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Rotavirus is the leading cause of severe dehydrating gastroenteritis among children younger than 5 years in low-income and middle-income countries. Two vaccines-Rotavac and Rotasiil-are used in routine immunisation in India. The safety and immunogenicity of these vaccines administered in a mixed regimen is not documented. We therefore aimed to compare the safety and seroresponse of recipients of a mixed regimen versus a single regimen.We did a multicentre, open-label, randomised, controlled, phase 4, non-inferiority trial at two sites in India. We recruited healthy infants aged 6-8 weeks. Infants with systemic disorders, weight-for-height Z scores of less than minus three SDs, or a history of persistent diarrhoea were excluded. Eligible infants were randomly allocated to six groups in equal numbers to receive either the single vaccine regimen (ie, Rotavac-Rotavac-Rotavac [group 1] or Rotasiil-Rotasiil-Rotasiil [group 2]) or the mixed vaccine regimen (ie, Rotavac-Rotasiil-Rotavac [group 3], Rotasiil-Rotavac-Rotasiil [group 4], Rotavac-Rotasiil-Rotasiil [group 5], or Rotasiil-Rotavac-Rotavac [group 6]). Randomisation was done using an online software by site in blocks of at least 12. The primary outcome was seroresponse to rotavirus vaccine, measured using rotavirus-specific serum IgA antibodies 4 weeks after the third dose. The seroresponse rates were compared between recipients of the four mixed vaccine regimens (consisting of various combinations of Rotavac and Rotasiil) with recipients of the single vaccine regimens (consisting of Rotavac or Rotasiil only for all three doses). The non-inferiority margin was set at 10%. Safety follow-ups were done for the duration of study participation. This trial was registered with the Clinical Trials Registry India, number CTRI/2018/08/015317.Between March 25, 2019, and Jan 15, 2020, a total of 1979 eligible infants were randomly assigned to receive a single vaccine regimen (n=659; 329 in group 1 and 330 in group 2) or a mixed vaccine regimen (n=1320; 329 each in groups 3 and 4, and 331 each in groups 5 and 6). All eligible participants received the first dose, 1925 (97·3%) of 1979 received the second dose, and 1894 (95·7%) received all three doses of vaccine. 1852 (93·6%) of 1979 participants completed the follow-up. The immunogenicity analysis consisted of 1839 infants (1238 [67·3%] in the mixed vaccine regimen and 601 [32·7%] in the single vaccine regimen; 13 samples were insufficient in quantity) who completed vaccination and provided post-vaccination sera. The seroresponse rate in the mixed vaccine regimen group (33·5% [95% CI 30·9-36·2]) was non-inferior compared with the single vaccine regimen group (29·6% [26·1-33·4]); the seroresponse rate difference was 3·9% (95% CI -0·7 to 8·3). The proportion of participants with any type of solicited adverse events was 90·9% (95% CI 88·4-93·0) in the single vaccine regimen group and 91·1% (89·5-92·6) in the mixed vaccine regimen group. No vaccine-related serious adverse events or intussusception were reported during the study.Rotavac and Rotasiil can be safely used in an interchangeable manner for routine immunisation since the seroresponse was non-inferior in the mixed vaccine regimen compared with the single vaccine regimen. These results allow for flexibility in administering the vaccines, helping to overcome vaccine shortages and supply chain issues, and targeting migrant populations easily.Ministry of Health and Family Welfare, Government of India.For the Hindi translation of the abstract see Supplementary Materials section.

Cholera remains a major contributor of diarrheal diseases and leads to substantial morbidity and mortality, particularly in low socioeconomic settings. Nonavailability of a national cholera control plan in India, compounded by underreporting of cholera cases and deficient accurate cholera hotspot estimates, has made cholera control a challenge. Obstacles in the programmatic introduction of oral cholera vaccine (OCV) lie within the infrastructure—stockpile, costing, distribution system, cold-chain mechanism, vaccine logistics, and lack of strengthened surveillance systems for adverse events following immunization. Sustained political commitment along with collaboration of people working in the media will also determine the policy outcome of OCV introduction in India.

Background\udNeonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs.\udMethods\udThe Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality.\udFindings\udBetween Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69–234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04–74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37–2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life.\udInterpretation\udFindings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs.\udFunding\udBill & Melinda Gates Foundation.

The serogroups O1 and O139 of the marine bacterium Vibrio cholerae are responsible for causing cholera in humans. The pentose sugar arabinose is nonmetabolizable by the pathogen and is present in environmental niches as well as in the human intestine. In this study, arabinose-mediated V. cholerae growth interference was assessed in M9 minimal medium containing gluconate as the sole carbon source in the light of Entner-Doudoroff (ED) pathway, an obligatory metabolic route for gluconate utilization. V. cholerae O1 and O139 strains failed to grow in the presence of ≥ 0.3% arabinose in M9 with 0.2% gluconate, but there was no growth inhibition in the presence of arabinose in M9 with 0.2% glucose. Transcriptional analysis of edd and eda, the genes constituting the ED pathway, showed ~100- and ~17-fold increases, respectively, in M9-gluconate. Minor increases of ~4- and ~2-fold for edd and eda, respectively, were noted in AKI medium supplemented with 0.5% arabinose. The observed arabinose-mediated growth inhibition can contribute toward deepening the understanding of altered phenotypes, if any, via complementation/expression studies in V. cholerae with pBAD vectors and arabinose as an inducer.

Background Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin–gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. Methods In BARNARDS, consenting mother–neonates aged 0–60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic–pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. Findings Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin–gentamicin, ceftazidime–amikacin, piperacillin–tazobactam–amikacin, and amoxicillin clavulanate–amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime–amikacin than for neonates treated with ampicillin–gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14–0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin–gentamicin; 286 (73·3%) to amoxicillin clavulanate–amikacin; 301 (77·2%) to ceftazidime–amikacin; and 312 (80·0%) to piperacillin–tazobactam–amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin–gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate–amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime–amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin–tazobactam–amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis. Interpretation Our data raise questions about the empirical use of combined ampicillin–gentamicin for neonatal sepsis in LMICs because of its high resistance and high rates of frequency of resistance and low probability of target attainment. Accessibility and affordability need to be considered when advocating antibiotic treatments with variance in economic health structures across LMICs. Funding The Bill & Melinda Gates Foundation.

Purpose. Two natural epidemic biotypes of Vibrio cholerae O1, classical and El Tor, exhibit different patterns of sensitivity against the antimicrobial peptide polymyxin B. This difference in sensitivity has been one of the major markers in biotype classification system for several decades. A recent report regarding the emergence of polymyxin B-sensitive El Tor V. cholerae O1 in Kolkata has motivated us to track the spread of the strains containing this important trait, along with Haitian-like genetic content, in different parts of India. Methodology. We have collected 260 clinical V. cholerae O1 strains from 12 states in India and screened them for polymyxin B susceptibility. Genetic characterization was also performed to study the tcpA, ctxB and rtxA genotypes by allele-specific polymerase chain reaction (PCR) and nucleotide sequencing. Results. Interestingly, 88.85 % of the isolates were found to be sensitive to polymyxin B. All of the states, with the exception of Assam, had polymyxin B-sensitive V. cholerae strains and complete replacement with this strain was found in eight of the states. However, from 2016 onwards, all the strains tested showed sensitivity to polymyxin B. Allele-specific PCR and sequencing confirmed that all strains possessed Haitian-like genetic traits. Conclusion. Polymyxin B-sensitive strains have begun to spread throughout India and may lead to the revision of the biotype classification. The dissemination of these new variant strains needs to be carefully monitored in different endemic populations through active holistic surveillance to understand their clinical and epidemiological consequences.

We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

Culture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae. Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as ‘no known aetiology’. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower Ct values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated.

Orthologs search identified that the Vibrio cholerae gluconate (Gnt) utilization system minimally consisted of the Entner-Doudoroff (ED) pathway (edd and eda) and three other genes, namely gntU, gntK and gntR This system appeared unique by genomic organization of component genes into two operons transcribed in opposite directions. In silico analysis indicated GntU as an inner-membrane protein functioning for transport and GntK as a kinase with cytosolic localization that generates Gnt6P, which is then metabolized through the ED pathway. Enzyme 6-phosphogluconate dehydratase encoded by edd converts Gnt6P to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is metabolized by the action of KDPG-aldolase encoded by eda Transcriptional upregulation of the Gnt utilization genes in the gntR mutant matched well to a predicted repressor role of GntR. GntR displayed DNA binding to a region in the promoters of two bi-directionally transcribed operons. Growth defect of mutants in Gnt-supplemented media confirmed obligate involvement of these genes in Gnt utilization and such defect was restored upon complementation. Defective Gnt utilization resulted in attenuation of colonization potential and reduction of cholera toxin secretion in V. cholerae The ED pathway mutants showed the highest level of virulence attenuation. Overall, this study established a minimal requirement of the V. cholerae Gnt utilization system, which played a critical role in pathogenesis.

Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline (75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).

A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .

Developing countries are burdened with Shigella diarrhea. Understanding mucosal immune responses associated with natural Shigella infection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens, p an- S higella s urface p rotein antigen 1 (PSSP1) and PSSP2, common to all virulent Shigella strains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α 4 β 7 + ) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive for Shigella infection by microbiological and real-time PCR assays, respectively. The frequency of α 4 β 7 + IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α 4 β 7 + ASC responses to Ipas may be considered an indirect marker of Shigella infection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by natural Shigella infection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude.

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae , the causative agent of cholera. In this study, activation of the genes edd and eda , encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae . Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes ( ctxA and tcpA ) and their regulator ( toxT ). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δ edd mutation [Δ edd N16961 strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

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

Objectives: In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. Methods: The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. Results: Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. Conclusions: This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages.

A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men aged between 16 and 50 years from Kolkata, India. A dose of 5 x 10(9)CFU (n=186) or a placebo (n=116) containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal antibody response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively. In a subgroup, anti-CT antibody rose (or =2-folds) in 23/30 (77%) and 6/19 (32%) in vaccine and placebo recipients, respectively. These studies demonstrate that VA1.3 at a dose of 5 x 10(9) is safe and immunogenic in adults from a cholera endemic region.

The incidence of Vibrio cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% ( n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher colonization in an animal model compared to those lacking tcpA . PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA , rtxA , Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.

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 chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N -acetyl- d -glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 μM. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-κB nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae .

Two genes of temperate mycobacteriophage L5, namely, gp63 and gp64, were hypothesized to be toxic to M. smegmatis. An identical L5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage L1 and characterized at length here. As expected, hlg1 affected the growth of M. smegmatis when overexpressed from a resident plasmid. HLG1 (the protein encoded by hlg1) in fact caused growth retardation of M. smegmatis and the region encompassing its 57-114 C-terminal amino acid residues was found indispensable for its growth-retardation activity. Both nucleic acid and protein biosynthesis were severely impaired in M. smegmatis expressing HLG1. Interestingly, HLG1 also affected E. coli almost similarly. This putative delayed early lipoprotein did not participate in the lytic growth of L1.

Summary Objectives To study the phenotypic and genotypic characterization of newly emerged V. cholerae O1, Inaba strains isolated from patients with diarrhoea. Methods Bacterial characterization was made using polymerase chain reaction, ribotyping, PFGE and RFLP. Results After its first appearance in July 2004, O1 Inaba became the dominant serotype by March 2005 and totally replaced the former dominant serotype, Ogawa from May 2005. Most of the Inaba isolates belong to a new ribotype RIV. Ogawa and also some Inaba strains isolated during the same period were identified as RIII. Similarly, the majority of the Inaba isolates belong to ‘H1' pulsotype and one isolate is type ‘H', while the Ogawa isolates were mostly ‘H' pulsotype. Presence of CTX prophage was detected in a single site of the chromosome with at least two RS elements. Conclusions There has been a switch of dominant serotype from Ogawa to Inaba in the Kolkata region. This is not necessarily due to emergence of a new clone but does serve as an epidemiological marker. Further analysis at the molecular level will be required to define this trend and to monitor future spread to other regions.

SUMMARYIn this study, we have analysed the epidemiological significance of the concurrent infections caused by Vibrio cholerae and intestinal parasites among different age groups of hospitalized diarrhoeal patients in Kolkata. A total of 3556 stool samples collected during 1996–2004 were screened for vibrios and parasites. The seasonality of V. cholerae and parasitic infections were studied in detail. The detection rates for Ascaris lumbricoides and Giardia lamblia infection were more than for the hookworm, Trichuris trichiura and Entamoeba histolytica. V. cholerae O1 was identified as the predominant serogroup among diarrhoeal patients. The highest rates for V. cholerae infection were in the 2–5 years age group and combined infection of V. cholerae and parasites was recorded among children aged between 2 and 10 years.

Background The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine. Methods 199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation. Results An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p

In developing countries,Shigellais a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multipleShigellaserotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigellasurface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection againstShigella flexneriserotypes 2a, 5a, and 6,Shigella boydii,Shigella sonnei, andShigella dysenteriaeserotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conservedShigellaprotein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine againstShigella.

The cholera toxin (CT) is a critical determinant of the virulence of epidemic Vibrio cholerae strains. The ctxAB operon encoding CT is part of the genome of a filamentous bacteriophage CTXΦ, which may integrate as a single copy or as multiple copies in the genome of V. cholerae. The CTXΦ genome is composed of RS2 (2.4 kb) and core (4.5 kb) regions. In the present study extensive genetic mapping analyses indicated that two copies of tandemly arrayed CTX prophages are integrated in the small chromosome of an environmental V. cholerae strain, VCE232, belonging to serogroup O4. Further mapping revealed that the integration of prophages has occurred in the same genetic locus of the small chromosome of VCE232 as that of V. cholerae O1 biotype El Tor strains. Interestingly, a new type of RS2-like element 3.5 kb in size was found in the CTX prophage genome in the small chromosome of VCE232. Cloning followed by sequencing of the new RS2-like element of VCE232 revealed the presence of three ORFs, which probably encode highly divergent types of phage regulatory proteins. Furthermore, the strain VCE232 also harbours two copies of a tandemly arranged CTX prophage devoid of the ctxAB genes, called pre-CTX prophage, in its large chromosome. The presence of multiple copies of diverse CTX prophages in both the chromosomes of VCE232 suggests that toxigenic environmental V. cholerae non-O1, non-O139 strains could play a role in the emergence of new epidemic clones.

Summary The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXφ. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXφ in V. cholerae, is comprised of the ‘RS2’ and the ‘Core’. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.

A large outbreak of acute watery diarrhoea involving all age groups of mongoloid tribal aborigines occurred during October-November, 2002 in the Nancowry group of Andaman and Nicobar Islands in the Indian Ocean. Twenty-one of the 67 stool samples from 67 patients were positive for toxigenic Vibrio cholerae O1, serotype Ogawa biotype El Tor, which showed striking similarity in its antibiogram with some of the strains of V. cholerae O1 Serotype Ogawa biotype El Tor isolated in Kolkata. The Nancowry and Kolkata isolates were compared with molecular tools involving random amplified polymorphic DNA (RAPD) fingerprinting, ribotyping and pulsed-field gel electrophoresis (PFGE). RAPD fingerprinting and ribotyping techniques revealed that all the V. cholerae strains associated with the outbreak in these islands were clonal in nature and identical to a population of isolates obtained from Kolkata since 1993. PFGE could discriminate within these Kolkata isolates further and established that a particular subtype of this population reached the remote Nancowry islands and was responsible for the outbreak.

We have determined the magnitude of flexural rigidity of flagellar hooks of peritrichous and single polar flagellated bacteria. Several physical parameters, such as contour length and straight length, were measured from electron micrographs of curved flagellar hooks. We correlated the thermal bending motion of the flagellar hooks with their elastic properties and by employing the measured parameters we determined the elastic parameters of the flagellar hooks, such as bend persistence length and flexural rigidity. The extremely low magnitude of the flexural rigidity of the flagellar hooks clearly indicates that the flagellar hooks can flex easily in any desired direction. Interestingly, the flagellar hooks of single polar flagellated bacteria also possess an extremely low flexural rigidity. These results will help elucidate the role of flagellar hooks in the swimming process of single polar flagellated bacteria.

Sixteen of the 18 Vibrio cholerae rough strains isolated from hospitalised diarrhoea patients were found to contain O1 serotype-specific (wbe) genes and all currently known virulence genes. Expression of the regulatory element ToxR was evident in these strains. Cholera toxin production ability of the rough strains was found to be higher (c. three- to five-fold) as compared to the smooth counterparts and this was transcriptionally regulated. Strains exhibiting the rough phenotype were more amenable to the uptake of CTXphi, which led us to consider that the rough phenotype could play a role in the generation of genetic diversity among V. cholerae strains.

We investigated the presence of cagA- like gene of Helicobacter pylori in environmental isolates of Aeromonas spp. from different water samples of Calcutta, India, by colony hybridization using a cagA -specific DNA probe and by PCR with cagA -specific primers. Nucleotide sequencing of five PCR products revealed 97 to 98% homology to canonical cagA of H. pylori 26695 as well as to four clinical H. pylori strains from Calcutta. The cagA -like gene of the environmental isolates was unstable in laboratory conditions and tended to be lost upon subculturing.

Diarrhoeal infection caused by Escherichia coli is common in India with occasional outbreaks. However, association of different pathotypes of diarrhoeagenic E. coli (DEC) with the disease and its phenotypic and genotypic characteristics are not fully demonstrated. In this study, E. coli strains from sporadic cases and outbreaks of diarrhoea during 2000-2001 were confirmed as DEC by polymerase chain reaction (PCR) targeting the specific virulence genes. DEC represented by enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAggEC) were mostly belonged to O serogroups 25, 86a, 114 and 146. The gene astA was frequently detected among ETEC and EAggEC than EPEC. After initial screening of 200 DEC strains with serology and antibiotic susceptibility test, 32 strains representing ETEC, EPEC, and EAggEC isolated from different areas of India were included in the pulsed-field gel electrophoresis (PFGE) analysis. Using the PFGE results, the hierarchical representation of different linkage levels between the DEC strains were determined by unweighed pair-group arithmetic mean (UPGAMA) method. Except for few strains, clonotyping by PFGE revealed no correlation between pathotypes and serogroups as well as the place of isolation of the DEC strains. The prevailing clonal diversity among the different categories of DEC strains suggests that the pathotypes of DEC belonged to diverse clones.

A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes ( aldA , tagA , tcpP/H , toxT , acfB/C , and int ) and intergenic regions ( tcpF to toxT and tcpH to tcpA ) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30°C and pH 6.5) led to transcriptional activation of tcpP/H , toxR , toxT , and tcpA genes, but not of ctxA , as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB . Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT , ctxA , and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly (∼1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT , ctxA , or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region., Author Summary Vibrio parahaemolyticus has been associated with several epidemics of foodborne diarrheal infection. Recent observations in several counties have shown the emergence of pandemic strains of V. parahaemolyticus with unique genetic features and their role in diarrheal outbreaks. Unlike other enteric pathogens, the appearance of pandemic strains of V. parahaemolyticus has not been associated with the economic/hygiene status of the population. The pandemic strains of V. parahaemolyticus continue to prevail in Kolkata, India since its appearance during 1996. The present communication describes not only the prevalence of pandemic serovars of V. parahaemolyticus, but also the appearance of novel serovars under the pandemic strain category. In addition, the trh gene was detected in some of the pandemic strains for the first time. In the newly emerged serovars genetic changes have occurred, as evidenced from the PFGE analysis. Overall, the antimicrobial susceptibility of pandemic strains remains unchanged for the past 20 years. The observations made in this study re-emphasize the importance of this pathogen and shows the recent genetic and serovar changes in the epidemiology of V. parahaemolyticus-mediated diarrhea.

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.

This study examined established enteric pathogens, Arcobacter species and enterotoxigenic Bacteroides fragilis (ETBF), in 201 U.S. and European travelers with acute diarrhea acquired in Mexico, Guatemala, and India. Arcobacter butzleri and ETBF were detected in 8% and 7% of diarrhea cases, respectively.

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (∼98%) of 233 were positive for toxR . None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW - or toxR -specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW - and toxR -specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one ( V. mimicus ) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus , V. splendidus , V. alginolyticus , V. fluvialis , V. proteolyticus , V. aestuarianus , V. salmonicida , V. furnissii , and V. parahaemolyticus ) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

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

Since 1996 infections caused by Vibrio parahaemolyticus have increased globally. Outbreak and/or sporadic cases of diarrhea caused by V. parahaemolyticus have been reported from India Indonesia Japan Taiwan the USA Laos Korea Chile Bangladesh Russia Thailand Spain France and Vietnam. This increase in incidence appears to be related to the emergence of a new clone belonging to the O3:K6 serovar which has pandemic potential. Due to its spread in many countries with identical phenotypic and genotypic features the recently emerged V. parahaemolyticus has now been termed a pandemic strain which can be identified by group-specific GSPCR based on the sequence variation in the toxRS gene. In addition the pandemic strains have a novel open reading frame orf8 which corresponds to a filamentous phage f237. On September 14 2003 an outbreak of diarrhea occurred near Dumdum in Kolkata India. About 200 people had diarrhea after consumption of rice with meat served at the blood donation camp. Twenty-one patients were admitted to the Infectious Diseases Hospital (IDH) Kolkata from this outbreak-affected area. Stool specimens were collected from all the patients and screened for common enteric pathogens within two hours of collection using standard methods. In addition 3--5 Escherichia coli colonies grown in MacConkey agar from each stool specimen were screened for diarrheagenic E. coli (DEC) by PCR.4 In this assay all the tested E. coli isolates were negative for common DEC such as enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. (excerpt)

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA+ strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed.

Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR , rtxC , and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

Vibrio cholerae O139 Bengal strain was the causative agent of the recent epidemics of cholera in India and Bangladesh. We studied antibacterial and antitoxin immune responses in acute and convalescent phase paired sera collected from seven of these cholera patients. Significant rise in the levels of both antibacterial and antitoxin antibodies was demonstrable in the sera of convalescent cholera patients. Antibacterial antibodies, directed primarily against O139 lipopolysaccharides (LPS), belonged to IgM class, while antitoxin antibodies were of IgG and IgA class and neutralized cholera toxin. The convalescent sera, however, showed no increase in the reactivity towards V. cholerae O1 whole cells or their LPS preparation. Immunoblotting experiments revealed that the convalescent, but not the acute, phase serum recognized the truncated form of LPS characteristics of O139 strains. Convalescent serum also induced definite protection against O139, but not O1, challenge in experimental animal model. Further studies showed that such protection was probably mediated by antibodies inhibiting intestinal colonization of O139 organisms. These results suggest that critical difference(s) exists between the immunogenic somatic components of V. cholerae O1 and O139 organisms that are of considerable importance in protection against cholera.

Summary. Vibrio cholerae 0139 organisms isolated from different parts of India and from Bangladesh were characterised with respect to their haemagglutination (HA) activity, plasmid content, cholera toxin (CT) production, cell surface protein and lipopolysaccharide (LPS) profiles, and antigenic properties. Of 28 V. cholerae 0139 isolates tested, 14 (50 YO) were shown to agglutinate chicken erythrocytes; the HA activity was sensitive to D-mannose 0.1 %. In parallel experiments, 12 (92.3%) of 13 V. cholerae 01 (El Tor) and 12 (75%) of 16 non-0 1, non-0 139 strains agglutinated chicken erythrocytes. Plasmid analysis of 32 0 139 isolates showed that 12 (37.5%) carried one or more plasmids of 35-8-2.6 MDa. Plasmids were not detected in any of the V. cholerae 01 strains, although plasmids were demonstrable in 35% of the non-01, non-0139 strains tested. V. cholerae 0139 isolates showed an ability to produce CT that depended on media composition and other cultural conditions. A comparison of envelope and outer-membrane protein profiles between 0 1 and 0 139 isolates failed to show any significant differences. LPS analysis of 0139 isolates revealed that these organisms were devoid of long " 0 " side-chain polysaccharides. Some of the non-0 1, non-0139 strains also showed similar LPS profiles whereas others showed the presence of long repetitive " 0 " side-chain polysaccharides similar to those seen in 0 1 organisms. An antiserum raised against V. cholerae 01 strain 0395 did not show any significant reactivity towards 0139 and non-01, non-0139 strains although it reacted with other 01 strains. Furthermore, the anti-01 serum induced marked protection against challenge with an 01 strain but not with an 0 139 strain in passive protection experiments. All these results indicate that, despite sharing some common properties with V. cholerae 01, the 0139 isolates posses certain characteristics that make them distinct from their 0 1 counterparts.