Antimicrobial resistance, virulence profiles and molecular subtypes of Salmonella enterica serovars Typhi and Paratyphi A blood isolates from Kolkata, India during 2009-2013.

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (blaTEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.

Hospital based surveillance and genetic characterization of rotavirus strains in children (<5 years) with acute gastroenteritis in Kolkata, India, revealed resurgence of G9 and G2 genotypes during 2011-2013.

INTRODUCTION: India accounts for an estimated 457,000-884,000 hospitalizations and 2 million outpatient visits for diarrhea. In spite of the huge burden of rotavirus (RV) disease, RV vaccines have not been introduced in national immunization programme of India. Therefore, continuous surveillance for prevalence and monitoring of the circulating genotypes is needed to assess the disease burden prior to introduction of vaccines in this region. METHODS: During January 2011 through December 2013, 830 and 1000 stool samples were collected from hospitalized and out-patient department (OPD) patients, respectively, in two hospitals in Kolkata, Eastern India. After primary screening, the G-P typing was done by multiplex semi-nested PCR using type specific primers followed by sequencing. Phylogenetic analysis for the VP7 gene of 25 representative strains was done. RESULTS: Among hospitalized and OPD patients, 53.4% and 47.5% cases were positive for rotaviruses, respectively. Unlike previous studies where G1 was predominant, in hospitalized cases G9 rotavirus strains were most prevalent (40%), followed by G2 (39.6%) whereas G1 and G12 occurred at 16.4% and 5.6% frequency. In OPD cases, the most prevalent strain was G2 (40.3%), followed by G1, G9 and G12 at 25.5%, 22.8%, 9.3%, respectively. Phylogenetically the G1, G2 and G9 strains from Kolkata did not cluster with corresponding genotypes of Rotarix, RotaTeq and Rotavac (116E) vaccine strains. CONCLUSION: The study highlights the high prevalence of RV in children with gastroenteritis in Kolkata. The circulating genotypes have changed over the time with predominance of G9 and G2 strains during 2011-2013. The current G2, G9 and G1 Kolkata strains shared low amino acid homologies with current vaccine strains. Although there is substantial evidence for cross protection of vaccines against a variety of strains, still the strain variation should be monitored post vaccine introduction to determine if it has any impact on vaccine effectiveness.

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.

Hemagglutinating activity is directly correlated with colonization ability of shigellae in suckling mouse model.

The aim of the present study was to explore a new approach based on the hemagglutination (HA) assay to understand the colonization ability of Shigella spp. To study colonization ability, an animal model of 4-day-old suckling mouse, was exploited. We characterized the HA activity of 48 Shigella strains, with erythrocytes collected from rabbit, guinea pig, chicken, and sheep. Only rabbit and guinea pig erythrocytes showed positive HA reactions in most of the cases. On the basis of HA pattern, 4 strains from each serogroup were selected for in vivo colonization studies. Our results showed a positive correlation between HA activity and colonization ability of the strains belonging to different serogroups (groups A, B, C, and D) of Shigella. In all 4 serogroups, high HA titer was associated with greater intestinal colonization.

Zinc, vitamin A, and micronutrient supplementation in children with diarrhea: a randomized controlled clinical trial of combination therapy versus monotherapy.

OBJECTIVE: To compare the clinical efficacy of supplementation of zinc, zinc plus vitamin A, and zinc plus combination of micronutrients and vitamins (iron, copper, selenium, vitamin B(12), folate, and vitamin A) on acute diarrhea in children. STUDY DESIGN: This was a double-blind, randomized, placebo-controlled trial. Children aged 6 to 24 months with diarrhea and moderate dehydration were randomized to receive zinc plus placebo vitamin A (group 1), zinc plus other micronutrients plus vitamin A (group 2), zinc plus vitamin A (group 3), or placebo (group 4) as an adjunct to oral rehydration solution. Duration, volume of diarrhea, and consumption of oral rehydration solution were compared as outcome variables within the supplemented groups and with the placebo group. RESULTS: The 167 study subjects included 41 in group 1, 39 in group 2, 44 in group 3, and 43 in group 4. All 3 supplemented groups demonstrated a significant reduction in outcome variables (P < .0001) compared with the placebo group. Group 3 had the lowest reduction of outcome variables and group 2 had a speedy recovery, but differences among the supplemented groups were not statistically significant. CONCLUSIONS: Supplementation with a combination of micronutrients and vitamins was not superior to zinc alone, confirming the clinical benefit of zinc in children with diarrhea.

Randomised controlled clinical trial of Lactobacillus sporogenes (Bacillus coagulans), used as probiotic in clinical practice, on acute watery diarrhoea in children.

OBJECTIVE: To assess the clinical efficacy of Lactobacillus sporogenes (Bacillus coagulans), as probiotic preparation, against dehydrating diarrhoea in children. METHODS: Double-blind, randomised, placebo-controlled, hospital-based clinical trial with children aged 6-24 months who had diarrhoea with some dehydration. Children received tablets of L. sporogenes (B. coagulans) or placebo (control group) and oral rehydration salt solution for correction of initial dehydration as well as maintenance therapy. Duration, frequency, volume of diarrhoea and intake of ORS of two groups were compared as outcome variables. RESULTS: One hundred and forty-eight children participated, of whom 78 (Study group) received L. sporogenes (B. coagulans) and 70 received placebo (Control group). Differences in recovery rate (P=0.2), duration (P=0.5), frequency (P=0.05), volume (P=0.1) of diarrhoea, intake of ORS (P=0.2) and other fluids (P=0.1) were not significant between both groups. Neither did a subgroup analysis of children who had rotavirus as sole enteropathogens show any significant differences in duration (P=0.5), frequency (P=0.6), volume (P=0.8) of diarrhoea, intake of ORS (P=0.8) and other fluids (P=0.8) among both groups. CONCLUSION: L. sporogenes (B. coagulans), as an adjunct to ORS, had no therapeutic impact on management of acute dehydrating diarrhoea of diverse etiology including rotavirus associated diarrhoea in children.

An adhesion protein of Salmonella enterica serovar Typhi is required for pathogenesis and potential target for vaccine development.

More than half of all Salmonella enterica serovar Typhi genes still remain unannotated. Although pathogenesis of S. Typhi is incompletely understood, treatment of typhoid fever is complicated by the emergence of drug resistance. Effectiveness of the currently available vaccines is also limited. In search of novel virulence proteins, we have identified several putative adhesins of S. Typhi through computational approaches. Our experiment shows that a 27-kDa outer membrane protein (T2544) plays a major role in bacterial adhesion to the host through high-affinity binding to laminin. Its role in bacterial pathogenesis is underscored by reduced systemic invasion and a 10-fold higher LD(50) of the mutant bacteria in mice. T2544 is strongly immunogenic as revealed by the detection of sustained high titers of serum IgG and intestinal secretory IgA in the immunized mice. In vitro, T2544 antiserum enhanced uptake and clearance of Salmonella by macrophages and augmented complement-mediated lysis, indicating a contribution of T2544-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with recombinant T2544 or passively immunized with T2544 antiserum against subsequent bacterial challenge, suggesting that T2544-specific antibodies are involved in protection. The present study describes an adhesion protein of S. Typhi that contributes to bacterial pathogenesis. Protective antibodies in mice, rapid seroconversion of naturally infected individuals with increasing titers of anti-T2544 IgG from acute to convalescent sera suggesting antibody response in humans, and wide distribution and conservation of the cell-surface adhesin in the clinical isolates of different Salmonella serovars make T2544 a potential vaccine candidate.

Subtype prevalence, plasmid profiles and growing fluoroquinolone resistance in Shigella from Kolkata, India (2001-2007): a hospital-based study.

OBJECTIVES: Shigellosis is a major public health problem, and increasing antimicrobial resistance has complicated its treatment. We report isolation frequency, plasmid profiles and antimicrobial resistance of Shigella subtypes in Kolkata, India, from a prospective hospital-based study. METHODS: Fresh stool or rectal swabs were collected from children (<5 years) attending the Diarrhea Treatment Unit of a governmental paediatric referral hospital in Kolkata. Samples were processed following standard methods over a 7-year period from January 2001 to December 2007. RESULTS: Of 4478 samples collected, 516 (11.5%) were positive for Shigella spp. S. flexneri (312; 6.9%) was the most frequently isolated serogroup, ranking before S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Although 10 subtypes of S. flexneri were identified, the common ones circulating locally were S. flexneri 2a (179), S. flexneri 6 (38) and S. flexneri 3a (36). Knowledge of Shigella subtypes is important for vaccine development. The majority of Shigella isolates (81.0%) were multidrug (two or more antimicrobial classes) resistant and showed high minimum inhibitory concentration (MIC) with commonly used drugs like ampicillin, tetracycline, co-trimoxazole and nalidixic acid. Emergence of fluoroquinolone (FQ)-resistant S. dysenteriae type 1 (100.0%) in 2002-2003 was followed by frequent isolation (>25.0%) of FQ-resistant S. flexneri 2a, and S. flexneri 3a in 2004, which restricted use of fluoroquinolones for treatment. A number of smaller plasmids (<20 kb) with distinct patterns have been observed for several years in predominant subtypes. CONCLUSION: Long-term surveillance of Shigellae and their antimicrobial resistance are mandatory in endemic areas to formulate treatment policy until any suitable candidate vaccine is available to control the disease.

A new variant of Norovirus GII.4/2007 and inter-genotype recombinant strains of NVGII causing acute watery diarrhoea among children in Kolkata, India.

BACKGROUND: Noroviruses (NoVs) are one of the major causal agents of acute gastroenteritis among different age groups. Some of the recent studies reveal that NoV genome is highly prone to mutation and recombination which often leads to emergence of new strains. OBJECTIVES: To explore the genetic diversity of human Caliciviruses (HuCVs) among diarrhoeic children in Kolkata. STUDY DESIGN: The HuCVs were detected by reverse transcription-polymerase chain reaction (RT-PCR) of the partial RNA dependent RNA polymerase gene (RdRp) and capsid gene and confirmed by sequencing. The sequences were analyzed and the recombination point was detected. RESULTS: Faecal specimens of children (n=111) visiting outpatient department of Dr B. C. Roy Memorial Hospital for Children with acute gastroenteritis were studied: 22 cases were HuCV positive with 21 NoVs. Of these, 12 NoV cases (54.5%) were GII.4 and six cases showed 99% identity with the new variant Japanese strain Hu/NoV/GII.4/OC07138/JP. Three novel NoV GII inter-genotype recombinant strains V1628/IND, V1656/IND and V1737/IND were also detected. The RdRp region of V1628 showed 96% identity with Pont de Roide 673/FRN whereas capsid region resembled GII.7/Osaka F140/JPN strain (98%); the strain V1656 showed 98% identity with RdRp region of GII.4/Monastir 375/TUN but capsid region resembled GII.8/Leverkusen 267/DE (91%); the strain V1737 showed 88% identity with RdRp of GII.5/Minato 6/N1/6/JPN whereas capsid region resembled the GII.12/Gifu 96/JPN (93%). During characterization of Caliciviruses two strains of NoV GII.b and one strain of each NoV GI.1/V1622/06/IND, GI.3/V1707/07/IND, GII.3/V1668/IND, GII.16/V1729/IND, Sapovirus GII.1/V1716/IND were also detected. CONCLUSIONS: The emergence of new variant of GII.4/2007, three novel NoV GII inter-genotype recombinant strains and various other NoVs, indicates the remarkable genetic diversity of the HuCVs as diarrhoeagenic viruses circulating in Kolkata, India.

Increase in prevalence of human group A rotavirus G9 strains as an important VP7 genotype among children in eastern India.

BACKGROUND: In addition to four globally important group A rotavirus (GARV) VP7 genotypes (G1-G4), recent surveillance studies have revealed importance of G9 strains as an aetiological agent of infantile diarrhoea. OBJECTIVE: Detection and genotyping of GARVs from children, admitted with gastroenteritis to Dr. B.C. Roy Memorial Hospital for Children, Kolkata, India. STUDY DESIGN: GARVs were detected in stool specimens by RNA electrophoresis in polyacrylamide gels. G- and P-genotyping were performed by seminested multiplex PCR assays. VP7 gene of rotavirus G9 and G12 strains were sequenced for further analysis. RESULTS: Of 249 GARV strains (n=668, May 2005-December 2006), G- and P-genotyping were successfully accomplished for 197 and 204 samples, respectively. G1 (41.6%) was most prevalent G-genotype followed by G2 (33%), G12 (14.2%), G9 (10.1%) and mixed genotype (1%). Prevalent P-genotypes were P[8] (54.4%), P[4] (31.4%), P[6] (7.3%) and mixed genotype (6.9%). Overall, G1P[8], G2P[4], G9P[8], G12P[8] and G12P[6] were identified as important G-P combinations. Phylogenetic analysis of 13 G9 strains revealed clustering within G9 lineage III. Nine of 28 G12 strains were sequenced and exhibited phylogenetic clustering with previously reported G12 strains from Kolkata. CONCLUSION: In comparison to our previous data (2003 to April 2005), G9 and G2P[4] strains established themselves in a short time span as important genotypes in eastern India.

Detection of Genogroup I and II human picobirnaviruses showing small genomic RNA profile causing acute watery diarrhoea among children in Kolkata, India.

Picobirnaviruses (PBVs) with bisegmented small RNA genome profile (1.75 and 1.55kbp for segment 1 and 2, respectively) were detected from 1999 to 2003 in faecal specimens of acute watery diarrhoea cases, largely children (n=20) and an adult in Kolkata, India. Varying degrees of dehydration necessitated their visit to hospital for further treatment and management of acute watery diarrhoea. PBV was associated with rotavirus (n=3) or astrovirus (n=3) and with both in one case. No co-infection with norovirus, sapovirus or adenovirus was detected in the picobirnavirus positive cases. No co-infection with parasites (Cryptosporidium spp., Giardia spp., Entamoeba spp., helminths) or bacteria (Vibrio spp., Shigella spp., Escherichia coli) was detected among the picobirnavirus positive cases. There was a single instance of co-infection with Salmonella spp. (n=1). PBVs not associated with serious diarrhoea illness and showing large genome profile (2.3-2.6 and 1.5-1.9kbp for segment 1 and 2, respectively) have earlier been reported in adult individuals and recently among children from a slum community in Kolkata, India. The short genome profile PBVs associated with acute watery diarrhoea may be another emerging diarrhoeagenic virus in Kolkata, India. Molecular characterization using reported primers PicoB25-PicoB43 for Genogroup I and PicoB23-PicoB24 for Genogroup II in RT-PCR showed the presence of Genogroup I PBVs (n=6) and Genogroup II PBVs (n=5), while some could not be amplified (n=3) with these primers. Sequence analysis of Genogroup I amplicons indicated remarkable sequence heterogeneity. After more than a decade, four PBV positives of Genogroup II were detected during this study. Phylogenetic analysis showed varying degree of genetic diversity amongst PBV strains from Kolkata and other countries.

Molecular epidemiology of human astrovirus infections in Kolkata, India.

UNLABELLED: The study is aimed to determine the seasonal distribution and clinical characteristics of astroviruses associated with acute watery diarrhoea among children in Kolkata and characterize them at the molecular level. METHOD OF STUDY: Faecal specimens of acute watery diarrhoea cases (n=857) and non-diarrhoeic samples (n=211) from the hospitals and a nearby field community were screened with IDEIA Astrovirus detection kit; astrovirus co-infections with rotavirus and/or picobirnavirus were detected by RNA-PAGE and silver staining. Further RT-PCR was carried out using specific primers, viz. Mon340 (+) and Mon348 (-) targeting a highly conserved domain of ORF1a (289 bp) of human astroviruses. RESULTS: Astrovirus infection was detected in 50 cases (50/857); astroviruses were detected mostly in children aged 6-12 months (50%); all non-diarrhoeic samples (n=211) were negative for astrovirus. In 52% of astrovirus positive cases, the virus was detected as the sole agent; mixed infections were also detected with other diarrhoeic pathogens such as rotavirus (32%), picobirnavirus (2%), rotavirus and picobirnavirus (2%), picobirnavirus and Enterotoxigenic E. coli (ETEC) (2%), rotavirus and ETEC (2%), rotavirus and Enteroaggregative E. coli (EAEC) (2%), Enteropathogenic E. coli (EPEC) (2%), Shigella flexneri type 3a (2%) and Ascaris (2%). RT-PCR and sequencing of amplicons of astroviruses from Kolkata, with specific primers targeted to the conserved domain of ORF1a (289 bp) of the astrovirus genome, showed maximum homology to the astrovirus strain ("5-158") from Seoul (98%). RESULTS AND CONCLUSIONS: Clinical characteristics of the diarrhoeic children in Kolkata indicated that astrovirus infections were detected throughout the year and were associated with varying degree of dehydration and acute watery diarrhoea. In-depth molecular epidemiological surveillance of astroviruses in Kolkata is essential for better understanding of their overall genetic nature.