensed oral cholera vaccine Dukoral®. We propose that such a strain may form the basis of a single strain killed whole cell cholera vaccine protecting against cholera caused by either the Inaba or Ogawa serotype of V. cholerae O1.

Cholera: a great global concern

Cholera, caused by the infection of toxigenic Vibrio cholerae (V. cholerae) to humans, is a life threatening diarrheal disease with epidemic and pandemic potential. The V. cholerae, both O1 and O139 serogroups, produce a potent enterotoxin (cholera toxin) responsible for the lethal symptoms of the disease. The O1 serogroup has two biotypes (phenotypes), classical and El Tor; each of which has two major serotypes (based on antigenic responses), Ogawa and Inaba and the extremely rare Hikojima. V. cholerae O1 strains interconvert and switch between the Ogawa and Inaba serotypes. Fluid and electrolyte replacement is the mainstay of treatment of cholera patients; the severe cases require antibiotic treatment to reduce the duration of illness and replacement of fluid intake. The antibiotic therapy currently has faced difficulties due to the rapid emergence and spread of multidrug resistant V. cholerae causing several outbreaks in the globe. Currently, cholera has been becoming endemic in an increasing number of geographical areas, reflecting a failure in implementation of control measures. However, the current safe oral vaccines lower the number of resistant infections and could thus represent an effective intervention measure to control antibiotic resistance in cholera. Overall, the priorities for cholera control remain public health interventions through improved drinking water, sanitation, surveillance and access to health care facilities, and further development of safe, effective and appropriate vaccines. Thus, this review describes the facts and phenomena related to the disease cholera, which is still a great threat mainly to the developing countries, and hence a grave global concern too.

Vibrio cholerae O1 Ogawa serotype outbreak in a village of Ambala district in Haryana, India

Vibrio cholerae O1 Ogawa serotype outbreak in a village of Ambala district in Haryana, India

Comparison of clinical features and immunological parameters of patients with dehydrating diarrhoea infected with Inaba or Ogawa serotypes of Vibrio cholerae O1

Vibrio cholerae O1, Ogawa and Inaba serotypes, both cause severe cholera. We compared clinical and immunological features in patients in Bangladesh infected with these 2 serotypes. Blood was collected from hospitalized Ogawa (N=146) or Inaba (N=191) patients at the acute stage (day 2) and 5 and 19 days later. Ogawa patients were younger than Inaba, presented with shorter duration of diarrhoea, and had more frequent abdominal pain, vomiting and need for intravenous fluids (p<0.05). Inaba patients more frequently had dark-field positive stools (p<0.01). Inaba strains were more susceptible to tetracycline and erythromycin than Ogawa strains (p<0.001). Ogawa infection produced higher plasma vibriocidal as well as IgG responses to cholera toxin B subunit, toxin-coregulated pilus subunit and lipopolysaccharide (LPS); higher IgA responses to LPS in 'antibody in lymphocyte supernatant' (ALS) specimens were also seen. These results suggest that a cholera vaccine based on the Ogawa serotype needs to be further investigated.

Hybrid El TorVibrio choleraeO1, Kuwait

To the Editor: The traditional causative agent of cholera, Vibrio cholerae O1, has 2 biotypes, classical and El Tor. The current seventh pandemic that began in 1961 and has spread to much of the world is caused by the El Tor biotype. This biotype has replaced the classical biotype responsible for the previous pandemics. The classical and El Tor biotypes are differentiated by phenotypic tests (1), and several nucleotide base differences occur at positions 115 and 203 in the ctxB gene (C in both positions in the classical and T in both positions in the El Tor biotype). These differences translate to histidine at amino acid position 39 and threonine at amino acid position 68 for the B subunit of cholera toxin (CT) in the classical biotype and tyrosine and isoleucine, respectively, for the corresponding amino acids in the El Tor biotype (2). Recently, 3 variants of the El Tor biotype have been found. These are the Matlab variants, which could not be biotyped because they have a mixture of classical and El Tor traits (1); the Mozambique variant, which has a typical El Tor genome but a tandem repeat of the classical CTX prophage located in the small chromosome (3); and the hybrid El Tor variant, which has a typical El Tor biotype and an El Tor CTX prophage but produces CT of the classical type (4). This hybrid El Tor variant has replaced the El Tor biotype in Dhaka and other parts of Bangladesh (4) and in India (5), Japan, Hong Kong, Zambia, the People’s Republic of China, Sri Lanka, and Vietnam (6). Kuwait was affected by cholera in the mid 1960s during the current seventh pandemic. Subsequently, cholera disappeared from Kuwait as living standards improved. Screening of ≈5,000 acute-phase diarrheal stool samples in the mid-1980s in a major hospital in Kuwait did not yield V. cholerae O1 (7). The occasional cholera cases detected in Kuwait are imported, mainly from Asia through expatriate workers. Two adult men, one who had just arrived from India and one who had just arrived from the Philippines, were admitted with severe watery diarrhea, vomiting, and dehydration to the Al-Adan Hospital, Kuwait, in November and December 2008, respectively. The patient from India reported eating in restaurants, and the patient from the Philippines had consumed fish soup just before the journey. Both patients were initially rehydrated with intravenous fluids. Stool cultures were performed by using a battery of media, including thiosulfate citrate bile salt sucrose (TCBS) agar. Yellow colonies from TCBS agar were tested by using MicroScan WalkAway 96 (Dade Behring, West Sacramento, CA, USA) panel NBPC 34 and API 20E strip (bioMerieux, Marcy l’Etoile, France), which suggested V. cholerae. In slide agglutination tests, the colonies agglutinated with V. cholerae O1 polyvalent antiserum and Ogawa serotype antiserum (Denka-Seiken, Tokyo, Japan). The isolate from the patient from India was susceptible to tetracycline and ampicillin and resistant to co-trimoxazole, but the isolate from the patient from the Philippines was susceptible to all 3 of these antimicrobial agents in MicroScan (Dade Behring) and disk diffusion tests. Both patients were successfully treated with rehydration therapy and intravenous vibramycin (a semisynthetic tetracycline). Both V. cholerae O1 Ogawa isolates showed positive results in Vogues-Proskauer, chicken cell agglutination, and tube hemolysin tests and were resistant to polymyxin B (50 international units), results that suggest the El Tor biotype (1). Both isolates were positive for the ctxA gene and El Tor–specific tcp gene but negative for classical-specific tcp gene by PCR (8). The genotype of the ctxB gene was determined by a mismatch amplification mutation assay PCR that detects polymorphism at nucleotide position 203; both isolates yielded a 186-bp amplicon with the classical biotype-specific primers and no amplicon with the El Tor biotype-specific primers (9). The classical ctxB genotype of the isolates was further confirmed by sequencing the ctxB gene by using the BigDye termination method (Applied Biosystems, Foster City, CA, USA) with specific primers (10). This sequencing showed that both isolates had histidine at position 39 and threonine at position 68 in CT-B subunit. Thus, the isolates are hybrid variants that are phenotypically El Tor but genotypically classical for the ctxB gene. Our findings suggest that cholera caused by the hybrid variant is present in the Philippines. The 2 cholera cases reported here were imported into Kuwait by travelers from cholera-endemic regions and were not endemic illnesses. The hybrid variant could possibly initiate the next cholera pandemic. Also, because CT is related to the major clinical sign of the disease, genetic changes in the molecule could result in alteration in the manifestation of the disease. The changes could also influence the effectiveness of cholera vaccines that contain CT-B as a component (6).

A duplex vibriocidal assay to simultaneously measure bactericidal antibody titers against Vibrio cholerae O1 Inaba and Ogawa serotypes

Currently available cholera vaccines are formulated with killed-whole cells of Vibrio cholerae O1 Inaba and Ogawa serotypes. A serum vibriocidal assay has been widely used to evaluate the immunogenicity of cholera vaccines in clinical trials. In this study, we developed a duplex vibriocidal assay to obtain vibriocidal antibody titers against both serotypes simultaneously. Initially, serial dilutions of serum from vaccinees were incubated with guinea pig complements along with both streptomycin-resistant Inaba and ampicillin-resistant Ogawa strains for 1 h. The mixture was then inoculated on separate agar plates containing each antibiotic to selectively culture each corresponding serotype and incubated at 37 °C for 16 to 20 h. Bacterial colonies were enumerated using an automated colony counting system to obtain the vibriocidal antibody titers defined by the reciprocal of serum dilution inhibiting bacterial growth by 50%. Performance of the duplex vibriocidal assay was examined by comparison with a single serotype vibriocidal assay using 20 clinical sera consisting of ten-paired sera prepared at pre- and post-vaccination. Both assays showed a good correlation for vibriocidal titers against the two serotypes, respectively, as determined by Pearson correlation coefficient ( r) and regression coefficient ( β) analyses; r = 0.998 , β = 1.003 for Inaba and r = 0.997, β = 0.999 for Ogawa, respectively. The duplex vibriocidal assay can diminish the amount of sera required for the assay and enhance assay efficiency in terms of time, labor intensity, and expenditure.

Characterization of a novel protective monoclonal antibody that recognizes an epitope common to Vibrio cholerae Ogawa and Inaba serotypes.

A novel protective monoclonal antibody (mAb) that recognizes a lipopolysaccharide (LPS) epitope common between serotypes Ogawa and Inaba of the O1 serogroup of Vibrio cholerae was characterized and the potential to develop peptide mimics of this protective LPS epitope was investigated. mAb 72.1 recognizes both Ogawa and Inaba LPS and it is vibriocidal and protective in passive immunization against infection by strains of both serotypes. The cDNA-derived amino acid sequence of mAb 72.1 is closely related to the previously characterized mAb ZAC-3, which is thought to recognize an epitope in the lipid A core region of O1 LPS. In an attempt to develop a peptide mimic-based vaccine against V. cholerae, phage display libraries were screened with mAb 72.1 and 11 peptide mimics were identified. Remarkably, all of the peptide sequences identified from linear phage display libraries contained two cysteine residues, suggesting that mAb 72.1 preferentially binds to peptides constrained with a disulphide bond. One of the peptide mimics was immunologically characterized. Although immunization of mice with this peptide mimic conjugated to KLH elicited antibodies against the peptide itself, these antibodies did not cross-react with Ogawa or Inaba LPS. Effectiveness of a peptide mimic as a vaccine may depend on how well the peptide can mimic the carbohydrate interactions when binding to the anti-carbohydrate antibody. Thus, investigating how peptides and LPS bind to mAb 72.1 may be useful in improving current peptide mimics or designing more effective peptide mimics. Identification and characterization of novel protective anti-LPS antibodies may be useful in studying protective epitopes of LPS, which may help develop LPS-based therapeutics against V. cholerae.

Molecular characterization of Vibrio cholerae isolates from cholera outbreaks in north India

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V. cholerae isolates carried the virulence-associated genes like ctxA, tcpA, ace, and zot. Molecular typing by repetitive sequence based ERIC, VCR1, and VC1 PCR's revealed similar DNA profile for both Inaba and Ogawa serotypes. A discrete VC1-PCR band identified among the El Tor strains had greater similarity (>97%) to the V. cholerae genome sequence and therefore has the potential to be used as a marker for the identification of the V. cholerae strains. Non-O1 strains recovered from Bhind region differed among themselves as well as from that of the O1 isolates. All the O1 serogroup isolates possessed SXT element and were uniformly resistant to the antibiotics nalidixic acid, polymyxin-B, furazolidone, cloxacilin, trimethoprim-sulfamethaxazole, and vibriostatic agent 0129. Inaba strains from both Delhi and Bhind differed from Ogawa strains by their resistance to streptomycin despite sharing similar DNA patterns in all the three rep-PCRs. Though Delhi and Bhind are separate geographical regions in Northern India, Inaba strains from both these places appear to be closely related owing to their similarity in antibiogram and genetic profile.

Isolation of Vibrio cholerae El Tor Serotype Inaba in 2006 and the most common phage type of V. cholerae in Mumbai

Sir, A total of 168 strains of Vibrio cholerae were isolated and tested over a period of 3 years (2004-2006). The strains were identified by standard methods(1) and were identified using a slide agglutination test with Vibrio cholerae O1 antisera and biotyped using a polymyxin B sensitivity test. The non-agglutinating strains were tested with V. cholerae O-139 antisera. Of 168 strains, 96 were isolated in 2004, 39 were isolated in 2005, and 33 were isolated in 2006. Eighty-three V. cholerae O1 isolates were sent to the National Institute of Cholera and Enteric Diseases (NICED) in Kolkata for serotyping and phagetyping. All the V. cholerae O1 isolates were of El Tor biotype. Among the two non-agglutinating vibrios isolated in 2005, both did not agglutinate with O-139 antisera. V. cholerae El Tor serotype Inaba was found only in 2006. The isolates of 2004 and 2005 were of the Ogawa serotype. In a previous study in the same institute, all isolates detected over a period of 5 years (1996-2000) were of the Ogawa serotype.(2) From 2001 to 2005, all isolates were V. cholerae El Tor Ogawa (unpublished). We had isolated serotype Inaba in 2006 for the first time. A shift in the occurrence of Ogawa and Inaba serotypes in a given area are thought to be a consequence of the genetic reversal that occurs in-vivo and in-vitro and is possibly mediated by the immune pressure in the population.(3) It appears that as an alternate to the Ogawa serotype, Inaba have appeared to aid the persistence of cholera and thus perpetuate the spread of Vibrio cholerae El Tor. All the isolates during 2004 to 2006 were Basu and Mukherjee phage type 2. Turbadkar et al.(4) from Mumbai have reported Ogawa serotypes in 2004 and all belonged to phage type 4. In the new phage typing scheme, out of 26 isolates sent to NICED in 2004, 14 were T26 and 12 were T27. Out of 39 isolates sent in 2005, 33 were T27 and 6 were other phage types (T13, T21, T22, T25, T23, or T15). In 2006, out of 18 isolates sent to NICED, 17 were T27 and only 1 was T26. Therefore, the most common phage type in this part of Mumbai is T27 (74.7%), followed by T26 (18.1%). In the study by Turbadkar et al.,(4) the majority belonged to phage type 27 (97.5%), which is in accordance with the present study. The 168 isolates of V.cholerae showed maximum sensitivity to amikacin (92.3%), followed by cefotaxime (89.9%). Tetracycline sensitivity was 91.1% followed by norfloxacin (86.3%). In the previous study during 1996-2000, tetracycline sensitivity was 39.6% and norfloxacin sensitivity was 46.2%.(2) Therefore, tetracycline and norfloxacin sensitivity have increased over the years. Co-trimoxazole and nalidixic acid susceptibility was only 2.4% and 1.2%, respectively in the present study. A decrease in sensitivity to nalidixic acid was observed in the present study (13.6% in previous study).(2)

Characterization of Diversity in wbeT Region of the O-Antigen Biosynthesis Gene Cluster in Ogawa and Inaba Serotypes of Vibrio cholerae

Characterization of Diversity in wbeT Region of the O-Antigen Biosynthesis Gene Cluster in Ogawa and Inaba Serotypes of Vibrio cholerae

Negative Matrix-Assisted Laser Desorption/Ionization Time-of-Flight/Time-of-Flight Tandem Mass Spectrometry Fragmentation of Synthetic Analogs of the O-Specific Polysaccharide of Vibrio Cholerae O:1 in the Presence of Anionic Dopants

Oligosaccharides (tri- to hexamers) that represent terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba, have been studied by negative matrix-assisted laser desorption/ionization time of flight/time-of-flight mass spectrometry (MALDI ToF/ToF MS). The [M - H(+)](-) ions are formed after expulsion of a proton from molecules studied under condition of MALDI MS analysis in the negative mode. Several ammonium salts (chloride, nitrate, hydrogencarbonate and hydrogensulfate) were used as additives to increase the formation of negative ions from saccharides. The most efficient was the addition of ammonium hydrogencarbonate, which increased the amount of [M - H(+)](-) ions more than six times. Between three fragmentation pathways, the new conjugated transfer of electrons within the second downstream unit of oligosaccharides was discovered. Production of these ions, which has not been observed in any other kinds of measurement, distinguishes substances belonging to Ogawa and Inaba serotypes. The negative MALDI ToF/ToF mass spectra are simpler and, at the same time, more informative, as compared with positive and negative electrospray ionization ion trap as well as with positive MALDI ToF/ToF analysis.

Crystal structure of a monoclonal antibody directed against an antigenic determinant common to Ogawa and Inaba serotypes of Vibrio cholerae O1

Crystal structure of a monoclonal antibody directed against an antigenic determinant common to Ogawa and Inaba serotypes of <i>Vibrio cholerae</i> O1

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.

B‐Cell Responses to Lipopolysaccharide Epitopes: Who Sees What First – Does It Matter?

Cholera is the paradigm for waterborne bacterial diseases. For over a 100 years, scientists have tried to develop a universally effective vaccine for cholera. We are hampered in our efforts because we do not know the details of the basic immune response to Vibrio cholerae antigens. What are the most proactive antigens? What special needs for immunization are engendered by previous exposure to cholera or the age of the individual? How long does immunity last, and is this immunity a classic immunologic memory or re-exposure and continual boosting? Immunization with synthetic derivatives of the carbohydrate moieties of V. cholerae lipopolysaccharide (LPS) coupled to different carrier proteins (neoglycoconjugates, NGC) has allowed dissection of the response to the disaccharide array of perosamine that represent either the Inaba or the Ogawa serotype. Studying serum anti-LPS endpoint titers and the serum vibriocidal response to NGC provides insight into the importance of LPS serotype-specific B-cell epitopes and how antibody response are influenced by the form of the LPS immunogen. We found that murine serum antibody responses to V. cholerae LPS are dynamic. The magnitude of serum anti-LPS antibody titers and the capacity to induced vibriocidal antibodies (immunoglobulin M) are influenced by the initial immunizing serotype of LPS, the structure of the LPS immunogen (native LPS versus NGC), and the order of serotype immunization in a prime boost immunization strategy. The dynamic of the immune response to LPS immunogens is typified by the fact that the host species can affect the immunization response. We found mice do not make vibriocidal antibody to Inaba NGC but rabbits do. This is in contrast to the Ogawa NGC that induced vibriocidal antibody in mice. The results suggest that the host's B-cell repertoire can influence the immunization efficacy; therefore, the development of the new generation of NGC V. cholerae vaccines should focus on human volunteers and their ability to mount protective responses.

Positive-Ion Fragmentation in Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry of Synthetic Analogs of the O-Specific Polysaccharide of Vibrio Cholerae O:1.

Saccharides (mono- to hexamers) that mimic the terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba have been studied by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI ToF/ToF) mass spectrometry (MS). Cationized adducts are the characteristic ions formed through the capture of sodium or potassium cations under MALDI MS conditions. Three characteristic pathways dominate in the fragmentation of model substances under MALDI ToF/ToF collision-induced dissociation MS/MS conditions of measurement. The first is the elimination which shortens the length of the oligosaccharide. In this way, conversion of the Ogawa to Inaba fragments takes place under the conditions of measurement. In the second, the conjugated transfer of electrons in the upstream unit of oligosaccharides takes place. The third route brings about the elimination of one alpha-hydroxy-gamma-butyrolactone molecule from the 4-(3-deoxy-L-glycero-tetronamido) group. The MALDI ToF/ToF MS/MS provided sufficient information about molecular mass, the number of saccharide residues, the structure of saccharides, the C(4)- amide of 3-deoxy-L-glycero-tetronic acid (DGT) of the compounds investigated, and allows to distinguish between Ogawa and Inaba serotypes.