Draft-genome analysis provides insights into the virulence properties and genome plasticity of Vibrio fluvialis organisms isolated from shrimp farms and Turag river in Bangladesh.

Vibrio fluvialis is an opportunistic waterborne and seafood-borne enteric pathogen capable of causing severe diarrhea leading to death. This pathogen is endemic to Bangladesh, a country which is a major producer of cultured shrimp and wild-caught prawns. In this study, we carried out whole-genome sequencing of three V. fluvialis organisms isolated from shrimp farm and river sediment showing strong pathogenic characteristics in vivo and in vitro and compared their genomes against other V. fluvialis and related pathogenic species to glean insights into their potential as pathogens. Numerous virulence-associated genes including hemolysins, cytolysins, three separate Type IV pili, Types II and VI secretion systems, biofilm, and the V. cholerae pathogenesis regulating gene, toxR, were identified. Moreover, we found strain S-10 to have the propensity to acquire antibiotic resistance genes through horizontal gene transfer. These findings indicate that shrimp farms and rivers could be potential sources of V. fluvialis organisms which are an infection threat of public health concern.

Analysis of Different Prognostic Indicators for Malnutrition and Shigella flexneri Infection Among the Children in Bangladesh.

The present study investigates into some socio-economic, demographic, and nutritional features that can predispose Bangladeshi children to malnutrition and Shigella flexneri infection. Significant prognostic indicators associated with malnutrition were mother's illiteracy (unadjusted odds ratio, OR = 2.580; 95% confidence interval, CI = 1.134-5.867 and adjusted odds ratio, ORa, 3.814; 95% CI = 1.124-12.943), low birth weight (OR = 3.143; 95% CI = 1.2-8.232 and ORa = 2.404; 95% CI = 0.870-6.644) and poor socioeconomic status (OR = 2.549; 95% CI = 1.382-4.701 and ORa = 1.808; 95% CI = 0.852-3.838). Age was the strongest predictor for the acquisition of S. flexneri infection in the studied population (OR = 5.472; 95% CI = 2.583-11.592 and ORa = 5.808; 95% CI = 2.420-13.942). The severity of malnutrition was significantly (P = 0.033) related to seroprevalence of S. flexneri infection. To reduce malnutrition emphasis should be given on controlling the incidence of low birth weight, improving the literacy status especially of mothers and narrowing the gap between different socio-economic levels. Malnourished children should be examined for severity and direct intervention therapy should be given when necessary.

Local bacteriophage isolates showed anti- Escherichia coli O157:H7 potency in an experimental ligated rabbit ileal loop model.

Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity (P < 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E. coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages.

Analysis of immune responses and serological cross reactivities among Vibrio cholerae O1, Shigella flexneri 2a and Haemophilus influenzae b.

Antigenic determinants expressed on the bacterial cell surface are of importance in the serological characterization and microbiological diagnosis. The bacterial strains carrying these identical or similar antigenic epitopes might react with antibodies produced against other strains. In this study, strong immunogenicity and antigenic cross reactivity were demonstrated among V. cholerae O1, S. flexnerii 2a and H. influenzae b surface components. The enzyme linked immunosorbent assay (ELISA) results were supported by Western blot analysis, where at least 20 antigenic bands, were obtained in each of the reactions, when the surface components were reacted with the homologous antisera. The indirect ELISA results also demonstrated high degree of antigenic relatedness between the surface components of these species, where each surface component was reacted with the heterologous antisera. Western blot analysis also revealed cross reactions between the surface components suggesting common distribution of antigens/epitopes in these bacterial species. This study, thus, gave a clear idea of the level of antigenic sharing and variations among the pathogenic V. cholerae O1, S. flexneri 2a and H. influenzae b strains, which in future, may help in selecting a proper candidate for vaccines and immunodiagnostics development.

Analysis of immune responses against H pylori in rabbits.

AIM: To investigate the immunogenicity of H pylori proteins, to evaluate the production rate of anti H pylori IgG antibodies in relation to time and to demonstrate the fidelity of newly optimized in-house enzyme-linked immunosorbent assay (ELISA) technique as an alternative for H pylori infection assay. METHODS: In the present study, 100 microg of formalin-fixed H pylori whole cell antigens was injected into an experimental animal (New Zealand white female rabbit) intramuscularly on d 0, 16, 27 and 36. The first two doses were injected with adjuvants. On d 0, a serum sample was collected from the rabbit before immunization and this pre-immunized serum was used as a negative control for the whole study. To evaluate the immunogenic responses of the injected antigen, serum samples were collected from the rabbit at regular intervals up to d 42. The sera were analyzed using in-house ELISA and Western blot techniques. RESULTS: The production of anti H pylori IgG antibodies in the rabbit in response to the injected antigen increased almost exponentially up to d 14 and after that it was maintained at the same level until the last day (d 42). By analyzing the immune profiles of immunized sera, 11 proteins were identified to be immunogenic, among them 2 (approximately 100 kDa and 85 kDa) were most prominent. CONCLUSION: Analysis of the immune responses against pathogenic microorganisms like H pylori is necessary for the development of various diagnostic and preventive approaches. The results of this experiment reveal that the formalin-fixed H pylori whole cell antigens injected into the rabbit are highly immunogenic. These prominent proteins (approximately 100 kDa and 85 kDa) might have higher immunogenic effects among humans infected with H pylori and some of these immunogenic proteins can be included in diagnostic approaches based on serology and also for vaccine formulation. The in-house ELISA is a promising alternative compared to invasive techniques.

Prevalence of genotypes and subtypes of hepatitis B viruses in Bangladeshi population.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergences in the entire genome of >8 %, HBV genomes have been classified into ten genotypes designated as A to J. The aim of this study was to determine HBV genotypes and subtype in samples of HBV infected patients in Bangladesh. The sera samples were collected from chronically infected HBV patients. At first the DNA positive HBV samples were screened by EIA in our laboratory and the 1063 bp region of surface gene was amplified, sequenced and genotyped by sequence analysis. The same sequences were also used for subtypes and mutational analyses. After that, genotyping was also carried out by nested PCR using genotype specific primers in the same region of HBV surface gene. A total of 39 samples were sequencing to find out the genotypes and subtypes. It was found that the prevalent genotype was genotype C (subgenotype C1) which accounted for 48.7 %. The other genotypes found were genotype A (23.1 %) and genotype D (28.2 %). Predominant subtypes in Bangladesh were adr (41 %) followed by subtype adw2 (28.2 %), ayw3 (25.6 %), and others. Additionally, genotyping was also done by nested PCR using type-specific primers. In this method, out of 17 samples 6 were found to be genotype C, followed by genotype D (4 of 17) and genotype A (3 of 17). In PCR-based genotyping system we also observed the mix genotypes; 3 samples contained both genotype A and D, and 2 samples contained both C and D. The genetic diversity of HBV and distribution of its genotypes and subtypes amongst Bangladeshi population were done in this study, which will help us to provide information regarding circulating genotypes in this region and also help physicians to prescribe proper antiviral/interferon therapy.

Distribution of coliphages against four e. Coli virotypes in hospital originated sewage sample and a sewage treatment plant in bangladesh.

The distribution of coliphages infecting different Escherichia coli virotypes (EHEC, EIEC, EPEC, ETEC) and an avirulent strain (K-12) in sewage system of a hospital and a sewage treatment plant (STP) was investigated by culture-based agar overlay methods. Coliphages were found in all the samples except stool dumping site in the sewage system of the hospital and lagoon of the STP. Bacteriophage count (pfu/ml) infecting E. coli strains showed the following ascending pattern (EHEC < EIEC < EPEC < ETEC < E coli K-12) in all the collected samples except one. Phages capable of infecting avirulent E. coli K-12 strains were present in the highest number among all the examined locations. Phages specific for E. coli K-12 presented high diversity in plaque size on the bacterial lawn. Virulent E. coli specific coliphages rarely produced plaques with diameter of 1-2 mm or over. Conventional agar overlay method was found to be not satisfactory for phage community analysis from primary stool dumping site of the hospital, probably due to the presence of high concentration of antimicrobial substances. The gradual decrease seen in the five groups of coliphage quantity with the ongoing treatment process and then the absolute absence of coliphages in the outlet of the examined treatment plant is indicative of the usefulness of the treatment processes practiced there.

Pregnant women in and around dhaka city: are their children at risk of developing congenital rubella syndrome?

Rubella Virus (RUBV) is a common cause of childhood rash and fever in non-immunized populations, and its public health importance relates to teratogenic effects of primary rubella infection in women with early pregnancy. Infection of the fetus may lead to congenital rubella syndrome (CRS). This work aimed to assess the degree of risk associated in acquiring rubella virus infection by the women during pregnancy and developing CRS among their children in Bangladesh. The study population (n = 275) included pregnant mothers (15-38 years) from various socioeconomic backgrounds attending a women health care based hospital. All subjects were personally interviewed, clinically examined and a standardized questionnaire was filled up for each of them. From each participant 3 ml blood was taken and serum was separated. Commercially available ELISA kit was used for the qualitative and quantitative determination of IgM and IgG class antibodies against RUBV in collected serum samples. 209 women were found to contain detectable level of antiRUBV IgG antibodies, but did not possess IgM antibodies against rubella. Only 9% participants were vaccinated previously against rubella virus among the whole antenatal population studied. Ninety-two percent of these vaccinated pregnant women contained serum anti-rubella IgG antibody which was significantly (P = 0.05) higher than that of the nonvaccinated study population (75%). Pregnant women from lower middle and poor socioeconomic class had significantly (P = 0.05) more intra uterine growth retardation (IUGR) of fetus than the upper middle class. 20% of the women of child bearing age examined in this work were not yet exposed to RUBV and at risk of acquiring this virus during pregnancy and subsequently transmitting the virus to the fetus. Our work demonstrates rubella attack rate among antenatal population in Bangladesh as 14.5 in 1000 during pregnancy. A proper and reliable vaccination policy against rubella virus is not yet adopted at the national level in many developing countries including Bangladesh. This work identifies the requirement of detailed study for the identification of intrauterine rubella infection and its related influence on perinatal morbidity and mortality. Thorough epidemiological studies are also considered necessary prior to the development and acceptance of national immunization program against rubella virus in Bangladesh.