OMICS InternationalActa Medica Okayama132017Intractable Seizure in a Case of Primary Amoebic Meningoencephalitis caused by the Free Living Amoeba Naegleria Fowleri1000114ENSandipanGangulyNational Institute of Cholera and Enteric Diseases, Indian Council of Medical ResearchAnilMalhotraKothari Medical CentreManishChowdhuriKothari Medical CentreAjantaGhosalNational Institute of Cholera and Enteric Diseases, Indian Council of Medical ResearchSanjib Kumar SardarNational Institute of Cholera and Enteric Diseases, Indian Council of Medical ResearchKeinosukeOkamotoCollaborative Research Center of Okayama University for Infectious Diseases in IndiaShantaDuttaNational Institute of Cholera and Enteric Diseases, Indian Council of Medical ResearchSujit KBhattacharyaGHSPL Sambhav KNJ Healthcares LLPNo potential conflict of interest relevant to this article was reported.Elsevier ScienceActa Medica Okayama15671348542017Characterization of Vibrio cholerae O1 strains that trace the origin of Haitian-like genetic traits4753ENPriyankaGhoshDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesDhirendraKumarMaharishi Valmiki Infectious Diseases HospitalGoutamChowdhuryDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesPuneetaSinghMaharishi Valmiki Infectious Diseases HospitalProsenjitSamantaDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesShantaDuttaDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesT.RamamurthyDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesN. C.SharmaMaharishi Valmiki Infectious Diseases HospitalPreetySinhaDepartment of Zoology, A.N. CollegeYogendraPrasadDepartment of Animal Science, MJP Rohilkhand UniversitySumioShinodaCollaborative Research Center of Okayama University for Infectious Diseases at NICEDAsish K.MukhopadhyayDivision of Bacteriology, National Institute of Cholera and Enteric Diseases Vibrio cholerae O1 is the etiological agent of the severe diarrheal disease cholera. The bacterium has recently been causing outbreaks in Haiti with catastrophic effects. Numerous mutations have been reported in V. cholerae O1 strains associated with the Haitian outbreak. These mutations encompass among other the genes encoding virulence factors such as the pilin subunit of the toxin-co-regulated pilus (tcpA), cholera toxin B subunit (ctxB), repeat in toxins (rtxA), and other genes such as the quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the alteration in the number of repeat sequences at the promoter region of ctxAB. Given the numerous genetic changes in those Haitian isolates, we decided to investigate the possible origins of those variations in the Indian subcontinent. Thus, we determined the genetic traits among V. cholerae O1 strains in Delhi, India. A total of 175 strains isolated from cholera patients during 2004 to 2012 were analysed in the present study. Our results showed that all the tested strains carried Haitian type tcpA (tcpACIRS) and variant gyrA indicating their first appearance before 2004 in Delhi. The Haitian variant rtxA and ctxB7 were first detected in Delhi during 2004 and 2006, respectively. Interestingly, not a single strain with the combination of El Tor rtxA and ctxB7 was detected in this study. The Delhi strains carried four heptad repeats (TTTTGAT) in the CT promoter region whereas Haitian strains carried 5 such repeats. Delhi strains did not have any deletion mutations in the rstB like Haitian strains. Overall, our study demonstrates the sequential accumulation of Haitian-like genetic traits among V. cholerae O1 strains in Delhi at different time points prior to the Haitian cholera outbreak.No potential conflict of interest relevant to this article was reported.PLOSActa Medica Okayama1122017Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, Indiae0005386ENDaisukeImamuraCollaborative Research Center of Okayama University for Infectious Diseases in IndiaMasatomoMoritaDepartment of Bacteriology I, National Institute of Infectious DiseasesTsuyoshiSekizukaPathogen Genomics Center, National Institute of Infectious DiseasesTamakiMizunoCollaborative Research Center of Okayama University for Infectious Diseases in IndiaTaichiroTakemuraVietnam Research Station, Institute of Tropical Medicine, Nagasaki UniversityTetsuYamashiroVietnam Research Station, Institute of Tropical Medicine, Nagasaki UniversityGoutamChowdhuryDivision of Bacteriology, National Institute of Cholera and Enteric Diseases Gururaja P.PazhaniDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesAsish K.MukhopadhyayDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesThandavarayanRamamurthyTranslational Health Science and Technology InstituteShin-ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMakotoKurodaPathogen Genomics Center, National Institute of Infectious DiseasesSumioShinodaCollaborative Research Center of Okayama University for Infectious Diseases in IndiaMakotoOhnishiDepartment of Bacteriology I, National Institute of Infectious Diseases Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years.No potential conflict of interest relevant to this article was reported.ELSEVIER GMBHActa Medica Okayama1438422130682016Role of a sensor histidine kinase ChiS of Vibrio cholerae in pathogenesis657665ENRhishitaChourashiDivision of Biochemistry, National Institute of Cholera and Enteric DiseasesMoumitaMondalDivision of Biochemistry, National Institute of Cholera and Enteric DiseasesRitamSinhaDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesAnusuyaDebnathDivision of Biochemistry, National Institute of Cholera and Enteric DiseasesSumanDasDivision of Biochemistry, National Institute of Cholera and Enteric DiseasesHemantaKoleyDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesNabenduSekhar ChatterjeeaDivision of Biochemistry, National Institute of Cholera and Enteric Diseases Vibrio cholera survival in an aquatic environment depends on chitin utilization pathway that requires two factors, chitin binding protein and chitinases. The chitinases and the chitin utilization pathway are regulated by a two-component sensor histidine kinase ChiS in V. cholerae. In recent studies these two factors are also shown to be involved in V. cholerae pathogenesis. However, the role played by their upstream regulator ChiS in pathogenesis is yet to be known. In this study, we investigated the activation of ChiS in presence of mucin and its functional role in pathogenesis. We found ChiS is activated in mucin supplemented media. The isogenic chiS mutant (ChiS-) showed less growth compared to the wild type strain (ChiS+) in the presence of mucin supplemented media. The ChiS- strain also showed highly retarded motility as well as mucin layer penetration in vitro. Our result also showed that ChiS was important for adherence and survival in HT-29 cell. These observations indicate that ChiS is activated in presence of intestinal mucin and subsequently switch on the chitin utilization pathway. In animal models, our results also supported the in vitro observation. We found reduced fluid accumulation and colonization during infection with ChiS- strain. We also found ChiS- mutant with reduced expression of ctxA, toxT and tcpA. The cumulative effect of these events made V. cholerae ChiS- strain hypovirulent. Hence, we propose that ChiS plays a vital role in V. cholerae pathogenesis.No potential conflict of interest relevant to this article was reported. Frontiers Media S.A.Acta Medica Okayama1664302X72016Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001-20061250ENRaikamalGhoshDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesNaresh C.SharmaMaharishi Valmiki Infectious Diseases HospitalKalpataruHalderInfectious Diseases and Immunology Division, Council of Scientific and Industrial Research-Indian Institute of Chemical BiologyRupak K.BhadraInfectious Diseases and Immunology Division, Council of Scientific and Industrial Research-Indian Institute of Chemical BiologyGoutamChowdhuryDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesGururaja P.PazhaniDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesSumioShinodaCollaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric DiseasesAsish K.MukhopadhyayDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesG. BalakrishNairCenter for Human Microbial Ecology, Translational Health Science and Technology InstituteThadavarayanRamamurthyCenter for Human Microbial Ecology, Translational Health Science and Technology Institute Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004–2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004–2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.No potential conflict of interest relevant to this article was reported.WILEY-BLACKWELLActa Medica Okayama0233111X56102016Regulation of Vibrio mimicus metalloprotease (VMP) production by the quorum-sensing master regulatory protein, LuxR10511058ENEl‐ShaymaaAbdel‐SattarGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shin‐ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityAbdelazizElgamlGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Vibrio mimicus is an estuarine bacterium, while it can cause severe diarrhea, wound infection, and otitis media in humans. This pathogen secretes a relatively important toxin named V. mimicus metalloprotease (VMP). In this study, we clarified regulation of the VMP production according to the quorum-sensing master regulatory protein named LuxR. First, the full length of luxR gene, encoding LuxR, was detected in V. mimicus strain E-37, an environmental isolate. Next, the putative consensus binding sequence of LuxR protein could be detected in the upstream (promoter) region of VMP encoding gene, vmp. Finally, the effect of disruption of luxR gene on the expression of vmp and production of VMP was evaluated. Namely, the expression of vmp was significantly diminished by luxR disruption and the production of VMP was severely altered. Taken together, here we report that VMP production is under the positive regulation of the quorum-sensing master regulatory protein, LuxR.No potential conflict of interest relevant to this article was reported.Oxford University PressActa Medica Okayama0305104844122016Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms56585672ENBhupesh Kumar ThakurDivision of Clinical Medicine, National Institute of Cholera and Enteric DiseasesNirmalyaDasguptaDivision of Clinical Medicine, National Institute of Cholera and Enteric DiseasesAtriTaDivision of Clinical Medicine, National Institute of Cholera and Enteric DiseasesSantasabujDasDivision of Clinical Medicine, National Institute of Cholera and Enteric Diseases Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.No potential conflict of interest relevant to this article was reported.Frontiers Research FoundationActa Medica Okayama1664302X72016Extraintestinal Infections Caused by Non-toxigenic Vibrio cholerae non-O1/non-O139144ENGoutamChowdhuryDepartment of Bacteriology, National Institute of Cholera and Enteric DiseasesSangeetaJoshiManipal HospitalSanjayBhattacharyaTata Medical CenterUmaSekarSri Ramachandra Medical CentreBalajiBirajdarMetropolis Healthcare Ltd-Global HospitalArpitaBhattacharyyaMetropolis Healthcare Ltd-Global HospitalSumioShinodaCollaborative Research Centre of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric DiseasesThandavarayanRamamurthyTranslational Health Science and Technology Institute, NCR Biotech Science Cluster Vibrio cholerae is an aerobic, sucrose fermentative Gram-negative bacterium that generally prevails in the environment. Pathogenic V. cholerae is well-known as causative agent of acute diarrhea. Apart from enteric infections, V. cholerae may also cause other diseases. However, their role in causing extraintestinal infections is not fully known as it needs proper identification and evaluation. Four cases of extraintestinal infections due to V. cholerae non-O1/non-O139 have been investigated. The isolates were screened for phenotypic and genetic characteristics with reference to their major virulence genes. Serologically distinct isolates harbored rtx, msh, and hly but lacked enteric toxin encoding genes that are generally present in toxigenic V. cholerae. Timely detection of this organism can prevent fatalities in hospital settings. The underlying virulence potential of V. cholerae needs appropriate testing and intervention.No potential conflict of interest relevant to this article was reported.Wiley-BlackwellActa Medica Okayama038556005952015Stepwise changes in viable but nonculturable Vibrio cholerae cells305310ENDaisukeImamuraCollaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric DiseasesTamakiMizunoCollaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric DiseasesShin‐ichiMiyoshiSumioShinodaCollaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric Diseases Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.No potential conflict of interest relevant to this article was reported.The Society for Antibacterial and Antifungal AgentsActa Medica Okayama134248152042015Role of the Histone-Like Nucleoid Structuring Protein (H-NS) in the Regulation of Virulence Factor Expression and Stress Response in Vibrio vulnificus263274ENAbdelazizElgamlGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityShin-ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Temperature is one of the important parameters regulating the expression of virulence factors in bacteria. The global regulator, a histone-like nucleoid structuring protein (H-NS), is known to play a crucial role in this regulation. In the present study, we first clarified the role of H-NS in the temperature-dependent regulation of virulence factor production in Vibrio vulnificus, including that of the cytolytic toxin (V. vulnificus hemolysin: VVH) and the proteolytic enzyme (V. vulnificus protease: VVP). The expression of hns itself was subjected to temperature regulation, where hns was expressed more at 26℃ than at 37℃. VVH production and the expression of its gene vvhA were increased by disruption of the hns gene. H-NS appeared to affect the vvhA expression by the well-documented transcriptional silencing mechanism. On the other hand, hns disruption resulted in the reduction of VVP production and the expression of its gene vvpE. H-NS was suggested to positively regulate vvpE expression through the increase in the level of the rpoS mRNA. Moreover, H-NS was found to contribute to the survival of V. vulnificus in stressful environments. When compared to the wild type strain, the hns mutant exhibited reduced survival rates when subjected to acidic pH, hyperosmotic and oxidative stress.No potential conflict of interest relevant to this article was reported.The Society for Antibacterial and Antifungal AgentsActa Medica Okayama134248152032015Presence of Nitric Oxide-Sensing Systems in the Human Pathogen Vibrio vulnificus199203ENAbdelazizElgamlGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityShin-ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Vibrio vulnificus is a halophilic estuarine bacterium, but this species causes fatal septicemia in humans. V. vulnificus may encounter many kinds of stresses either in the natural environment or in the human body. One of the striking stresses is the exposure to the reactive oxygen species including nitric oxide (NO). The present study revealed that NO could participate in the regulation of the V. vulnificus community behavior. When the bacterium was cultivated in the presence of sub-lethal doses of an NO donor, the expression of the genes encoding NO-detoxifying enzymes was significantly increased. The NO donor was also found to cause significant increase in production of a metalloprotease, a putative virulence factor, by the bacterium.No potential conflict of interest relevant to this article was reported.Elsevier ScienceActa Medica Okayama 0264410X32supplment 12014Hospital 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 2011-2013A20A28ENSatarupaMullickNational Institute of Cholera and Enteric DiseasesPaulamiMandalNational Institute of Cholera and Enteric DiseasesMukti Kant NayakNational Institute of Cholera and Enteric DiseasesSouvikGhoshDepartment of Hygiene, Sapporo Medical University School of MedicinePapiyaDeNational Institute of Cholera and Enteric DiseasesK.RajendranNational Institute of Cholera and Enteric DiseasesMihir K.BhattacharyaNational Institute of Cholera and Enteric DiseasesUtpalaMitraNational Institute of Cholera and Enteric DiseasesThandavarayanRamamurthyNational Institute of Cholera and Enteric DiseasesNobumichiKobayashiDepartment of Hygiene, Sapporo Medical University School of MedicineMamtaChawla-SarkarNational Institute of Cholera and Enteric DiseasesINTRODUCTION:
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.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama009511375232014Haitian variant tcpA in Vibrio cholerae O1 El Tor strains in Kolkata, India10201021ENPriyankaGhoshDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)ArindamNahaDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)SurajitBasakDepartment of Molecular Biology & Bioinformatics, Tripura UniversitySantanuGhoshDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)T.RamamurthyDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)HemantaKoleyDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)RanjanK NandyDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)SumioShinodaCollaborative Research Center of Okayama University for Infectious Diseases at NICEDHaruoWatanabeNational Institute of Infectious DiseasesAsish K.MukhopadhyayDivision of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED)No potential conflict of interest relevant to this article was reported.Wiley-BlackwellActa Medica Okayama20458827322014Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state239246ENMitsutoshiSenohCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama UniversityJayeetaGhosh-BanerjeeNational Institute of Cholera and Enteric Diseases,TamakiMizunoCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama UniversitySumioShinodaCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama UniversityShin-ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakashiHamabataResearch Institute, National Center for Global Health and MedicineG. BalakrishNairTranslational Health Science and Technology InstituteYoshifumiTakedaCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama University Previously, we reported that viable but nonculturable (VBNC) Vibrio cholerae was converted into a culturable state by coculture with several eukaryotic cell lines including HT-29 cells. In this study, we found that a factor converting VBNC V. cholerae into a culturable state (FCVC) existed in cell extracts of eukaryotic cells. FCVC was nondialyzable, proteinase K-sensitive, and stable to heating at <60°C for 5 min. We prepared thiosulfate citrate bile salts sucrose (TCBS) plates with FCVC (F-TCBS plates). After confirming that VBNC V. cholerae O1 and O139 formed typical yellow colonies on F-TCBS plates, we tried to isolate cholera toxin gene-positive VBNC V. cholerae from environmental water samples collected in urban slum areas of Kolkata, India and succeeded in isolating V. cholerae O1 El Tor variant strains harboring a gene for the cholera toxin. The possible importance of VBNC V. cholerae O1 as a source of cholera outbreaks is discussed.No potential conflict of interest relevant to this article was reported.SpringerActa Medica Okayama095939933022014Effects of temperature, growth phase and luxO-disruption on regulation systems of toxin production in Vibrio vulnificus strain L-180, a human clinical isolate681691ENAbdelazizElgamlGraduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayama UniversityKazutakaHigakiGraduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayama UniversityShin-ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayama University Vibrio vulnificus is a halophilic estuarine bacterium while it causes fatal septicemia or necrotizing wound infections in humans. This pathogen secretes the metalloprotease (V. vulnificus protease: VVP) and the cytolysin (V. vulnificus hemolysin: VVH) as protein toxins; however, their production was coordinated in response to the bacterial cell density. This regulation is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). In the present study, we investigated effects of disruption of luxO encoding a central response regulator of the QS circuit, as well as effects of temperature and growth phase, on the toxin production by V. vulnificus. Disruption of luxO was found to increase VVP production and expression of its gene vvpE. The expression of smcR, crp and rpoS, of which products positively regulate vvpE expression, and luxS encoding the AI-2 synthetase were also significantly increased. On the other hand, the luxO disruption resulted in reduction of VVH production and expression of its gene vvhA. Expression of other two genes affecting the QS circuit, luxT and rpoN, were also significantly decreased. The regulation systems of VVP production were found to exert their action during the stationary phase of the bacterial growth and to be operated strongly at 26 °C. By contrast, those of VVH production apparently started at the log phase and were operated more effectively at 37 °C.No potential conflict of interest relevant to this article was reported.BioMed CentralActa Medica Okayama17574749512013Inflammatory diarrhea due to enteroaggregative Escherichia coli: evidence from clinical and mice model studies36ENDhira Rani SahaDivision of Histology & Electron microscopy, National Institute of Cholera and Enteric DiseasesSucharitaGuinNatl Inst Cholera & Enter Dis, Div BacteriolRajendranKrishnanNatl Inst Cholera & Enter Dis, Div Data ManagementDhrubajyotiNagNatl Inst Cholera & Enter Dis, Div BacteriolHemantaKoleyNatl Inst Cholera & Enter Dis, Div BacteriolSumioShinodaOkayama Univ Infect Dis India, Collaborat Res CtrThandavarayanRamamurthyNatl Inst Cholera & Enter Dis, Div BacteriolBackground
This study was conducted to determine the role of enteroaggregative Escherichia coli (EAEC) in inflammatory diarrhea among hospitalized patients in Kolkata. The inflammatory pathogenesis of EAEC was established in mice model and histopathological studies. Presence of fecal leucocytes (FLCs) can be suspected for EAEC infection solely or as a mixed with other enteric pathogens.
Methods
Active surveillance was conducted for 2 years on 2 random days per week with every 5th patient admitted to the Infectious Diseases Hospital (IDH). Diarrheal samples were processed by conventional culture, microscopy, ELISA and molecular methods. Two EAEC isolated as sole pathogens were examined in mice after induced intestinal infection. The intestinal tissue samples were processed to analyze the histological changes.
Results
Of the 2519 samples screened, fecal leucocytes, erythrocytes and occult blood were detected in 1629 samples. Most of the patients had acute watery diarrhea (75%) and vomiting (78%). Vibrio cholerae O1 was the main pathogen in patients of 5–10 years age group (33%). Shigellosis was more in children from 2–5 years of age (19%), whereas children <2 years appeared to be susceptible for infection caused by EAEC (16%). When tested for the pathogenicity, the EAEC strains colonized well and caused inflammatory infection in the gut mucosa of BALB/C mice.
Conclusion
This hospital-based surveillance revealed prevalence of large number of inflammatory diarrhea. EAEC was the suspected pathogen and <2 years children appeared to be the most susceptible age group. BALB/C mice may be a suitable animal model to study the EAEC-mediated pathogenesis.No potential conflict of interest relevant to this article was reported.MARY ANN LIEBERT, INCActa Medica Okayama1535314110102013An outbreak of foodborne gastroenteritis caused by dual pathogens, Salmonella enterica serovar Weltevreden and Vibrio fluvialis in Kolkata, India904906ENGoutamChowdhuryNational Institute of Cholera and Enteric Diseases AnirbanSarkarNational Institute of Cholera and Enteric DiseasesGururaja P.PazhaniNational Institute of Cholera and Enteric DiseasesAsish K.MukhopadhyayNational Institute of Cholera and Enteric DiseasesMihir K.BhattacharyaNational Institute of Cholera and Enteric DiseasesThandavarayanRamamurthyNational Institute of Cholera and Enteric Diseases Salmonella enterica serovar Weltevreden and Vibrio fluvialis were identified as etiological agents of a foodborne gastroenteritis outbreak after an Iftar feast in North Dumdum. Of the 278 cases admitted to the Infectious Diseases Hospital, Kolkata, 44 stool samples were tested for the enteric pathogens. Six were positive for Salmonella Weltevreden, 5 for Vibrio fluvialis, and 8 contained both of the pathogens. Consumption of mutton-ghogni might have been the likely vehicle of this outbreak. In the pulsed-field gel electrophoresis, Salmonella Weltevreden was identified as a single clone but the V. fluvialis strains were heterogeneous.No potential conflict of interest relevant to this article was reported.Mary Ann LiebertActa Medica Okayama153531411042013Isolation and characterization of pandemic and nonpandemic strains of Vibrio parahaemolyticus from an outbreak of diarrhea in North 24 Parganas, West Bengal, India338342ENGoutamChowdhuryNational Institute of Cholera and Enteric DiseasesSantanuGhoshNational Institute of Cholera and Enteric DiseasesGururaja P.PazhaniNational Institute of Cholera and Enteric DiseasesBimal K.PaulIntegrated Disease Surveillance Program, Directorate of Health ServicesDipankarMajiIntegrated Disease Surveillance Program, Directorate of Health ServicesAsish K.MukhopadhyayNational Institute of Cholera and Enteric DiseasesThandavarayanRamamurthyNational Institute of Cholera and Enteric Diseases Strains of the enteric pathogen Vibrio parahaemolyticus harboring the thermostable hemolysin (TDH) encoding gene tdh is known to cause epidemic and pandemic diarrhea. In industrialized countries, this pathogen causes sporadic or outbreaks of diarrheal illness associated with consumption of raw or improperly cooked seafood. This report describes a foodborne outbreak of gastroenteritis caused by V. parahaemolyticus in June 2011 following consumption of food served at a funeral reception held at Habra, North 24 Parganas, West Bengal, India. About 650 people attended the function, of whom 44 had acute watery diarrhea with other clinical symptoms; 35 of them were admitted to the District Hospital for the rehydration treatment. Stool specimens collected from three hospitalized cases were positive for V. parahaemolyticus, of which two strains were identified as an O4:K8 serovar and one was identified as O3:K6 serovar. The O3:K6 strain also possessed the pandemic group-specific toxRS gene target (GS), whereas the O4:K8 strains were negative. All strains were polymerase chain reaction-positive for tdh but were polymerase chain reaction-negative for trh. All of the strains were resistant to ampicillin but were pansensitive to other antimicrobials tested. Pulsed-field gel electrophoresis (PFGE) analysis using NotI showed that the O3:K6 strain was similar to that of a recent clinical strain from Kolkata, but had diverged from other strains during previous years. In contrast, PFGE analysis showed that the O4:K8 strains were closely related but differed from the Kolkata strain.No potential conflict of interest relevant to this article was reported.SCIENCEDOMAIN internationalActa Medica Okayama22310886322013Multi-locus Genotyping Reveals High Occurrence of Mixed Assemblages in Giardia duodenalis within a Limited Geographical Boundary190197ENAvik KumarMukherjeeDepartment of Parasitology, National Institute of Cholera and Enteric DiseasesSumallyaKarmakarDepartment of Parasitology, National Institute of Cholera and Enteric DiseasesDibyenduRajDepartment of Parasitology, National Institute of Cholera and Enteric DiseasesSandipanGangulyDepartment of Parasitology, National Institute of Cholera and Enteric DiseasesAim: To determine the common genotypes of Giardia duodenalis causing diarrhea in the study region and to assess the extent of genetic polymorphism among them.
Study Design: Stool samples were collected from the patients attending IDBG Hospital, Kolkata with diarrheal complaints through a systemic sampling technique and were screened for Giardia duodenalis. The G. duodenalis positive samples were subjected to molecular genotyping through ‘PCR - Direct DNA sequencing’ procedure. All the sequence data obtained were incorporated into MEGA 4 software for multiple alignment and validation followed by phylogenetic analysis. The genotyping data obtained are stored in Excel spreadsheets and incorporated into EpiInfo 3.1 for analyzing possible association of genotype outcome with common physical factors such as age, sex etc.
Place and Duration of Study: Department of parasitology, National Institute of Cholera and Enteric Diseases, Kolkata, India from July 2009 to November 2011.
Methodology: A total of 68 Giardia duodenalis positive stool samples were identified from the diarrhea patients attending IDBG hospital in the city and were subjected to multi-locus genotyping. Fragments of ß-giardin, Glutamate-dehydrogenase and Triosephosphate-isomerase genes of Giardia were amplified from those samples with specific primers and sequenced. All the sequences were analyzed using MEGA 4 software for obtaining the genotyping results.
Results: Multi-locus genotyping identified 13 isolates as assemblage A and 41 as assemblage B, whereas 14 of them could not be assigned in a particular group. Detailed phylogenetic analysis revealed that multiple genotypes were observed in those 14 isolates depending upon the marker loci.
Conclusion: The study could produce a preliminary idea about the G. duodenalis genotypes found in Kolkata city. High percentage of mixed assemblages in the study population also revealed the presence of genetic diversity among a small population of diarrheal patient within a limited geographical boundary. It has also hypothesized the possibility of inter-assemblage genetic exchange among Giardia.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama009511375132013Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania10401045ENA.Naha1National Institute of Cholera and Enteric DiseasesG.Chowdhury1National Institute of Cholera and Enteric DiseasesJ.Ghosh-Banerjee1National Institute of Cholera and Enteric DiseasesM.SenohCollaborative Research Center of Okayama University for Infectious Diseases at NICED,T.TakahashiCollaborative Research Center of Okayama University for Infectious Diseases at NICED,B.LeyThe International Vaccine InstituteK.ThriemerThe International Vaccine InstituteJ.DeenThe International Vaccine InstituteL. V.SeidleinMenzies School of Health ResearchS. M.AliMinistry of Health and Social WelfareA.KhatibMinistry of Health and Social WelfareT.Ramamurthy1National Institute of Cholera and Enteric DiseasesR. K.Nandy1National Institute of Cholera and Enteric DiseasesG. B.Nair1National Institute of Cholera and Enteric DiseasesY.TakedaCollaborative Research Center of Okayama University for Infectious Diseases at NICED,A. K.Mukhopadhyay1National Institute of Cholera and Enteric Diseases 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.No potential conflict of interest relevant to this article was reported.National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC)Acta Medica Okayama10806040 1932013Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India464467ENDevaratiDuttaational Institute of Cholera and Enteric Diseases, KolkataGoutamChowdhuryational Institute of Cholera and Enteric Diseases, KolkataGururaja P.Pazhaniational Institute of Cholera and Enteric Diseases, KolkataSucharitaGuinational Institute of Cholera and Enteric Diseases, KolkataSanjuctaDuttaational Institute of Cholera and Enteric Diseases, KolkataSantanuGhoshational Institute of Cholera and Enteric Diseases, KolkataK.Rajendranational Institute of Cholera and Enteric Diseases, KolkataRanjan K.Nandyational Institute of Cholera and Enteric Diseases, KolkataAsish K.Mukhopadhyayational Institute of Cholera and Enteric Diseases, KolkataMihir K.Bhattacharyaational Institute of Cholera and Enteric Diseases, KolkataUtpalaMitraational Institute of Cholera and Enteric Diseases, KolkataYoshifumiTakedaational Institute of Cholera and Enteric Diseases, KolkataG. BalakrishNairTranslational Health Science and Technology InstituteThandavarayanRamamurthyational Institute of Cholera and Enteric Diseases, Kolkata 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.No potential conflict of interest relevant to this article was reported.Public Library of ScienceActa Medica Okayama19326203822013Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, Indiae56068ENSanjuctaDuttaDivision of Bacteriology, National, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaSucharitaGuinClinical Division, Institute of Cholera and Enteric Diseases, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaSantanuGhoshDivision of Bacteriology, National, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaGururaja P.PazhaniDivision of Bacteriology, National, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaKrishnanRajendranDivision of Bacteriology, National, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaMihir K.BhattacharyaClinical Division, Institute of Cholera and Enteric Diseases, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaYoshifumiTakedaNational Institute of Cholera and Enteric DiseasesG. BalakrishNairTranslational Health Science and Technology InstituteThandavarayanRamamurthyDivision of Bacteriology, National, Collaborative Research Centre of Okayama University for Infectious Diseases in IndiaBACKGROUND:
To analyse the trends in the prevalence of different pathogroups of diarrheagenic Escherichia coli (DEC) among hospitalized acute diarrheal patients.
METHODOLOGY/PRINCIPAL FINDINGS:
From the active surveillance of diarrheal disease at the Infectious Diseases Hospital, Kolkata, 3826 stool specimens collected during 2008-2011 were screened for DEC and other enteric pathogens. PCR was used in the detection of enterotoxigenic, enteropathogenic and enteroaggregative E. coli and 10 major colonization factor antigens (CFs) of enterotoxigenic E. coli. The relationship between DEC infected patient's age group and clinical symptoms were also investigated. Multiplex PCR assay showed that the prevalence of EAEC was most common (5.7%) followed by ETEC (4.2%) and EPEC (1.8%). In diarrheal children >2 year of age, EAEC and EPEC were detected significantly (p = 0.000 and 0.007, respectively). In children >2 to 5 and >5 to 14 years, ETEC was significantly associated with diarrhea (p = 0.000 each). EAEC was significantly associated with diarrheal patients with age groups >14 to 30 and >30 to 50 years (p = 0.001, and p = 0.009, respectively). Clinical symptoms such as vomiting, abdominal pain, watery diarrhea, were recorded in patients infected with ETEC. Dehydration status was severe among patients infected by ST-ETEC (19%) and EPEC (15%). CS6 was frequently detected (37%) among ETEC.
CONCLUSIONS/SIGNIFICANCE:
Hospital based surveillance reviled that specific pathogroups of DEC are important to certain age groups and among ETEC, CS6 was predominant.No potential conflict of interest relevant to this article was reported.Elsevier B.VActa Medica Okayama1198743X1922013Culture-independent real-time PCR reveals extensive polymicrobial infections in hospitalized diarrhoea cases in Kolkata, India173180ENA.SinhaNational Institute of Cholera and Enteric Diseases (NICED)S.SenGuptaNational Institute of Cholera and Enteric Diseases (NICED)S.GuinNational Institute of Cholera and Enteric Diseases (NICED)S.DuttaNational Institute of Cholera and Enteric Diseases (NICED)S.GhoshNational Institute of Cholera and Enteric Diseases (NICED)P.MukherjeeNational Institute of Cholera and Enteric Diseases (NICED)A. K.MukhopadhyayNational Institute of Cholera and Enteric Diseases (NICED)T.RamamurthyNational Institute of Cholera and Enteric Diseases (NICED)Y.TakedaCollaborative Research Centre of Okayama University for Infectious Diseases in India, NICEDT.KurakawaYakult Central Institute for Microbiological ResearchK.NomotoYakult Central Institute for Microbiological ResearchG. B.NairNational Institute of Cholera and Enteric Diseases (NICED)R. K.NandyNational Institute of Cholera and Enteric Diseases (NICED) 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 C(t) 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.No potential conflict of interest relevant to this article was reported.Longdom PublishingActa Medica Okayama23195584232013THE SPLICEOSOMAL PROTEIN SnRNP F BINDS TO BOTH U3 AND U14 CLASS OF snoRNA IN Giardia lamblia178184ENArjunGhosh Division of Parasitology, National Institute of Cholera & Enteric DiseasesSumallyaKarmakar Division of Parasitology, National Institute of Cholera & Enteric DiseasesAvik K.Mukherjee Division of Parasitology, National Institute of Cholera & Enteric DiseasesDibyenduRaj Division of Parasitology, National Institute of Cholera & Enteric DiseasesKoushikDas Division of Parasitology, National Institute of Cholera & Enteric DiseasesSrimantiSarkar Department of Biochemistry, Bose InstituteT.NozakiDepartment of Parasitology, National Institute of Infectious Diseases, Japan and Graduate School of Life and Environmental Sciences, University of TsukubaSandipanGanguly Division of Parasitology, National Institute of Cholera & Enteric Diseases Small nuclear Ribonucleo Protein F (snRNP F) is a spliceosomal protein that binds with U1, U2, U4/U6 and U5 small nuclear RNA (snRNA) to form spliceosomal complexes responsible for pre mRNA processing. This study reports the unusual interaction of giardial snRNP F with small nucleolar RNAs (snoRNA) that are responsible for pre rRNA processing. Electrophoretic Mobility Shift Assay was used to demonstrate the interaction of this protein with U3 and U14 class snoRNA of the early branching eukaryote Giardia lamblia. It was also evident from our study that snRNP F in Giardia is evolutionary distinct from its other eukaryotic orthologues.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1080-604018112012Vibrio fluvialis in Patients with Diarrhea, Kolkata, India18681871ENGoutamChowdhuryGururajaP. PazhaniDevaratiDuttaSucharitaGuinSanjuctaDuttaSantanuGhoshHidemasaIzumiyaMasahiroAsakuraShinjiYamasakiYoshifumiTakedaEijiArakawaHaruoWatanabeAsish K.MukhopadhyayMihir K.BhattacharyaK.RajendranGopinath BalakrishNairThandavarayanRamamurthyWe identified 131 strains of Vibrio fluvialis among 400 nonagglutinating Vibrio spp. isolated from patients with diarrhea in Kolkata, India. For 43 patients, V. fluvialis was the sole pathogen identified. Most strains harbored genes encoding hemolysin and metalloprotease; this finding may contribute to understanding of the pathogenicity of V. fluvialis.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0022-26156192012Significant association of the dupA gene of Helicobacter pylori with duodenal ulcer development in a South-east Indian population12951302ENJawedAlamSankarMaitiPrachetashGhoshRonitaDeAbhijitChowdhurySuryasnataDasRaginiMacadenHarshadDevarbhaviT.RamamurthyAsish K.MukhopadhyayA novel virulence factor, duodenal ulcer-promoting gene A (dupA), in Helicobacter pylori has been found to be associated with disease in certain populations but not in others. This study analysed a South-east Indian population as part of the debate about the relevance of dupA for the prediction of clinical outcomes. A total of 140 H. pylori strains isolated from duodenal ulcer (DU) (n=83) and non-ulcer dyspepsia (NUD) patients (n=57) were screened by PCR and dot-blot hybridization to determine the presence of the ORFs jhp0917 and jhp0918. Part of jhp0917-jhp0918 was sequenced to search for the C/T insertion that characterizes dupA and the levels of dupA transcripts were also assessed. The PCR and dot-blot results indicated the presence of jhp0917 and jhp0918 in 37.3% (31/83) and 12.2% (7/57) of H. pylori strains isolated from DU and NUD patients, respectively. Sequencing analysis showed insertion of a C at nt 1386 in the 3' region of jhp0917, forming the dupA gene in 35 strains. RT-PCR analysis detected the dupA transcript in 28 of these 35 strains. The expression level of the dupA transcript varied from strain to strain, as shown by real-time PCR. The results demonstrated that analysis based on PCR only for dupA may produce an erroneous interpretation. The prevalence of dupA was significantly greater among strains isolated from patients with DU than from patients with NUD in this population (P=0.001, odds ratio=4.26, confidence interval=1.60-11.74). Based on these findings, dupA can be considered a biomarker for DU patients in India. The reported discrepancies for this putative virulence marker in different populations may be due to the genome plasticity of H. pylori.No potential conflict of interest relevant to this article was reported.BioMed Central Ltd.Acta Medica Okayama1757-474942012Distinct repeat motifs at the C-terminal region of CagA of Helicobacter pylori strains isolated from diseased patients and asymptomatic individuals in West Bengal, IndiaENSantanuChattopadhyayRajashreePatraRaghunathChatterjeeRonitaDeJawedAlamT.RamamurthyAbhijitChowdhuryG. BalakrishNairDouglas E.BergAsish K.MukhopadhyayBackground: Infection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India.
Results: Seventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants.
Conclusion: Our analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama0095-11375052012Development and Evaluation of a PCR Assay for Tracking the Emergence and Dissemination of Haitian Variant ctxB in Vibrio cholerae O1 Strains Isolated from Kolkata, India17331736ENArindamNahaG. P.PazhaniMouGangulySantanuGhoshT.RamamuranthyRanjan K.NandyG. BalakrishNairYoshifumiTakedaAsish K.MukhopadhyayA 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.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama0095-11375042012Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls13081312ENSubrataSabuiSanjuctaDuttaAnusuyaDebnathAvishekGhoshT.HamabataK.RajendranT.RamamurthyJames P.NataroDipikaSurMyron M.LevineNabendu SekharChatterjeeEnterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.No potential conflict of interest relevant to this article was reported.SpringerActa Medica Okayama0959-39932842012An extracellular serine protease produced by Vibrio vulnificus NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel16331639ENShin-ichiMiyoshiWangJiyouKeizoKatohMitsutoshiSenohTamakiMizunoYokoMaeharaVibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in immunocompromised humans, cultured eels or shrimps. An extracellular metalloprotease VVP/VvpE has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA, but not VVP/VvpE. In the present study, we found that these strains had lost the 80 kb genomic region including the gene encoding VVP/VvpE. We also purified VvsA from the culture supernatant through ammonium sulfate fractionation, gel filtration and ion-exchange column chromatography, and the enzyme was demonstrated to be a chymotrypsin-like protease, as well as those from some vibrios. The gene vvsA was shown to constitute an operon with a downstream gene vvsB, and several Vibrio species were found to have orthologues of vvsAB. These findings indicate that the genes vvp/vvpE and vvsAB might be mobile genetic elements.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0882-40105142011Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence243249ENTakeakiWajimaSubrataSabuiMegumiFukumotoShigeyukiKanoThandavarayanRamamurthyNabendu SekharChatterjeeTakashiHamabataEnterotoxigenic Escherichia coli (ETEC) produces a variety of colonization factors necessary for attachment to the host cell, among which CS6 is one of the most prevalent in ETEC isolates from developing countries. The CS6 operon is composed of 4 genes, cssA, cssB, cssC, and cssD. The molecular mechanism of CS6 assembly and cell surface presentation, and the contribution of each protein to the attachment of the bacterium to intestinal cells remain unclear. In the present study, a series of css gene-deletion mutants of the CS6 operon were constructed in the ETEC genetic background, and their effect on adhesion to host cells and CS6 assembly was studied. Each subunit deletion resulted in a reduction in the adhesion to intestinal cells to the same level of laboratory E. coli strains, and this effect was restored by complementary plasmids, suggesting that the 4 proteins are necessary for CS6 expression. Bacterial cell fractionation and western blotting of the mutant strains suggested that the formation of a CssA–CssB–CssC complex is necessary for recognition by CssD and transport of CssA–CssB to the outer membrane as a colonization factor.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0304-40171783-42011Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm workers in West Bengal, India342345ENShahbaz ManzoorKhanChanchalDebnathAmiya KumarPramanikLihuaXiaoTomoyoshiNozakiSandipanGangulyNo study in the past has examined the genetic diversity and zoonotic potential of Giardia duodenalis in dairy cattle in India. To assess the importance of these animals as a source of human G. duodenalis infections and determine the epidemiology of bovine giardiasis in India, fecal samples from 180 calves, heifers and adults and 51 dairy farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the β-giardin gene of G. duodenalis followed by DNA sequencing of the nested PCR products. The overall prevalence of G. duodenalis in cattle was 12.2% (22/180), the infection being more prevalent in younger calves than in adult cattle. Zoonotic G. duodenalis Assemblage A1 was identified in both calves and workers although the most prevalent genotype detected in cattle was a novel Assemblage E subgenotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of G. duodenalis infections between cattle and humans on dairy farms in India.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0041-01015762011Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain904908ENShin-ichiMiyoshiYukiAbeMitsutoshiSenohTamakiMizunoYokoMaeharaHiroshiNakaoVibrio vulnificus is an etiological agent causing serious systemic infections in the immunocompromised humans or cultured eels. This species commonly produces a hemolytic toxin consisting of the cytolysin domain and the lectin-like domain. For hemolysis, the lectin-like domain specifically binds to cholesterol in the erythrocyte membrane, and to form a hollow oligomer, the toxin is subsequently assembled on the membrane. The cytolysin domain is essential for the process to form the oligomer. Three-dimensional structure model revealed that two domains connected linearly and the C-terminus was located near to the joint of the domains. Insertion of amino acid residues between two domains was found to cause inactivation of the toxin. In the C-terminus, deletion, substitution or addition of an amino acid residue also elicited reduction of the activity. However, the cholesterol-binding ability was not affected by the mutations. These results suggest that mutation of the C- or N-terminus of the lectin-like domain may result in blockage of the toxin assembly.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0022-13179222011Whole-genome characterization of human group C rotaviruses: identification of two lineages in the VP3 gene361369ENDaiYamamotoSouvikGhoshMitsutakaKuzuyaYuan-HongWangXuanZhouMamtaChawla-SarkarShyamal KumarPaulMasahoIshinoNobumichiKobayashiGroup C rotavirus (GCRV) is distributed worldwide as an enteric pathogen in humans and animals. However, to date, whole-genome sequences are available only for a human strain (Bristol) and a porcine strain (Cowden). To investigate the genetic diversity of human GCRVs, nearly full-length sequences of all 11 RNA segments were determined for human GCRVs detected recently in India (v508), Bangladesh (BS347), China (Wu82 and YNR001) and Japan (OH567 and BK0830) and analysed phylogenetically with sequence data for GCRVs published previously. All the RNA segments of human GCRV strains except for the VP3 gene showed high levels of conservation (>93 % nucleotide sequence identity, >92 % amino acid sequence identity), belonging to a single genetic cluster distinct from those of animal GCRVs. In contrast, the VP3 genes of human GCRVs could be discriminated into two clusters, designated M2 and M3, that were distinguished phylogenetically from those of porcine and bovine GCRVs (clusters M1 and M4, respectively). Between M2 and M3, amino acid sequence identity of the VP3 gene was 84.1–84.7 %, whereas high identities were observed within each cluster (92.3–97.6 % for M2, 98.2–99.3 % for M3). Sequence divergence among the four VP3 clusters was observed throughout the amino acid sequence except for conserved motifs, including those possibly related to enzyme functions of VP3. The presence of obvious genetic diversity only in the VP3 gene among human GCRVs suggested that either the M2 or M3 VP3 gene of human GCRVs might have been derived through reassortment from an animal GCRV or from an unidentified human GCRV strain belonging to a novel genogroup.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama1567-13481112011Full genomic analysis of a simian SA11-like G3P[2] rotavirus strain isolated from an asymptomatic infant: Identification of novel VP1, VP6 and NSP4 genotypes5763ENSouvikGhoshZipporahGatheruJamesNyangaoNoriakiAdachiNorikoUrushibaraNobumichiKobayashiWe report here the full genomic analysis of a simian SA11-like G3P[2] group A rotavirus (GAR) strain, B10, isolated from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the VP7–VP4–VP2–VP3–NSP1–NSP2–NSP3–NSP5 genes of strain B10 exhibited maximum genetic relatedness to those of the different isolates of simian strain SA11, and were assigned to the G3–P[2]–C5–M5–A5–N5–T5–H5 genotypes, respectively. On the other hand, the VP1, VP6 and NSP4 genes of strain B10 did not belong to any of the established GAR genotypes, and therefore, were assigned to new genotype numbers R8, I16 and E13, respectively, by the Rotavirus Classification Working Group. These observations suggested that strain B10 might have originated from reassortment event/s involving simian SA11-like strains and GAR strains from unknown animal host species (possibly other wild animals) preceding transmission to humans. Alternatively, considering the lack of data on simian GARs, it might be also possible that the VP1, VP6 and NSP4 genes of strain B10 are those of unknown simian strains, and that strain B10 might be a typical simian strain that was directly transmitted to humans. Therefore, either hypothesis pointed towards a rare instance of possible direct transmission of GARs from an animal host (possibly a monkey or some other wild animal) to humans. This was corroborated by the presence of different species of wild animals including non-human primates, and unhygienic conditions at the sampling site. To our knowledge, the present study is the first report on the detection of a simian SA11-like G3P[2] GAR strain in humans.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0168-160514332010Prevalence and antimicrobial resistance of Salmonella in retail foods in northern China230234ENHeYanLinLiM. JahangirAlamSumioShinodaShin-ichiMiyoshiLeiShiA total of 387 retail meat, seafood and milk powder samples were collected from nine cities in northern China in 2005 and screened for the presence of Salmonella. Salmonella strains isolated were subjected to serotyping and antimicrobial susceptibility testing. Salmonella was isolated from 81 (20.9%, 81/387) samples and classified into 23 serotypes. The isolates were frequently resistant to sulfamethoxazole (86.4%), sulfamethoxazole/trimethoprim (48.1%), nalidixic acid (30.9%), tetracycline (19.8%), carboxybenzylpenicillin (17.3%), amoxicillin (17.3%) and ampicillin (16.0%). The multiple resistance (resistance to ≥ 3 antibiotics) was found in 29.6% (n = 24) isolates. Additionally, 4 isolates from chicken displayed the ACSSuTNx profile, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline and nalidixic acid, in particular, strain HBS084 showing the resistance to as many as 20 antibiotics. Salmonella from chicken showed the higher frequency of antimicrobial resistance. Our findings indicate that in northern China food products of animal origin can be a source of exposure for consumers to multiresistant Salmonella strainsNo potential conflict of interest relevant to this article was reported.BioMed Central Ltd.Acta Medica Okayama1757-474922010Trend of Entamoeba histolytica infestation in KolkataENAvik KMukherjeeKaushikDasMihir KBhattacharyaTomoyoshiNozakiSandipanGangulyBackground:
Entamoeba histolytica infection is found almost all over the world and is highly endemic and a major cause of parasitic diarrhoea particularly in the developing countries.
Methods:
A systemic surveillance was set up at the Infectious Disease hospital, Kolkata, India between November 2007 and October 2009 for understanding the trend of E. histolytica infection in Kolkata. Fecal samples were collected from diarrhoeal patients attending the hospital, under the surveillance system and processed for detection of E. histolytica.
Results:
During the last two years about 2500 diarrhoeal samples were collected and screened for E. histolytica. About 3.6% were positive for E. histolytica. As compared to the earlier years, E. histolytica infection was observed to be less amongst patients screened during the last two years. No seasonality was observed in Kolkata although in the neighboring tropical country Bangladesh, a typical seasonality of E. histolytica infection was noticed.
Conclusion:
The study indicates that the detection rate of E. histolytica infection amongst diarrhoeal patients in Kolkata is decreasing during the last two years than that of Bangladesh.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama0095-113748112010Cholera Toxin Production by the El Tor Variant of Vibrio cholerae O1 Compared to Prototype El Tor and Classical Biotypes42834286ENJGhosh-BanerjeeMSenohTTakahashiTHamabataSBarmanHKoleyA. KMukhopadhyayTRamamurthySChatterjeeMAsakuraSYamasakiG. BNairYTakedaVibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0022-13179192010Complete genome constellation of a caprine group A rotavirus strain reveals common evolution with ruminant and human rotavirus strains23672373ENSouvikGhoshMohammed MahbubAlamMuzahed UddinAhmedRafiqul IslamTalukdarShyamal KumarPaulNobumichiKobayashiThis study reports the first complete genome sequence of a caprine group A rotavirus (GAR) strain, GO34. The VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of strain GO34, detected in Bangladesh, were assigned to the G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genotypes, respectively. Strain GO34 was closely related to the VP4, VP6–7 and NSP4–5 genes of bovine GARs and the NSP1 gene of GO34 to an ovine GAR. Strain GO34 shared low nucleotide sequence identities (<90 %) with VP2–3 genes of other GARs, and was equally related to NSP3 genes of human, ruminant and camelid strains. The VP1, VP6 and NSP2 genes of strain GO34 also exhibited a close genetic relatedness to human G2, G6, G8 and G12 DS-1-like GARs, whereas the NSP1 of GO34 was also closely related to human G6P[14] strains. All these findings point to a common evolutionary origin of GO34 and bovine, ovine, antelope, guanaco and human G6P[14] GARs, although phylogenetically GO34 is not particularly closely related to any other rotavirus strains known to date.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0304-40171711-22010Molecular characterization and assessment of zoonotic transmission of Cryptosporidium from dairy cattle in West Bengal, India4147ENShahbaz ManzoorKhanChanchalDebnathAmiya KumarPramanikLihuaXiaoTomoyoshiNozakiSandipanGangulyFew studies in the past have examined the genetic diversity and zoonotic potential of Cryptosporidium in dairy cattle in India. To assess the importance of these animals as a source of human Cryptosporidium infections, fecal samples from 180 calves, heifers and adults and 51 farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the 18S rRNA gene of Cryptosporidium followed by DNA sequencing of the PCR products. Phylogenetic analysis was carried out on the DNA sequences obtained in the study and those available in GenBank. The overall prevalence of Cryptosporidium in cattle was 11.7% though the infection was more prevalent in younger calves than in adult cattle. The occurrence of Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium andersoni in cattle followed an age-related pattern. A Cryptosporidium suis-like genotype was also detected in a calf. Farm workers were infected with Cryptosporidium hominis, C. parvum and a novel C. bovis genotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of Cryptosporidium infections between cattle and humans on dairy farms in India.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0022-13179172010Analysis of genetic diversity and molecular evolution of human group B rotaviruses based on whole genome segments17721781ENDaiYamamotoSouvikGhoshBalasubramanianGaneshTriveniKrishnanMamtaChawla-SarkarMohammed MahbubAlamTin SabaiAungNobumichiKobayashiGroup B rotavirus (GBR) is a rare enteric pathogen that causes severe diarrhoea, primarily in adults. Nearly full-length sequences of all 11 RNA segments were determined for human GBRs detected recently in India (IDH-084 in 2007, IC-008 in 2008), Bangladesh (Bang117 in 2003) and Myanmar (MMR-B1 in 2007), and analysed phylogenetically with the sequence data of GBRs reported previously. All RNA segments of GBR strains from India, Bangladesh and Myanmar showed >95 % nucleotide sequence identities. Among the 11 RNA segments, the VP6 and NSP2 genes showed the highest identities (>98 %), whilst the lowest identities were observed in the NSP4 gene (96.1 %), NSP5 gene (95.6 %) and VP8*-encoding region of the VP4 gene (95.9 %). Divergent or conserved regions in the deduced amino acid sequences of GBR VP1–VP4 and NSP1–NSP5 were similar to those in group A rotaviruses (GARs), and the functionally important motifs and structural characteristics in viral proteins known for GAR were conserved in all of the human GBRs. These findings suggest that, whilst the degree of genetic evolution may be dependent on each RNA segment, human GBR may have been evolving in a similar manner to GAR, associated with the similar functional roles of individual viral proteins.No potential conflict of interest relevant to this article was reported.BioMed Central Ltd.Acta Medica Okayama1757-474922010Emerging trends in the etiology of enteric pathogens as evidenced from an active surveillance of hospitalized diarrhoeal patients in Kolkata, IndiaENGopinath BalakrishNairThandavarayanRamamurthyMihir KumarBhattacharyaTriveniKrishnanSandipanGangulyDhira RaniSahaKrishnanRajendranByomkeshMannaMrinmoyGhoshKeinosukeOkamotoYoshifumiTakedaBackground: This study was conducted to determine the etiology of diarrhoea in a hospital setting in Kolkata. Active
surveillance was conducted for 2 years on two random days per week by enrolling every fifth diarrhoeal patient
admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata.
Results: Most of the patients (76.1%) had acute watery diarrhoea in association with vomiting (77.7%) and some
dehydration (92%). Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of diarrhoea. Among
Shigella spp, S. flexneri 2a and 3a serotypes were most predominantly isolated. Enteric viruses, EPEC and EAEC were
common in children <5 year age group. Atypical EPEC was comparatively higher than the typical EPEC. Multidrug
resistance was common among V. cholerae O1 and Shigella spp including tetracycline and ciprofloxacin. Polymicrobial
infections were common in all age groups and 27.9% of the diarrhoea patients had no potential pathogen.
Conclusions: Increase in V. cholerae O1 infection among <2 years age group, resistance of V. cholerae O1 to tetracycline,
rise of untypable S. flexnerii, higher proportion of atypical EPEC and G. lamblia and polymicrobial etiology are some of
the emerging trends observed in this diarrhoeal disease surveillance.No potential conflict of interest relevant to this article was reported.SpringerActa Medica Okayama0304-860815522009Molecular characterization of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of bovine group B rotaviruses: identification of a novel VP4 genotype159167ENSGhoshNKobayashiSNagashimaMChawla-SarkarTKrishnanBGaneshTNNaikStudies on bovine group B rotaviruses (GBRs) are limited. To date, only the VP6 gene of a single bovine GBR strain and the VP7 and NSP5 genes of a few bovine GBR strains have been sequenced and analyzed. In the present study, using a single-primer amplification method, we have determined the full-length nucleotide sequences of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of three bovine GBR strains from eastern India. In all six of these genes, the bovine GBR strains shared high genetic relatedness among themselves but exhibited high genetic diversity with cognate genes of human, murine and ovine GBRs. Interestingly, as with group A rotaviruses, the bovine GBR VP1, VP2, VP6 and NSP2 genes appeared to be more conserved than the VP4 and NSP1 genes among strains of different species. The present study provides important insights into the genetic makeup and diversity of bovine GBRs, and also identifies a novel GBR VP4 genotype.No potential conflict of interest relevant to this article was reported.