Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries.

BACKGROUND: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). METHODS: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015-2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. RESULTS: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. CONCLUSIONS: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome.

Influence of maternal and socioeconomic factors on breast milk fatty acid composition in urban, low-income families.

The lipid composition of breast milk may have a significant impact on early infant growth and cognitive development. Comprehensive breast milk data is lacking from low-income populations in the Indian subcontinent impeding assessment of deficiencies and limiting development of maternal nutritional interventions. A single breast milk specimen was collected within 6 weeks postpartum from two low-income maternal cohorts of exclusively breastfed infants, from Dhaka, Bangladesh (n = 683) and Kolkata, India (n = 372) and assayed for percentage composition of 26 fatty acids. Mature milk (>15 days) in Dhaka (n = 99) compared to Kolkata (n = 372) was higher in total saturated fatty acid (SFA; mean 48% vs. 44%) and disproportionately lower in ω3-polyunsaturated fatty acid (PUFA), hence the ω6- and ω3-PUFA ratio in Dhaka were almost double the value in Kolkata. In both sites, after adjusting for days of lactation, increased maternal education was associated with decreased SFA and PUFA, and increasing birth order or total pregnancies was associated with decreasing ω6-PUFA or ω3-PUFA by a factor of 0.95 for each birth and pregnancy. In Dhaka, household prosperity was associated with decreased SFA and PUFA and increased ω6- and ω3-PUFA. Maternal height was associated with increased SFA and PUFA in Kolkata (1% increase per 1 cm), but body mass index showed no independent association with either ratio in either cohort. In summary, the socioeconomic factors of maternal education and household prosperity were associated with breast milk composition, although prosperity may only be important in higher cost of living communities. Associated maternal biological factors were height and infant birth order, but not adiposity. Further study is needed to elucidate the underlying mechanisms of these effects.

Carriage and within-host diversity of mcr-1.1-harbouring Escherichia coli from pregnant mothers: inter- and intra-mother transmission dynamics of mcr-1.1.

Exchange of antimicrobial resistance genes via mobile genetic elements occur in the gut which can be transferred from mother to neonate during birth. This study is the first to analyse transmissible colistin resistance gene, mcr, in pregnant mothers and neonates. Samples were collected from pregnant mothers (rectal) and septicaemic neonates (rectal and blood) and analysed for the presence of mcr, its transmissibility, genome diversity, and exchange of mcr between isolates within an individual and across different individuals (not necessarily mother-baby pairs). mcr-1.1 was detected in rectal samples of pregnant mothers (n = 10, 0.9%), but not in neonates. All mcr-positive mothers gave birth to healthy neonates from whom rectal specimen were not collected. Hence, the transmission of mcr between these mother-neonate pairs could not be studied. mcr-1.1 was noted only in Escherichia coli (phylogroup A & B1), and carried few resistance and virulence genes. Isolates belonged to diverse sequence types (n = 11) with two novel STs (ST12452, ST12455). mcr-1.1 was borne on conjugative IncHI2 bracketed between ISApl1 on Tn6630, and the plasmids exhibited similarities in sequences across the study isolates. Phylogenetic comparison showed that study isolates were related to mcr-positive isolates of animal origin from Southeast Asian countries. Spread of mcr-1.1 within this study occurred either via similar mcr-positive clones or similar mcr-bearing plasmids in mothers. Though this study could not build evidence for mother-baby transmission but the presence of such genes in the maternal specimen may enhance the chances of transmission to neonates.

Comparison of the immunogenicity and safety of Euvichol-Plus with Shanchol in healthy Indian adults and children: an open-label, randomised, multicentre, non-inferiority, parallel-group, phase 3 trial.

Background: Considering the cholera menace in India and to seek licensure of the oral cholera vaccine (OCV), Euvichol-Plus, we conducted a clinical trial to compare the immunogenicity and safety of Euvichol-Plus with Shanchol in healthy Indian adults and children. Methods: This phase 3, open-label, multicentre, randomised, non-inferiority, parallel-group, comparative study was conducted at seven sites across India involving 416 healthy adults (aged ≥18-60 years) and children (aged ≥1 to <18 years). Healthy individuals who agreed to participate through a voluntary written informed consent form along with oral or written assent (for children aged 7-18 years) were included. No assent was required for those <7 years, as consent was given by the legally acceptable representatives (LAR). Participants were randomised 1:1 to receive two doses of either Euvichol-Plus or Shanchol orally, 14 days apart. The first dose (1.5 ml) was administered on visit 1, and the second dose at 2 weeks after the first dose during visit 2. Participants were followed up telephonically for 3 consecutive days after each visit and returned for final assessment at 2 weeks after the second dose (visit 3). Blood samples were collected for immunogenicity assessment, and safety analyses were done during all the visits. The primary immunogenicity endpoint was the percentage of participants with ≥4-fold increase in anti-Vibrio cholerae (V. cholerae) O1 Ogawa and O1 Inaba (vibriocidal) antibody titres at 2 weeks after the second dose as compared to baseline titres prior to dosing. The secondary immunogenicity endpoints included the percentage of participants with ≥4-fold increase in anti-V. cholerae O139 antibody titres at 2 weeks after the second dose as compared to baseline titres, and geometric mean titres (GMT) and geometric mean ratios (GMR) as measured by anti-V. cholerae O1 Ogawa, O1 Inaba, and O139 antibody titres at 2 weeks after the second dose as compared to baseline titres. The safety endpoints included assessment of solicited, unsolicited adverse events (AEs), and serious adverse events (SAEs). The clinical trial was registered with the Clinical Trials Registry of India (CTRI/2021/08/035344). Findings: The study was performed in two age cohorts: cohort 1 (aged ≥18-60 years, 208 participants [104 in Euvichol-Plus group and 104 in Shanchol group]), and cohort 2 (aged ≥1 to <18 years, 208 participants [104 in Euvichol-Plus group and 104 in Shanchol group]). A total of 414 participants (Euvichol-Plus: 206 and Shanchol: 208) who completed the study (intention-to-treat and per-protocol set) were analysed to compare the vibriocidal titre as an index for immunogenicity. At 2 weeks after the second dose, the percentage of participants in the Euvichol-Plus group who reported a ≥4-fold increase in anti-V. cholerae antibody titres were 68.93% (O1 Ogawa) [95% CI 62.13%-75.18%], 66.02% (O1 Inaba) [95% CI 59.11%-72.46%], and 59.71% (O139) [95% CI 52.67%-66.47%] as compared to 63.94% (O1 Ogawa) [95% CI 57.01%-70.47%], 65.87% (O1 Inaba) [95% CI 58.99%-72.28%], and 56.25% (O139) [95% CI 49.22%-63.10%] in the Shanchol group. The lower limit of 95% CI for treatment difference for all the antibody titres was ≥10% (non-inferiority margin), demonstrating that Euvichol-Plus was non-inferior to Shanchol. The post-vaccination GMT (Day 14 and 28) were more than the pre-vaccination GMT for all three serotypes in both groups. The GMR obtained for Euvichol-Plus over Shanchol for O1 Ogawa, O1 Inaba, and O139 serotypes was >1, indicating non-inferiority of Euvichol-Plus to Shanchol. The safety cohort included 416 participants. Headache was the most common solicited AE, whereas cold and cough were the most common unsolicited AEs in both groups. Interpretation: Euvichol-Plus appears to be non-inferior to Shanchol in terms of immunogenicity and safety in healthy Indian adults and children. Funding: Techinvention Lifecare Private Limited, Mumbai, India.

Nonmetabolizable Arabinose Inhibits Vibrio cholerae Growth in M9 Medium with Gluconate as the Sole Carbon Source.

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.

Emergence of tetracycline-resistant Vibrio cholerae O1 serotype Inaba, in Kolkata, India.

Out of 2,235 diarrheal stool samples collected from patients admitted to the Infectious Diseases Hospital, Kolkata, 343 cases were positive for Vibrio cholerae (341, V. cholerae O1 and 2, O139). During the year 2004, infections caused by V. cholerae serotype Ogawa and Inaba were 93 and 7%, respectively, while in 2005, the Inaba isolation rate rose to 88% as compared to 12% for Ogawa. Susceptibility to antimicrobial agents revealed that the O1 strains were resistant to multiple antibiotics (ampicillin, co-trimoxazole, furazolidone, nalidixic acid and streptomycin) with reduced susceptibility to ciprofloxacin. Increased isolation of tetracycline-resistant strains (27.3% for Ogawa and 15% for Inaba) was noted in 2005. It appears that the population might be at risk of infection by the Inaba serotype and that tetracycline may not be useful for the treatment.

Dissemination of newly emerged polymyxin B sensitive Vibrio cholerae O1 containing Haitian-like genetic traits in different parts of India.

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.

Antibody secreting B cells and plasma antibody response to rotavirus vaccination in infants from Kolkata India.

BACKGROUND: Assessing immune response after rotavirus vaccination consists in measuring serum or plasma IgA and IgG antibodies, but these assays provide very little information about the mucosal immune response. Thus the development of assays for detection of mucosal immune response following rotavirus vaccination is essential. We evaluate to assess circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immune responses to rotavirus vaccine. METHODS: 372 subjects, aged 6 weeks, were enrolled in the study. All the subjects were assigned to receive two doses of Rotarix® vaccine. Using a micro-modified whole blood-based ELISPOT assay, circulating rotavirus type-specific IgA- and IgG-ASCs, including gut homing ß7+ ASCs, were enumerated on week 6 before the first dose of Rotarix vaccination at 7 weeks of age and week 18 after the second vaccination at 17 weeks of age. Plasma samples collected before vaccination, and after two doses of Rotarix® vaccination were tested for plasma rotavirus IgA titers. RESULTS: Two doses of Rotarix® provided to induce sero-protective titer of ≥ 20 Units in 35% of subjects. Total blood IgA- ASC responses were detected in 26.4% of subjects who were non-responder before vaccination. Among responders, 47% of the subjects also have sero-protective plasma IgA titers. DISCUSSION: Our results suggest that virus-specific blood gut homing ASCs were detected and provide insight into mucosal immune response after rotavirus vaccination. Further studies are needed to evaluate the duration of such immune responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the rotavirus immunization program.

Phenotypic and genotypic traits and epidemiological implication of Vibrio cholerae O1 and O139 strains in India during 2003.

During 2003, Vibrio cholerae O1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identical wbeT region among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTX(El), CTX(Cal) and truncated CTX(Cal) (devoid of ctxAB) prophages was unique among the majority of these O139 strains.

Comparison of IPV to tOPV week 39 boost of primary OPV vaccination in Indian infants: an open labelled randomized controlled trial.

BACKGROUND: The final endgame strategy of global polio eradication initiative includes switching from trivalent oral poliovirus vaccines (tOPV) to bivalent oral polio vaccine (bOPV), and introduction of inactivated poliovirus vaccine (IPV). This study compares IPV with tOPV week 39 boost in Indian infants. METHODS: Starting 28 March 2012, we enrolled 372 Indian infant-mother pairs from Kolkata, India in an open-label, block-randomized, controlled trial comparing a 39 week tOPV to an IPV boost among infants immunized with three doses of tOPV. The primary outcome was mucosal immunity to poliovirus as measured by fecal polio virus shedding after OPV challenge. The secondary outcome was humoral response as defined by >1:8 titers for neutralizing antibodies at week 40. Seroconversion was measured by change in level of antibody titers from week 18 to week 40. The analyses were performed by both intention-to-treat (ITT) and per-protocol (PP) comparing the occurrences of outcomes between the arms of the study. FINDINGS: Both the study arms provided equivalent mucosal immunity at 52 weeks with a total shedding prevalence of 28%. Vaccination with IPV resulted in significantly higher seroconversion rates for Polio type 2 (p = 0.03) and Polio type 3 (p < 0.01). CONCLUSIONS: This study indicates that an IPV boost at week 39 is equivalent to tOPV in intestinal immunity, and provides higher seroconversion compared to tOPV. The major limitation of the study was the additional OPV doses receive by infants during pulse polio immunization resulted in additional mucosal boosting, diminishing the impact of IPV or tOPV boost at week 39. However, IPV for OPV boost should prove to be a step forward in the global polio eradication initiative to reduce the problem of circulating vaccine-derived poliovirus (cVDPV).

Use of the data system for field management of a clinical study conducted in Kolkata, India.

BACKGROUND: Designing an appropriate data system is important to the success of a clinical study. However, little information is available on this topic. We share our experiences on designing, developing, and implementation of a data system for management of data and field activities of a complex clinical study. METHODS: The data system was implemented aiming at determining the biological basis for the underperformance of oral vaccines, such as polio and rotavirus vaccines in children at a site in Kolkata, India. The system included several functionalities to control data and field activities. It was restricted to authorized users based on their access privileges. A relational database platform was chosen, and Microsoft Visual FoxPro 7.0 (Microsoft Corporation, Seattle, WA, USA) was used to develop the system. The system was installed at the clinic and data office to facilitate both the field and data management activities. RESULTS: Data were doubly entered by two different data operators to identify keypunching errors in the data. Outliers, duplication, inconsistencies, missing entries, and linkage were also checked. Every modification and users log-in/log-out information was auto-recorded in an audit trail. The system offered tools for preparation of visit schedule of the participants. A visit considered as protocol deviation was documented by the system. The system alerted field staff to every upcoming visit date to organize the field activities and to inform participants which day to come. The system also produced a growth chart for evaluating nutritional status and referring the child to a specialized clinic if found to be severely malnourished. CONCLUSION: The data system offered unique features for controlling for both data and field activities, which led to minimize drop-out rates as well as protocol deviations. Such system is warranted for a successful clinical study.

An Open Label Non-inferiority Trial Assessing Vibriocidal Response of a Killed Bivalent Oral Cholera Vaccine Regimen following a Five Year Interval in Kolkata, India.

BACKGROUND: The bivalent killed oral cholera vaccine (OCV) provides 65% cumulative protection over five years. It remains unknown whether a boosting regimen can maintain protection in previously immunized populations. This study examines the immunogenicity and safety of an OCV regimen given five years following initial dosing. METHODOLOGY/PRINCIPAL FINDINGS: An open label controlled trial was conducted in 426 healthy Indian participants previously enrolled in a large efficacy trial. To assess whether an OCV regimen given after five years can elicit an antibody response equal to that of a primary series, we compared vibriocidal antibody titers in previously immunized participants receiving a two dose booster regimen to participants receiving a primary two dose immunization series. Among participants receiving a two dose primary series of OCV (n = 186), 69% (95% CI 62%-76%) seroconverted. In the intervention arm (n = 184), 66% (95% CI 59%-73%) seroconverted following a two dose boosting schedule given five years following the initial series. Following a single boosting dose, 71% (95% CI 64%-77%) seroconverted. Children demonstrated 79% (95% CI 69%-86%) and 82% (95% CI 73%-88%) seroconversion after primary and boosting regimens, respectively. CONCLUSIONS/SIGNIFICANCE: Administration of an OCV boosting regimen elicits an immune response similar to those receiving a primary series in endemic areas. Though a single boosting dose induces a strong immune response, further investigations are needed to measure if these findings translate to clinical protection.

Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

In developing countries, Shigella is 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 multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface 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 against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 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 conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella.

Flexibility of oral cholera vaccine dosing-a randomized controlled trial measuring immune responses following alternative vaccination schedules in a cholera hyper-endemic zone.

BACKGROUND: A bivalent killed whole cell oral cholera vaccine has been found to be safe and efficacious for five years in the cholera endemic setting of Kolkata, India, when given in a two dose schedule, two weeks apart. A randomized controlled trial revealed that the immune response was not significantly increased following the second dose compared to that after the first dose. We aimed to evaluate the impact of an extended four week dosing schedule on vibriocidal response. METHODOLOGY/PRINCIPAL FINDINGS: In this double blind randomized controlled non-inferiority trial, 356 Indian, non-pregnant residents aged 1 year or older were randomized to receive two doses of oral cholera vaccine at 14 and 28 day intervals. We compared vibriocidal immune responses between these schedules. Among adults, no significant differences were noted when comparing the rates of seroconversion for V. cholerae O1 Inaba following two dose regimens administered at a 14 day interval (55%) vs the 28 day interval (58%). Similarly, no differences in seroconversion were demonstrated in children comparing the 14 (80%) and 28 day intervals (77%). Following 14 and 28 day dosing intervals, vibriocidal response rates against V. cholerae O1 Ogawa were 45% and 49% in adults and 73% and 72% in children respectively. Responses were lower for V. cholerae O139, but similar between dosing schedules for adults (20%, 20%) and children (28%, 20%). CONCLUSIONS/SIGNIFICANCE: Comparable immune responses and safety profiles between the two dosing schedules support the option for increased flexibility of current OCV dosing. Further operational research using a longer dosing regimen will provide answers to improve implementation and delivery of cholera vaccination in endemic and epidemic outbreak scenarios.

Overexpression of a delayed early gene hlg1 of temperate mycobacteriophage L1 is lethal to both M. smegmatis and E. coli.

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.

PCR-based identification of common colonization factor antigens of enterotoxigenic Escherichia coli.

Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.

Molecular tracking of the lineage of strains of Vibrio cholerae O1 biotype El Tor associated with a cholera outbreak in Andaman and Nicobar Islands, India.

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.