Safety and immunogenicity of the Rotavac and Rotasiil rotavirus vaccines administered in an interchangeable dosing schedule among healthy Indian infants: a multicentre, open-label, randomised, controlled, phase 4, non-inferiority trial.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Ministry of Health and Family Welfare, Government of India. TRANSLATION: For the Hindi translation of the abstract see Supplementary Materials section.

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study.

BACKGROUND: Neonatal 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. METHODS: The 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. FINDINGS: Between 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. INTERPRETATION: Findings 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. FUNDING: Bill & Melinda Gates Foundation.

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS).

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.

Virulence Regulation and Innate Host Response in the Pathogenicity of Vibrio cholerae.

The human pathogen Vibrio cholerae is the causative agent of severe diarrheal disease known as cholera. Of the more than 200 "O" serogroups of this pathogen, O1 and O139 cause cholera outbreaks and epidemics. The rest of the serogroups, collectively known as non-O1/non-O139 cause sporadic moderate or mild diarrhea and also systemic infections. Pathogenic V. cholerae circulates between nutrient-rich human gut and nutrient-deprived aquatic environment. As an autochthonous bacterium in the environment and as a human pathogen, V. cholerae maintains its survival and proliferation in these two niches. Growth in the gastrointestinal tract involves expression of several genes that provide bacterial resistance against host factors. An intricate regulatory program involving extracellular signaling inputs is also controlling this function. On the other hand, the ability to store carbon as glycogen facilitates bacterial fitness in the aquatic environment. To initiate the infection, V. cholerae must colonize the small intestine after successfully passing through the acid barrier in the stomach and survive in the presence of bile and antimicrobial peptides in the intestinal lumen and mucus, respectively. In V. cholerae, virulence is a multilocus phenomenon with a large functionally associated network. More than 200 proteins have been identified that are functionally linked to the virulence-associated genes of the pathogen. Several of these genes have a role to play in virulence and/or in functions that have importance in the human host or the environment. A total of 524 genes are differentially expressed in classical and El Tor strains, the two biotypes of V. cholerae serogroup O1. Within the host, many immune and biological factors are able to induce genes that are responsible for survival, colonization, and virulence. The innate host immune response to V. cholerae infection includes activation of several immune protein complexes, receptor-mediated signaling pathways, and other bactericidal proteins. This article presents an overview of regulation of important virulence factors in V. cholerae and host response in the context of pathogenesis.

Characterization of the gluconate utilization system of Vibrio cholerae with special reference to virulence modulation.

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.

Rugose atypical Vibrio cholerae O1 El Tor responsible for 2009 cholera outbreak in India.

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).

Circulating Gut-Homing (α4ß7+) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea.

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, pan-Shigella surface protein 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 (α4ß7 (+)) 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 α4ß7 (+) IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α4ß7 (+) 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.

Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae.

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 .

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus.

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 α4ß7+ 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<0.001). In the IPV group, α4ß7+ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4ß7+ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4ß7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus. DISCUSSION: Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.

Safety and immunogenicity of a live oral recombinant cholera vaccine VA1.4: a randomized, placebo controlled trial in healthy adults in a cholera endemic area in Kolkata, India.

BACKGROUND: A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18-60 years from Kolkata, India. METHOD: A lyophilized dose of 1.9×109 CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14. RESULT: The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%-79.5%) at both 7 days (i.e. after 1st dose) and 21 days (i.e. after 2nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine. CONCLUSION: This study demonstrates that VA 1.4 at a single dose of 1.9×109 is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen. TRIAL REGISTRATION: Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582.

Trends in the epidemiology of pandemic and non-pandemic strains of Vibrio parahaemolyticus isolated from diarrheal patients in Kolkata, India.

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 ß-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.

High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae.

BACKGROUND: Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen. RESULTS: A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 µM and a minimal bactericidal concentration (MBC) of 3.2 µM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype. CONCLUSION: The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target.

Case-control study on the role of enterotoxigenic Bacteroides fragilis as a cause of diarrhea among children in Kolkata, India.

A total of 874 fecal specimens (446 diarrheal cases and 428 controls) from diarrheal children admitted in the Infectious Diseases Hospital, Kolkata and age and sex matched asymptomatic subjects from an urban community were assessed for the prevalence of enterotoxigenic Bacteroides fragilis (ETBF). Isolates of B. fragilis were tested for the presence of enterotoxin gene (bft) by PCR. The detection rate of ETBF was 7.2% (63 of 874 specimens) that prevailed equally in diarrheal cases and controls (7.2% each; 32 of 446 cases and 31 of 428 controls). Male children up to one year age group was significantly (p<0.05) associated with ETBF infection as compared to children > 2 years of age in cases and controls. In 25 ETBF isolates, the bft gene was genotyped using PCR-RFLP and only two alleles were identified with prevalence rate of 40% and 60% for bft-1 and bft-3, respectively. All the ETBF isolates were susceptible for chloramphenicol and imipenem but resistant to clindamycin (48%), moxifloxacin (44%) and metronidazole (32%). Resistance of ETBF to moxifloxacin (44%) and metronidazole is an emerging trend. Pulsed-field gel electrophoresis (PFGE) revealed that majority of the ETBF isolates are genetically diverse. In the dendrogram analysis, two clusters were identified, one with ETBF resistant to 5-8 antimicrobials and the other cluster with metronidazole and moxifloxacin susceptible isolates from diarrheal cases. To our knowledge, this is the first detailed report on ETBF from India indicating its clinical importance and molecular characteristics.

Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India.

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.

The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae.

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 Δedd mutation [Δedd(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.

Development and evaluation of a PCR assay for tracking the emergence and dissemination of Haitian variant ctxB in Vibrio cholerae O1 strains isolated from Kolkata, India.

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains.

Transmission of imipenem resistance determinants during the course of an outbreak of NDM-1 Escherichia coli in a sick newborn care unit.

OBJECTIVES: This study reports a cluster of septicaemic newborns with imipenem-resistant Escherichia coli in the blood and delineates the possible mechanisms of transmission of imipenem resistance with respect to the New Delhi metallo-ß-lactamase (NDM-1) gene. METHODS: During a point prevalence survey, attempts were made to isolate Gram-negative bacilli (GNB) from the environment of a sick newborn care unit (SNCU) and body sites of neonates. Subsequently, four fresh neonates admitted to the SNCU developed sepsis with E. coli. E. coli isolates from body sites and blood of the newborns were analysed in terms of clonality, carbapenemases, integrons, virulence factors, porins and transmissibility. RESULTS: During the survey, both imipenem-resistant and imipenem-susceptible E. coli were isolated from the body sites of neonates, but none from the environment. None of these neonates developed sepsis. The freshly admitted septicaemic neonates had imipenem-resistant E. coli in their blood, which were similar to the imipenem-susceptible E. coli obtained from the body sites (during the survey) in terms of clonality, phylogroup, virulence and other resistance genes, except possession of bla(NDM-1). Imipenem-resistant E. coli from blood and body sites were not clonal, though both had bla(NDM-1). Besides E. coli, other GNB isolated from the environment and body sites also harboured bla(NDM-1). Imipenem-resistant and imipenem-susceptible E. coli from the blood and body sites respectively, possessed a novel AmpC ß-lactamase, bla(CMY-59). The plasmid carrying bla(NDM-1) was transferable. CONCLUSIONS: The time frame of isolation and clonal identity indicated a possible transfer of bla(NDM-1) from imipenem-resistant GNB to the imipenem-susceptible E. coli, which subsequently caused septicaemia. This establishes the promiscuous nature of bla(NDM-1) and emphasizes the need for the early recognition of similar isolates.

In vitro antimicrobial susceptibility of bacterial enteropathogens isolated from international travelers to Mexico, Guatemala, and India from 2006 to 2008.

The incidence rates of travelers' diarrhea (TD) have remained high for the last 50 years. More recently, there have been increasing recommendations for self-initiated therapy and use of prophylactic drugs for TD. We last examined the in vitro susceptibilities of commonly used antibiotics against TD pathogens in 1997. We now examine 456 enteropathogens isolated from adult travelers to Mexico, India, and Guatemala with diarrhea acquired between 2006 and 2008 to determine changes in susceptibility against 10 different antimicrobials by the agar dilution method. Traditional antibiotics, such as ampicillin, trimethoprim-sulfamethoxazole, and doxycycline, continue to show high levels of resistance. Current first-line antibiotic agents, including fluoroquinolones and azithromycin, showed significantly higher MICs than in our earlier study, and MIC(90) levels were above the Clinical and Laboratory Standards Institute cutoffs for resistance. There were significant geographical differences in resistance patterns when Central America was compared with India. Entertoxigenic Escherichia coli (ETEC) isolates showed increased resistance to ciprofloxacin (P = 0.023) and levofloxacin (P = 0.0078) in India compared with Central America. Enteroaggregative E. coli (EAEC) isolates from Central America showed increased resistance to nearly all of the antibiotics tested. Compared to MICs of isolates 10 years prior, there were 4- to 10-fold increases in MIC(90) values for ceftriaxone, ciprofloxacin, levofloxacin, and azithromycin for both ETEC and EAEC. There were no significant changes in rifaximin MICs. Rising MICs over time imply the need for continuous surveillance of susceptibility patterns worldwide and geographically specific recommendations in TD therapy.

Evaluation of a rapid immunochromatographic dipstick kit for diagnosis of cholera emphasizes its outbreak utility.

We evaluated the Crystal VC, a commercially produced dipstick, for the rapid detection of Vibrio cholerae serotypes O1 and O139 directly from the stool samples of hospitalized diarrheal patients using the conventional bacteriological method as gold standard. The sensitivity and specificity of the dipsticks were about 92 and 73%, respectively. Introduction of the PCR-based method along with the classical bacteriological method as the gold standard for the evaluation of a kit may improve the sensitivity and specificity of the assay. The dipstick method requires minimal technical skill, and the test can be read in about 10 min. This dipstick test has the potential to act as an early warning system for cholera in many developing countries, especially during the start of an outbreak, which would ultimately lead to a decrease in the spread of the disease as well as the case fatality rate. Furthermore, the use of a rapid detection test will improve surveillance and thus reduce the burden of disease estimates, especially in remote settings.