Department of Microbiology

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Epidemiology and molecular characterization of circulating foot-and-mouth disease viruses of cattle and vaccine development in Bangladesh
(University of Dhaka, 2000-05-19) Ullah, Huzzat
Foot-and Mouth Disease (FMD) is the major impediment of livestock production and trade worldwide. Currently there are seven FMDV serotypes, namely O, A, C, Asia-1 and Southern African Territories (SAT) 1-3, which infect cloven-hoofed animals. Due to huge local demand for meat, milk and sacrificing animals, livestock industries are rapidly growing in Bangladesh. Furthermore, there is a great prospect of healthy meat export in the Middle East. To control FMD, mass vaccination of cattle every six months with a trivalent FMD vaccine (locally produced or imported from type O, A, and Asia-1) is practiced in Bangladesh. But the current vaccination strategy does not provide sufficient protection of the herd. Although FMD vaccines need to be adequately matched to the field virus to ensure sufficient protection against a challenge with a field virus, but limited/ no step is adopted in Bangladesh so far to match vaccines before implementation. Moreover, FAO and OIE have proposed a time-frame strategy for the progressive control of FMD (PCP-FMD) road map and accordingly, Bangladesh will achieve stage 2 in 2016- provided that epidemiological studies, risk identification, and fixed riskbased control plan are completed. Unfortunately, due to poor/no reporting system by the appropriate authorities of FMD to the OIE, inappropriate vaccination, and poor veterinary service care, Bangladesh is placed at stage 0 (non-reporting stage). It is hypothesized that proper epidemiological studies and characterization of circulatory FMDVs to select the appropriate vaccine candidate in Bangladesh is the prime step to fix risk-based strategies to implement FMDcontrol program and achieve the PCP-FMD goal. To address the hypothesis, demographic data were collected using a questionnaire at field level infected farms; 283 FMDV-infected tissues were sampled from the 39 individual FMD outbreaks within May 2012 to April 2016 and characterization was done by PCR based identification of VP1 gene, isolation of viruses in BHK21 cell-culture, genome sequencing and analysis. The results demonstrated that the disease is endemic in Bangladesh. The husbandry systems, practiced in the investigated herds were intensive, semi-intensive with free animal movement or extensive. Demographic data revealed that the average morbidity rate of FMD was 53.8% with a higher incidence in indigenous cattle (55.7%) than that of crossbred cattle (49.2%). The highest number of outbreaks occurred in October (23.1%), followed by December (12.8%) and March or September (10.3%) which was decreased gradually up to mid-August, and in April (0%). Among seven serotypes distributed worldwide, FMDV type O, A and Asia-1 were circulating in Bangladesh and type O FMDV accounted for the most outbreaks (87%), followed by Asia-1 (8%) and A (5%) type virus. Phylogenetic analysis revealed a single lineage and topotype of FMDV serotypes O (Ind2001 lineage and O/ME-SA topotype), A (genotype VII of Asia topotype) and Asia-1 (genetic lineage C) circulatory in Bangladesh; and intrusion of FMDV occurred from India and vice versa. The complete genome of serotype O [KF985189] and A [KJ754939] were retrieved to be 8131 nucleotides (nt) and 8220 nt in length, respectively. Comparative genome analyses with reference sequence or vaccine strain revealed that within serotype O, 82 nt deletion in S-fragment and 43 nt insertion in 5'-UTR resulting introduction of an extra pseudoknot structure, whereas a 84 nt insertion within the 5′-UTR, a lengthened polyC tract was observed in serotype A. Within VP1, variation in B-C loop (40~60), G-H loop (133~160) in both serotypes and a 10 amino acid insertion (position 92~101) in 3A protein within serotype O were found. In summary-(i) three types of FMDVs are prevailing in Bangladesh (ii) intrusion of FMDV occurred from neighboring countries and (iii) inappropriate vaccination with mismatch virus strain caused vaccine failure. Finally, for effective control and prevention of FMD, proper epidemiology data, disease reporting, animals‟ movement quarantine, appropriate vaccination and strong political will of the government are required.
Characterization of the immune response to vibrio cholerae O1 antigens and its impact on vaccine development strategies
(University of Dhaka, 2014-07-13) Ahmed, Firoz
Cholera persists as one of the major causes of mortality and morbidity globally. Prevention using different approaches are needed, one of which is vaccination. Improving immunogenicity of oral cholera vaccines and production of effective single dose schedules suitable for all age groups and populations is the prerequisite. Historically injectable cholera vaccines have been replaced in the last three decades by oral mucosal vaccines but neither fulfills all the requisites. The aim was to evaluate potential vaccine immunogens which have not been tested earlier for generating protective immunity. The concept of a one dose vaccine using bacterial antigen(s) through a “needle free approach” of immunization via the transcutaneous route (TCI) for enteric vaccines was explored in this work. The lipopolysaccharide (LPS) of Vibrio cholerae O1 is the most consistently described protection associated immunogen and has been identified as the most important of the antigens described so far. Epidemiological studies of natural infection in cholera suggest that Inaba serotype is more protective against subsequent attacks of cholera than the Ogawa serotype. Crystallographic studies have revealed antigenic determinants of LPS Ogawa and binding pattern of monoclonal antibodies (mAbs) using synthetic fragment of Ogawa in the O-specific polysaccharide (O-PS) region. Previous studies with other mAbs have indicated involvement of both the core and the O-specific polysaccharide as common antigenic determinants. This dissertation describes the first crystal structure of a mAb F-22-30 that recognizes a common epitope present in both the Ogawa and Inaba serotypes of V. cholerae O1. Modeling calculations and preliminary NMR results suggest that binding pocket at the center of antibody recombination site could bind side-chain of a perosaminyl residue from the O-PS with higher accessibility compared with core-PS implying a lateral recognition in contrast to antibodies specific for the Ogawa serotype. Mechanism of the immune response elicited by transcutaneous route of immunization (TCI) with potential V. cholerae antigens was evaluated as alternative modes of vaccination compared with oral (ORI) and subcutaneous route (SCI) with the same antigens i.e. recombinant cholera toxin B subunit (CTB), cholera toxin (CT), recombinant toxin co-regulated pilus A (TcpA), LPS Ogawa and Inaba and in-house prepared membrane extract in Balb/c mice to determine the immunoglobulin isotype patterns and its properties linked with protection. Both humoral and cellular immune responses induced by the immunization were studied using ELISA and flow cytometric procedures to assess and phenotypic changes in the mucosal and systemic compartments were analyzed. Among the three routes of immunization, SCI showed robust elevation pattern with superiority of IgG compared toIgM and IgA respectively. TcpA in the TCI route did not up-regulate IgA, while CT and CTB were seen to respond with all three antibody isotypes. CTB immunized mice showed reduced serum antibody titer in the zinc supplemented but CT and TcpA individually as well as co-administered together had no effect. When CT in the TCI and SCI route generated all four IgG subclasses, TcpA and LPS-specific antibody response was only IgG1 subclass where membrane extract has overcome the inherent limitation of LPS. However, sera obtained from both CT and TcpA immunized were negative for vibriocidal antibody responses. Membrane extract displayed more than 10 times elevated IgM and IgG titers with vibriocidal response than LPS with comparable positive effect of zinc supplementation. Moreover, ALS antibody titers were comparable with ELISA and vibriocidal assays and expressed the recently formed status of the humoral response. Expression of increased CD19+ve B cells in blood and lamina propria as well as CD4+ cells in the lamina propria were detected following TCI. Significant elevations of the co-stimulatory marker CD40 in the CD19+ve B cell population as well as CD28 and CD154 in the CD4 population were observed in spleen and lamina propria. The expression of homing markers, cutaneous lymphocyte antigen (CLA) was increased in blood and lamina propria (CD19, CD4), whereas an increase of the gut homing marker (β7) was not seen in blood or the mucosal surface in the different cell types. The results of TCI immunization were compared with those obtained after subcutaneous (SCI) where similar or discordant results were seen but interestingly the magnitude of responses by TCI was equal to that seen by SCI. The isotype switch and antibody maturation patterns elicited by the TCI route of immunization incorporating zinc as another confounder with the potential immunogens, applying a novel in-house immunogenetic method, were studied. Finally, bioinformatics approaches were applied to compare position specific similarities and dissimilarities of LPS specific 'membrane extract' immunized antibody sequences with experimentally characterized mAb F-22-30 for better understanding the binding affinity of the carbohydrate antigen. Considering the percentages of antigen-specific cellular representation at different compartments, CTB specific cells was the highest compared to TcpA and LPS specific pool. With the novel in situ positive selection immunogenetic technique, first murine B-cell VH and VL chain repertoire specific for protein and polysaccharide antigens were reported. VH and VL amino acid sequence comparison demonstrated variable expression in co-administration than individual immunogens. Amino acids expressed through immunization of polysaccharide immunogens were more stable than protein immunogens specific sequences. Moreover, CDRH3 length of antibody sequences to protein immunogens varied between 8 and 19 amino acids with CTB specific shorter sequences containing more charged and hydrophobic residues. Further bioinformatics approaches with Inaba specific sequences obtained through TCI confirmed ligand binding with similar nature amino acids to be identical groove type binding pattern. Findings presented here suggest that the newer, needle-free TCI route is suitable for inducing humoral and cellular immune responses in the systemic and mucosal compartments and should be considered as an option in vaccine development strategies.
The impact of biofilm formation on the survival of Vibrio cholerae in aquatic environment and its role in pathogenicity
(University of Dhaka, 2015-01-28) Sultana, Marzia
Vibrio cholerae, the causative agent of epidemic cholera, has been established as an autochthonous flora of the aquatic environment, persisting predominately as non-culturable cells within clusters of biofilms and in association with plankton. Although biofilm has been proposed to serve as an important reservoir for V. cholerae, very little is known about the factors responsible for biofilm formation in the natural aquatic ecosystem, including its role in the active growth and survival, especially how the bacterium turns actively growing to initiate the seasonal outbreaks of cholera. Laboratory microcosm prepared with water collected from estuarine ecosystem of Mathbaria, Bangladesh showed slower biofilm formation and extended culturability for 68 days at 4°C, as compared to microcosms that were maintained at higher temperatures (30°C, 37°C, and 45°C), suggesting biofilm formation to be temperature dependent and negatively linked to loss of culturability. Mathbaria water (MW) microcosm supported the active growth of V. cholerae O1 for an extended period up to 54 days under higher pH (8.0-9.0), whereas microcosms having pH 6 and pH 7 supported the active growth for only 26 days and 40 days, respectively. Instant ocean (IO; salinity 1%) microcosms maintained at room temperature supported the active growth of V. cholerae for 54 days, while the IO microcosms with 0%, 2%, and 4% salinity supported the active growth for only 26 days. The biofilm formation was robust in Luria Bertani (LB) broth with 2% salinity and pH range 7.0-9.0 in presence of calcium chloride (10mM) and magnesium chloride (40mM). MW microcosm supplemented with dehydrated shrimp chitin chips (MW-CC) as the single source of nutrient supported both active growth of toxigenic V. cholerae O1 for up to six months and biofilm formation by the bacteria. V. cholerae O1 cells in chitin-associated biofilms remained metabolically active even in a high acidic environment without losing either viability or virulence. Biofilm-bound coccoid, nonculturable V. cholerae O1 cells maintained in MW microcosm for 495 days were able to be culturable upon passage through rabbit ileal loop (RIL). Genetic screening by polymerase chain reaction (PCR) of V. cholerae cells in MW microcosm revealed loss of several virulence and related genes including ctxA, ace, zot, rstR, tcpA, acf, toxT and two of four mshA genes. The mutant strains lacking mshA genes produced significantly less amount of biofilm in the air-water interface of the borosilicate glass tubes when compared with the parent strain. Pulsed-field gel electrophoresis (PFGE) analysis of the genomic DNA (NotI-digested) of the mshA-mutant daughter strains revealed them to be clonal, as they had identical PFGE pattern, but they differ from the parent strain. Results obtained from the field-based investigations in estuarine ecosystem of Mathbaria, Bangladesh have shown the presence of non-culturable V. cholerae cells as a component of the bacterial community of which only a negligible proportion could be enumerated when conventional culture methods were employed. In conclusion, V. cholerae biofilms formed in situ in response to physicochemical, and related biotic and abiotic factors of aquatic environment thus serve as an important reservoir where the bacterium can take refuge to, and persist as dormant cell until the favorable season returns for them to be actively growing again to increase the probability of infecting human and spreading cholera epidemics.
Identification and characterization of Neurotoxic factor(s) present in Shigella
(University of Dhaka, 2015-05-12) Azmi, Ishrat Jahan
Infection by Shigella is associated with a significant number of human deaths worldwide. Complications from Shigellosis include leukemoid reaction, hemolytic uremic syndrome (HUS), seizure, encephalopathy, abdominal cramps, rectal pain and septicemia, which significantly increase morbidity and mortality. Abnormalities in the central nervous system (CNS) and HUS are more common in children under 5 years of age. The majority of Shigella associated neurological findings were reported in patients with S. sonnei and S. flexneri. But both the species do not usually produce shiga toxin, as both are lacking the structural gene encoding shiga toxin production. However, neurotoxic principle(s) in S. flexneri has not yet been discovered. In this study an attempt was taken to identify neurotoxic factor(s) present in Shigella to understand the molecular basis of toxin induced mammalian cell death. A total of 385 clinical isolates of different serotypes of Shigella isolated between 1997 and 2013 from diarrhoeal patients attending the Dhaka treatment centre of icddr,b were included in this study. Of these, 238 were S. flexneri, 80 S. dysenteriae, 42 S. boydii and 25 S. sonnei. All these strains were characterized following standard Microbiological and Biochemical methods. These strains were screened for the presence of known toxin and virulence genes such as ipaH, ial, ipaBCD, set, sen and stx by PCR and plasmid profiling. Cytotoxic and neurotoxic activity of the toxin were determined using HeLa cells and cerebellar granule neurons from rat pups respectively. Fluid accumulation in Rabbit ileum and histopathological changes of the rabbit loop segments was studied for enterotoxic activity. DNA fragmentation and chromatin condensation assay were used as markers of apoptosis. The biological status of cells at different phases of cell cycle was analyzed by FACS at different time intervals. The toxin(s) preparations involved 40%, 60% and 80% saturation of the culture supernatant with ammonium sulphate. Toxin(s) containing fraction was prepared by column chromatography using sephadex G-75. Of 385 isolates, PCR and plasmid analysis showed that almost all serotype of Shigella contain the 140MDa plasmid and sen gene except 17 S. flexneri 1c strains. All S. dysenteriae 1 contained stx gene whereas the set gene is only present in S. flexneri 2a and rarely in S. flexneri 2b. Seventeen 140 MDa lacking S. flexneri 1c strains were also negative for the common toxin gene. These 17 strains were noninvasive by sereny and Congo red binding test. Therefore, these 17 S. flexneri 1c strains were extensively used for comparative analysis. S. flexneri 1c (K-314 and K-915) were used as a representative of these 17 strains for partially purified toxin fractions and source of novel toxic factor(s) in this study. The degree of cytotoxicity differs in different species of Shigella. Around 72% (172/238) strains of S. flexneri possessed the cytotoxic activity. Of S. flexneri, serotype 1c had severe effect (more than 85% cell death) on HeLa cell. Serotype 2a, 2b, X variant and type 4 had almost similar cytotoxic effect (>70% cell death) while serotype 3a, Y variant, 4X, 6a and 6b showed less cytotoxic effect (40-45% cell death). In case of S. dysenteriae (n=80), only serotype 1 and some strains of serotype 4 showed cytotoxic effect (>90% cell death). In contrast, S. boydii (n=42) supernatant caused elongation of the HeLa cells whereas S. sonnei (n=25) caused the rounding of the cell with less frequency. It is interesting to note that the 17 S. flexneri 1c strains lacking 140 MDa plasmid and toxin gene showed strong cytotoxic effect (>90% cell death) on HeLa cells in dose dependant manner. Further analysis showed that toxic factors in these S. flexneri 1c exhibited biochemical properties similar to those of protein. Heat treatment significantly reduced cytotoxic activity of S flexneri 1c (K-314). DNase and RNase treatment did not affect cytotoxic activity while protease treatment significantly reduced its cytotoxic activity. HeLa cells were also treated with 40%, 60% and 80% fractions of S. flexneri 1c K-314 strain. However, only 60% fraction showed strong cytotoxic activity. This fraction contains an active protein component molecular mass of 100–125kDa which is sensitive to heat and trypsin. In the rabbit ileal loops assay, S. flexneri 1c (K-314) was unable to accumulate fluid. But the histo-pathological report of the loop segments showed moderate inflammatory infiltrate in lamina propria, submucosa of the wall of the intestine and sheared off of tips villi in some places with Grade-3 inflammation. However, lysates S. dysenteriae 1 caused fluid accumulation and enterocyte necrosis and shearing of tip of villi with Grade-4 inflammation. In this report, the culture supernatant of S. flexneri exhibited not only the cytotoxic activity, but also impaired neurological function in mice. The LD50 dose was found to be 1 µg. The sign of toxic effect appeared after 30h and finally hind limb paralysis and rapid breathing were observed and all mice died within 36h. In primary rat brain (cerebellar) neurons culture model it was found that these strains showed strong neurotoxic activity. The neurite length (at least 100 neurons from random fields) was determined from both toxin treated and untreated neurons. Reduction of neurite length indicated the presence of neurotoxic factor. S. flexneri induced apoptosis in HeLa cells. The toxin fraction(s) has been shown to induce apoptosis as determined by morphological changes (rounding) inhibiting polymerization of actin filament, chromatin condensation, and chromosomal DNA fragmentation in HeLa cells. Phosphorylation of the H2AX and release of cytochrome C indicated that toxin from S. flexneri might activate additional pathways leading to apoptosis. The progression of HeLa cell was arrested at G 0 /G phase as the percentage of cells in that phase increased gradually from 17.25 to 51.52 with time. This study represents the first attempt for identification and characterization of neurotoxic factor present in S. flexneri. The toxin produced by S. flexneri 1c showed cytotoxic, enterotoxic and neurotoxic activity. The above evidences suggested that toxic factors in S. flexneri are different from that of stx, set, and sen. Identification and characterization of toxic factors in S. flexneri may provide critical information in understanding the molecular basis of Shigella-induced neurotoxic effect on different types of human cells.
Characterization of microbial agents in diabetic foot infection and its correlation with chronic subclinical inflammation
(University of Dhaka, 2015-05-24) Karmaker, Mousumi
Diabetes mellitus (DM) is a metabolic disorder with chronic physiological complication increased risk of developing Diabetic Foot Infection (DFI) by 25% in diabetics’ patients. The pathophysiological differences between DFI and non-DFI patients may alter microbial compositions in infections. The present study aims at culture based comparative analysis of microbes’ colonized in DFI and non-DFI patients in Bangladesh and explore their correlation with chronic subclinical inflammation (CSI). Pus specimens were collected from 67 DFI and 10 non-DFI patients. Most of them were from rural area; belong to lower middle income group and age between 28-75 years. Enterococcus spp. (9%), and Klebsiella spp. (8%) occurred only in DFI and the other predominant bacteria in DFI over non-DFI patients were Enterobacter spp. (22%) and Bacillus spp. (12%). In contrast, non-DFI patient’s samples predominated Pseudomonas spp. (29%) and Citrobacter spp. (29%). In both cases, Staphylococcus spp. (13%) and Acinetobacter spp. (10%) contained same percentages. The statistical calculation (Comparative bacterial significance analysis: at 90% confidence level) revealed that the rate of occurrence of three organisms; Enterococcus spp. |Z|=2.2125, Klebsiella spp. |Z|= 1.732, Bacillus spp. |Z| =1.9034 is significance in DFI patients compare to non-DFI patients. However in non-DFI patients rate of occurrence of Citrobacter spp. |Z|=14.1901 were significance. Most of the isolates from DFI patients were commonly resistant to cephalosporins (ceftazidime, ceftriaxone, cefuroxime) and monobactam (azteronam) groups of antibiotics. Plasmid profile analysis of the isolates revealed that most of the multidrug resistant (MDR) isolates were plasmid free implicating chromosome mediated MDR properties. A plasmid free, P. studzeri isolated from DFI showed resistant to more than 12 groups of antibiotics. DFI patients had comparatively higher C-reactive protein (CRP) values than non-DFI patients; and there were positive correlation with MDR with CRP values. A significant correlation (p<0.01) were observed between high serum CRP and infection with MDR organism in DFI patients. In conclusion, this investigation showed that there is a clear demarcation of resistant microbiomes composition between DFI and Non-DFI patients’ samples and a positive correlation of CRP values and resistant microbiomes in DFI patients. These results have implication the need for an alternate treatment of DFI patients in Bangladesh.
Development of a PCR based diagnostic system for kala azar
(University of Dhaka, 2015-05-25) Khatun, Mahbuba
Kala-azar or black fever is a severe form of Leishmaniasis, a disease caused by protozoan parasite, Leishmania spp. one of the largest parasites-borne illnesses, this disease could be fatal, if untreated, as a consequence of infection to different internal organs such as liver, spleen, and bone marrow resulting in death. The conventional approach for the diagnosis of the disease relies on collecting samples from bone marrow and splenic tissues from suspected patients, a method which is not only painful, stressful and risky for patients, but also cumbersome. This prompted us to develop a simple, rapid PCR-based diagnostic assay for the detection of Leishmania using patient‟s venous blood sample. A total of thirty five clinically suspected febrile patients who had sign and symptoms for the disease, and produced positive reaction for rk39 immunochromatographic test were taken for the diagnosis. In addition to the blood samples, specimens from bone marrow/ splenic aspirations were collected from those individuals. While presence of Leishmania spp. was confirmed in twenty six patients either by microscopy and by culture on Nicolle Novy McNeal (NNN) culture media from the samples collected from bone marrow/spleen aspirations of patients with Kala-Azar, however their buffy coat preparations of blood samples could not produce the same, indicating the unsuitability of the blood samples to be used as a specimen for diagnosis by conventional methods. In an attempt to address whether those blood samples could be used for diagnosis, a pair of oligonucleotides was designed using conserved sequences of kinetoplast DNA minicircles of Leishmania spp and was used as primers to detect the presence of Leishmania genome in a polymerase chain reaction. In addition, blood samples collected from sixteen and twenty five healthy individuals from the endemic and non-endemic regions respectively, and twenty five patients with other similar diseases (TB, malaria and dengue) were used as controls. All the twenty six blood samples from patients with Kala-azar yielded specific amplifications in PCR, while blood samples taken as controls produced no amplicon, producing the sensitivity and specificity records as 98% and 100% respectively. Such an analysis was found superior when compared with two other sets of primers reported in the literature. The identity of the amplicon was subsequently confirmed as Leishmania by DNA sequencing and BLAST search. Therefore, the primers designed for this study could be used to score the presence of Leishmania in a PCR-based detection system.
Genetic mechanisms of persistence of the Culturable Vibrio cholerae in aquatic environment
(University of Dhaka, 2015-12-01) Jubair, Mohammad
Cholera, an ancient disease caused by toxigenic strain of Vibrio cholerae, remains a human scourge killing millions of people worldwide in the past through pandemics and epidemics, and still continues to be a major public health threat to countries where sanitation and clean drinking water are not optimal. According to World Health Organization (WHO), 48 countries (mostly from Africa, Asia and South Americas) reported 2-3 million cases of cholera with 100-120 thousand deaths. Although V. cholerae is autochthonous to aquatic environments, the physiologic and genetic basis of persistence of the organism is not clearly understood despite decades of research. In other bacterial species, it has been reported that a subpopulation of a bacterium can shift to a “persister” phenotype by stochastic mechanism in response to environmental stressors, including antibiotic and nutrient-poor/limitation stresses. Furthermore, reports are also available that bacteria persisting in stationary phase can assume a growth advantage stationary phase (GASP) where a subpopulation of bacterium sustains positive selection mutation. Both persister and GASP phenotype are shown to play major role in the persistence of bacteria in adverse survival conditions. I hypothesis that, like other bacterial species, V. cholerae can assume both persister and GASP phenotypes and that such phenotypes can promote environmental persistence of V. cholerae. As microcosms mimic environmental conditions, particularly relative to nutrient-poor environmental conditions, I used filtered sterilized lake water microcosms (FSLW) to test my hypothesis. My data presented in this work have indeed proved that a subpopulation of V. cholerae can assume persister and GASP phenotype and that both phenotypes can response to supplementation of complex nutrients, including chitin and phosphate. My data also exhibited that GASP phenotype promotes a biofilm specific to FSLW but not in nutrient rich L-broth. In summary, I demonstrate that V. cholerae can survive in culturable form for years in nutrient-poor conditions and that, persister and GASP phenotypes underwent many changes, including morphologic, genetic and physiologic changes that presumably contributed to their persistence in microcosms.
Clinical significance and molecular characterization of Extended Spectrum -Lactamases (ESBLs) producing escherichia coli associated with Urinary tract infection in Bangladesh
(University of Dhaka, 2015-12-01) Ibne Mahmood, Belal Ahmed
Patients having urinary tract infections (UTIs) with multidrug resistance bacteria suffer from various complications. This is due to -lactamase-production by certain group of bacteria. This study was undertaken to explore the molecular mechanisms of drug resistance caused by ESBL-producing Escherichia coli isolated from UTI patients in the Sylhet community of Bangladesh. A total of 100 urine samples from symptomatic UTI patients were used in this study. Twenty three isolates were found to be Klebsiella, which were discarded. Remaining 77 isolates were identified as Escherichia coli by standard methods in the laboratory. Of these isolates, 25 were further confirmed as ESBL-producing Escherichia coli by double disc-diffusion synergy test and these 25 were used in the present study. The patients’ age ranged from 4 to 60 years. The highest age incidence of UTI patients was 21-30 years (28%), followed by 31-40 years (20%), 50 years and above (19%) and 41-50 years (15%). The antibiotic susceptibility test for non-ESBL-producing organisms showed 100% sensitivity to imipenem (IMP) and meropenem (MPM). There were multidrug resistance (MDR) to amoxyclavulanic acid (AMC, 30%), ceftazidime (CAZ, 40%), ceftriaxone (CRO, 35%) and ciprofloxacin (CIP, 60%). Some isolates were sensitive to AMC (70%), CAZ (60%), CRO (65%) and CIP (40%). The ESBL-producing isolates were also 100% sensitive to carbepenems and some were resistant to AMC (76%), CAZ (55%), CRO (66%) and CIP (73%). Serogrouping showed that all ESBL isolates were heterogeneous and did not match with non-ESBL isolates. Nine ESBL isolates could not be typed and they were designated as untypeable. Plasmid profile analysis was done both for ESBL and non-ESBL producing isolates. The plasmid profile showed both ESBL and non-ESBL exhibits high molecular weight plasmids (>140 MDa), which were 68 and 75% respectively. These findings suggest that both ESBL and non-ESBL-producing isolates harbor large size plasmid, indicating that the isolates carry transmissible plasmids and they are responsible for multidrug resistance (MDR) in Sylhet community. The investigations were further carried out about the detection of genes encoding ESBL types by polymerase chain reactions (PCRs). The ESBL positive strains showed blaCTX-M-1 gene (60%), blaOXA-1 gene (41%) and 45% isolates constituted blaTEM-1 gene. PCRs were also done in all these 20 ESBL isolates for virulent genes. Only 2 isolates showed pilus-associated pylonephritis (pap) genes and one isolate showed afrimbial adhesion (afa) gene. Further data analysis revealed that out of 20, 12 (60%) isolates were having single gene, 7 (35%) isolates had double genes and 1 (5%) isolate had triple gene. Interestingly, it was seen that isolates having single gene (60%) were harboring 140 MDa plasmids. These large-sized plasmids are transmissible in the community of Sylhet District. These results suggest that multidrug resistance (MDR) is probably due to TEM-1 gene harboring 140 MDa plasmids in the community. Pulsed-field gel electrophoresis results showed no clonal relatedness among the ESBL isolates. Same findings were found in non-ESBL-producing isolates. In conclusion, the ESBL uropathogenic E. coli isolates were responsible for multidrug resistance (MDR) in the community of Sylhet Town. They also harbor large sized transmissible plasmids and these plasmids are encoded by genes such as TEM-1, OXA-1 and CTX-M-1. It is CTX-M-1 that constitutes the highest percentage (60%) in the district of Sylhet in Bangladesh.
Characterization of cry genes and insecticidal proteins from indigenous bacillus thuringiensis to develop potential biopesticide
(University of Dhaka, 2016-05-31) Shishir, Md. Asaduzzaman
Indiscriminate use of chemical pesticides, the cause of serious public health problems and environment pollutions as well as the emergence of pest resistance in many developing countries, could be reduced with Bacillus thuringiensis (Bt) biopesticides for its eco-friendly properties. The work was aimed at controlling vegetable pests by foliar spraying of locally developed potential Bt biopesticides as the facts of immunological adverse effects and increasing insect resistance due to the continuous exposure to the Bt toxins were reported for transgenic crops. Bt isolates were, therefore, isolated from different eco-regions of Bangladesh and characterized based on their phenotype, genetics, proteomics, diversity, toxicity and field efficacy. Upon biochemical typing of 298 indigenous Bt isolates, Bt indiana (17%), kurstaki (16%), thuringiensis (12%) were found to be the most prevalent biotypes in Bangladesh and from the distribution index, an indication of the degree of ubiquity, Bt thuringiensis (0.5) and indiana (0.5) appeared to be more ubiquitous than all other biotypes followed by eleven (0.43), kurstaki, sotto and nine (0.4 for each). Variation in abundance and distribution pattern of 16 different biotypes in Bangladesh was demonstrated in comparison to the other parts of the world. Broad spectrum cytolytic activity was observed to vary with biotypes and 58.36% strains were presumed to insecticidal based on this attribution. The molecular weight based distribution of small plasmids was compared among the biotypes and the maximum diversity was observed among Bt thuringiensis, kurstaki, indiana and israelensis biotypes. The screening for insecticidal genes in the native Bt strains revealed their presence in varied proportion being cry1 to be the most prevalent (30.8%) followed by cry2 (25.5%), cry3 (22.2%) and cry9 (7.2%). Prevalence of cry4, cry8 and cry10 genes were less than 5% and cry11 gene was not found in any strain. The SDS-PAGE analysis of the partially purified Cry proteins revealed the presence of Cry1- type delta endotoxins including Cry1Aa, Cry1Ab, Cry1Af, Cry1B, Cry1C and Cry1M, Cry2, Cry3, Cry8 and Cry9 proteins which were also supported by the cry gene profiles. Genetic diversity among 177 Bt strains were analyzed by RAPD-PCR which revealed 15 genotypes and type 9 and type 11 were found to contain more than 25% of the strains. In combination of RAPD-PCR and cry gene profile analysis, genotype based distribution of cry genes was evaluated and Genotype 1, 6, 9, 11 and 12 were found to contain the most of the cry genes at different extent while cry genes with diversities were most prevalent in genotype 6, 9 and 1. Genetic diversity analyzed thus is highly relevant and significant in discovering novel insecticidal genes in indigenous Bt strains and to deal with the problems of emerging resistance towards Bt toxins. Novel toxicity of indigenous Bt strains was identified at significant level against the larvae of Bactrocera cucurbitae and the efficacy of Bt JSc1 was highly comparable with the reference strain Btk HD-73 with no statistically significant difference. The complete Open Reading Frame (ORF) of cry1A- type gene from Bt strain JSc1 was, therefore, obtained by PCR walking technique and was revealed to be identical to cry1Aa9 and cry1Aa13 genes. Five conserved block of Cry proteins were identified from the deduced amino acid sequence analysis and the 3-D protein structure constructed by homology modeling revealed its 3-domain protein fold. A cost effective medium MSeMC-AS was formulated with defatted soybean meal, molasses, marine water, cystine and ammonium sulfate which demonstrated more than 80% increase in yield during scaling up from shake flask to a 3.0 L bioreactor and the growth kinetics of indigenous Bt strain JSc1 was also revealed faster than the reference Btk HD-73 in this medium. Yield in δ- endotoxin production was enhanced by inhibiting the endogenous protease activity which was demonstrated to be the cause of protein degradation and loss in productivity. From the field trial of Bt biopesticide preparation in cabbage, cauliflower and organic tea farming, no significant differences were observed with the active treatments which indicated high feasibility of the prepared Bt biopesticide in replacing conventional pesticides with no yield loss. These results suggest that the Bt strains of Bangladesh with their unique and diverse properties as well as potentials as revealed in this study, could potentially be utilized not only in controlling the pests in agriculture but also the vectors, protozoa, nematodes etc of public health concern.
Characterization of indigenous bio-control agents against wilt complex pathogens of tomato
(University of Dhaka, 2016-06-16) Sultana, Nazneen
The main objective of the study was to find out potential indigenous bio-control agents active against tomato wilt complex pathogens like Ralstonia solanacearum and Fusarium oxysporum and various strains of the pathogens were first isolated from wilted tomato plants grown in different areas in Bangladesh. Then different indigenous strains of bacteria and fungi, known for their antagonistic activity against wilt complex pathogens, were isolated and tested against the pathogens. It was found that twenty strains of R. solanacearum and three strains of F. oxysporum demonstrated infectivity in tomato plants. Based on the ability to infect and severity of infections, one strain of each organism was then selected for further study. Bacillus spp., Pseudomonas spp. Rhizobium spp. and Trichoderma spp. are known to have potential antagonistic effect against R. solanacearum and F. oxysporum. To determine their ability and efficacy as bio-control agents, all these organisms were isolated from rhizosphere except Rhizobium spp. which were isolated from root nodule of lentils. In vitro screening test carried out for antagonistic property revealed that five Bacillus strains- Bacillus subtilis 1, Bacillus subtilis 2, Bacillus pumilus, Bacillus licheniformis, Paenibacillus polymixa, one strain of Pseudomonas fluorescens, one strain of Rhizobium leguminosorum and two strains of Trichoderma harzianum were effective against R. solanacearum and F. oxysporum f. sp. lycopersici as bio-control agents. The findings indicated that R. leguminosorum inhibited the maximum growth of F. oxysporum f. sp. lycopersici in dual culture method with 65.56% inhibition while in case of fungi versus fungi, T. harzianum and T. harzianum (BAU) showed strong antagonism against F. oxysporum f. sp. lycopersici pathogen inhibiting 78.37% and 83.78% of radial mycelial growth, respectively after 7 days of incubation. On the other hand, B. subtilis, and P. fluorescens were found to be more potent against R. solanacearum. Four different methods of inoculation viz. collar region, root dipping, soil drenching and sick bed were evaluated to determine the degree of pathogenicity of R. solanacearum and F. oxysporum f. sp. lycopersici. It was found that collar region inoculation method was best for evaluation of pathogenicity of both pathogens. In the field experiment B. subtilis 2, P. fluorescens and R. leguminosorum showed similar effect against bacterial wilt when seedling roots were treated separately before transplanting. In case of fungal wilt, B. subtilis 2 and T. harzianum BAU were found to be most effective in reducing wilt severity. However, when two pathogens were combined together with the same root treatments, B. subtilis 2, P. fluorescens, R. leguminosorum and T. harzianum showed better results. It results indices that the optimal temperature and pH for the growth of the indigenous bacterial bio-control agents were 350C and pH 7, respectively. Growth of the bacterial strains seemed to be affected by varying the temperatures and pHs. From the present study, it suggests that B. subtilis 2, P. fluorescens, R. leguminosorum and T. harzianum (BAU) have great potential as bio-control agents not only active against the tested tomato wilt complex pathogens but also they hold a promising future in the development or formulation of an effective bio-control strategy against other plant pathogens.
Occurrence of emerging food-borne pathogens in common fast foods and inactivation of pathogens with essential oil
(University of Dhaka, 2016-08-16) Taniya, Afroz Jahan
The present investigation can be classified into four major parts. The first part is the isolation and presumptive identification of Escherichia coli, Staphylococcus aureus and Bacillus cereus from fast food samples collected from various parts of Dhaka city of Bangladesh. This was done following the criteria put forth in Bergey’s manual of Determinative Bacteriology, 9 th edition. The second part is the detection of antibacterial activity of cinnamaldehyde against three food borne pathogens individually and combined by the disc diffusion method. The third part is to determine the effect of pH and temperature on antibacterial activity of cinnamaldehyde against three food borne pathogens. The fourth and the final part is inactivation of isolated organism applied in ground chicken meat with cinnamaldehyde. For isolation of Escherichia coli, Bacillus cereus and Staphylococcus aureus from food samples three types of selective media was used such as Sorbitol MacConkey agar (SMA), Bacillus cereus selective agar base medium supplemented with egg yolk emulsion, Polymyxin B sulphate, and Mannitol salt agar (MSA), From the selective medium, the selected colonies were isolated and then sub cultured to maintain pure culture. From the collected food samples, 14 Escherichia coli, 09 Bacillus cereus and 17 Staphylococcus aureus were subjected to cultural, morphological, and microscopic and biochemical study for their presumptive identification. Antibacterial activity of cinnamaldehyde, carvacrol and euginol was tested against Escherichia coli, Bacillus cereus and Staphylococcus aureus using disc diffusion method. Screening of cinnamaldehyde, carvacrol and euginol extracts showed antibacterial activity against the isolated organisms studied. The MIC values of cinnamaldehyde, carvacrol and euginol were determined by broth dilution method at 37 °C and pH 7. MIC values of cinnamaldehyde, carvacrol and euginol against the test bacteria ranged from 2.5 to 10%. The effect of temperature and pH on the antibacterial activity of cinnamaldehyde against Escherichia coli, Bacillus cereus and Staphylococcus aureus were determined. Cinnamaldehyde showed antibacterial activity after treatment at 100° C for 30 min treatment suggesting that a high temperature does not affect the activity but only reduced the activity insignificantly as showed by decreased zone diameter. The highest antibacterial activity was found at pH 7 for cinnamaldehyde against the test organisms. Cinnamaldehyde at each concentration of 2.5% (MIC level), 7.5% (three times of MIC) and 12.5% (five times of MIC) was applied separately in ground chicken meat inoculated with Escherichia coli, Bacillus cereus and Staphylococcus aureus and kept at -18° C for 10 days. The result showed that only a few log reduction of Escherichia coli, Bacillus cereus and Staphylococcus aureus was found in selective media used in case of MIC level of cinnamaldehyde (2.5%). 3 times (7.5%) and 5 times (12.5%) MIC level of cinnamaldehyde employed resulted 7 log reduction of Escherichia coli, Bacillus cereus and Staphylococcus aureus, respectively, growing on selective media after 24 hours of exposure. Therefore, cinnamldehyde could be useful to control Escherichia coli, Bacillus cereus and Staphylococcus aureus in ground chicken meat. From this experimental finding it was apparent that essential oils from herbs, sharps, trees and spices, especially cinnamldehyde from cinnamon could be fruitfully used to control food borne pathogens in various foods and food commodities.
Molecular detection of multi-drug resistance genes in Mycobacterium tuberculosis
(University of Dhaka, 2016-10-13) Sultana, Monika
The spread of multidrug resistant tuberculosis (MDR-TB) due to emergence of multidrug resistant M. tuberculosis isolates has increased worldwide and reached epidemic proportions in many countries. The emergence of MDR-TB has become a major threat for TB control in Bangladesh. MDR-TB is defined as resistant to at least rifampicin and isoniazid, which are the backbone of short-course chemotherapy for tuberculosis. Mutations in the 81-bp core region of rpoB were reported to be responsible for resistance in at least 95% of isolates. Resistance to isoniazid is due to mutations at one of two sites, in either the katG or inhA genes. The culture-based methods for detection of M. tuberculosis infection and drug susceptibility testing (DST) usually take more than 1 month due to the slow growth of this bacterium. The use of molecular methods for the identification of mutations in the resistance genes may offer the means for rapid screening of the drug resistance among the M. tuberculosis isolates and initiation of early treatment. In this study, we evaluated rapid detection of multidrug resistant strains using the molecular techniques from MTB strains and sputum directly. For this purpose, a total of 55 sputum samples were collected from 55 patients of National Tuberculosis Reference Laboratory (NTRL), Mohakhali, Dhaka- 1212, Bangladesh. Samples were tested for microscopic examination, conventional culture method, drug susceptibility testing, Gene Xpert assay and PCR amplification followed by DNA sequencing. All 55 samples (36 from MTB strain, 19 from direct sputum) were subjected to rpoB gene amplification and 21 samples (12 from MTB strain, 9 from direct sputum) for katG gene amplification by PCR. Then the samples were subjected to DNA sequencing for rpoB and katG gene mutation analysis. RIF-associated mutations were detected in (44/55) samples (80%), of which, mutations at codons 531 (54.54%), 526 (12.73%) and 516 (9.09%) were observed. Sixteen samples (76.19%) out of 21 had isoniazid associated mutations at codon 315 in the katG gene. Considering conventional DST as gold standard, sensitivity and specificity of DNA sequencing for the detection of Rif- resistance were 98% and 100%; whereas isoniazid resistance were 94% and 100%. Comparison between Xpert MTB/RIF and DST results showed that eight samples were sensitive to rifampicin by Genexpert assay, but not on DST. The reason may be due to the selection of resistant strains in the antibiotic added media for a prolonged incubation period. Therefore, falsepositive results of rifampicin resistance were observed in DST.
Enterotoxicity of Vibrio fluvialis
(University of Dhaka, 2016-10-17) Jainab, Tahsina
Vibrio fluvialis is a halophilic bacterial species which emerged as a diarrhoeal pathogen and has been a human public health hazard all over the world, especially in coastal areas of developing countries and regions with poor sanitation. In this study, we isolated, identified and characterized the enterotoxic and other toxic activities of five V. fluvialis organisms isolated from the environmental sources. Though they are halophilic in nature, however, these organisms were isolated from fresh environmental water sources having 0% salinity from rivers around Dhaka city and ponds from Khulna, and higher salinity in Satkhira area. All the V. fluvialis strains showed growth in 7% salinity and did not produce gas from glucose. In the toxicity assay, live cells and culture filtrates of all five isolates gave positive results in rabbit ileal loop assay indicating their enterotoxicity. Two strains which gave poor results in enterotoxin production initially, showed enhancement of enterotoxicity as judged by increased fluid outpouring after two consecutive passages in rabbit gut indicating that, the enteric environment probably influences toxin production. On the other hand, PCR analysis showed the absence of ctxA gene and other genes like tdh, trh, stx1 and stx2, which are commonly associated with enterotoxicity. The culture filtrate of the isolates showed cytotoxic activities on BHK-21 and HeLa cells lines, gave positive results for haemolysin in blood agar plate and tube methods, and caused paralysis of hind legs of Swiss Albino mice within 2-3 days. The toxic activities shown by culture filtrates of V. fluvilais were increased after concentration of culture filtrates by ammonium sulphate precipitation method. All these results suggested that these environmental V. fluvialis organisms, which lacked ctxA gene, are highly pathogenic. They also lacked other common enterotoxic genes. Although ctxA gene negative, these organisms showed rabbit ileal loop positive reactions, which indicated that they might produce a toxin which is different from the known cholera toxin. Apart from the enterotoxic activity, these organisms also produced neurotoxin, cytotoxin and haemolysin. Therefore, the presence of V. fluvialis organisms in aquatic environment of Bangladesh constitutes a threat to public and environmental health.
Molecular characterization of Burkholderia pseudomallei and its seroprevalence in Bangladesh
(University of Dhaka, 2017-05-08) Jilani, Md. Shariful Alam
The Gram negative bacillus, Burkholderia pseudomallei causes melioidosis, a rare but serious infection that can be fatal if untreated or misdiagnosed. The organism is widely distributed in the soil and water of tropical and subtropical countries. Melioidosis is naturally acquired through environmental contact with the bacterium. Although melioidosis is largely restricted to the Southeast Asia and Northern Australia, the disease has been increasingly reported from Bangladesh. However, its actual prevalence in Bangladesh is largely unknown due to the lack of systematic study and awareness of the medical community about the disease and the organism. So far, no systematic study has been done to find out the extent of exposure of B. pseudomallei infection among healthy individuals and to detect the source of this organism in environmental samples of Bangladesh. In order to address the issue, the first phase of the present study was designed to determine the magnitude of exposure by detecting antibodies to B. pseudomallei among the healthy population of selected regions of Bangladesh. In the second phase of the study, attempt was made to detect the organism in the soil by culture and molecular methods. The clinical and environmental isolates were further characterized by molecular techniques to determine the relatedness of the organisms. In the first phase of the study, blood samples were collected from healthy population residing in rural area of four districts (Mymensingh, Sylhet, Narayangange and Kishoregange) to determine the seroprevalence of B. pseudomallei infection. We have used both sonicated whole cell antigen (SWCA) and recombinant truncated flagellin antigen (RTFA) of B. pseudomallei in an in-house indirect enzyme linked immunosorbent assay (ELISA). The cut off optical density (OD) value for SWCA and RTFA was 0.8 and 0.4 respectively. The cut off OD value was determined by mean OD of negative control + 3x SD. In the second phase of the study, we have determined the source of the organism in the soil samples from four northeastern districts of Bangladesh by culture and polymerase chain reaction (PCR). Multiple soil samples from 5–7 sampling points of 3–5 sites were collected from rural areas of four northeastern districts (Mymensingh, Sylhet, Gazipur, and Narayangange) of Bangladesh from where culture confirmed melioidosis cases were detected earlier. Approximately 200 gm soil was taken from each point from a depth of about 20-30 cm using a shell augur disinfected with 70% alcohol in between soil collection. Collected soil was placed into a sterile plastic bag and sealed with rubber band to prevent moisture loss and was transported to the laboratory as soon as possible. In the second phase of the study, total 179 soil samples were collected and cultured in Ashdown selective media and sub-cultured in MacConkey`s agar medium. The suspected organisms which grew on MacConkey`s agar medium at 420C were identified as B. pseudomallei by typical colony morphology, Gram staining (bipolar staining), motility, biochemical tests (including API 20NE), arabinose assimilation and resistance to colistin and aminoglycoside. Monoclonal antibody based latex agglutination test (Melioidosis Research Center, Khon Kaen, Thailand) was performed for the final identification and confirmation of the suspected colonies of B. pseudomallei. Phenotypically suspected colonies of B. pseudomallei were further confirmed by PCR using 16s rRNA specific primers and orf2 gene of type three secretion system1 (TTS1). In the present study, molecular analysis was performed to characterize the strains isolated from clinical specimens and environmental sources. All these isolates were analyzed by real-time PCR assay targeting TTS1, Yersinia-like fimbrial (YLF) gene cluster and B. thailandensis-like flagellum and chemotaxis (BTFC) gene cluster. Real-time PCR assay targeting TTS1 was performed to confirm phenotypically identified organisms. YLF and BTFC gene clusters were examined to demonstrate the diversity of B. pseudomallei from various geographical regions and sources. Multilocus sequence typing (MLST) with seven house-keeping genes was performed with all the isolates to clarify the genetic relationships between B. pseudomallei isolated from clinical specimens and environmental sources and also between strains isolated from Bangladesh and other countries. The primers used in the PCR amplification and sequencing of the seven housekeeping gene fragments were ace-up and ace-dn, -gltB-up and gltB-dn, gmhD-up and gmhD-dn, lepA-up and lepA –dn, lipA-up and lipA –dn, narK-up and narK-dn, ndh-up and ndh-dn. Out of 940 blood samples, anti- B. pseudomallei IgG antibody against SWCA was detected in 21.5% individuals. However, ELISA using RTFA protein showed the seropositivity rate as 13.7%, which was lower than the seropositivity (21.5%) detected by ELISA using SWCA. Seropositivity rate by using SWCA was 22.6%-30.8% in three districts from where melioidosis cases were detected earlier, compared to 9.8% in a district (Kishoregange) where no melioidosis case was either detected or reported (p<0.01) previously. Seropositivity increased with the advancement of age from 5.3% to 30.4% among individuals aged 1–10 years and > 50 years respectively. The seropositivity rates were 26.0% and 20.6% in male and female respectively, while it was 20–27% among different occupational groups. No significant association was observed with gender (χ2 = 3.441, p = 0.064) or any occupational group (χ2 = 3.835, p = 0.280). Out of 179 soil samples, B. pseudomallei was isolated from two (2) samples from paddy field of Gazipur district, which is located 54 km north of capital Dhaka city. Both the isolates were phenotypically identical and arabinose negative and were positive for 16s rRNA and orf2 (TTS1) gene by conventional and real-time PCR assay respectively. Gene cluster analysis targeting YLF and BTFC gene demonstrated that all the isolates from Bangladesh contained YLF gene cluster. None of the isolates was positive for BTFC gene cluster. YLF gene cluster is predominantly found among B. pseudomallei derived from Southeast Asia. Phylogenetic analysis of 24 B. pseudomallei isolates by MLST revealed thirteen different sequence types (STs) of which 4 STs (ST- 1352, 1124, 761 and 756) were of novel types and identified for the first time. All these isolates were from Bangladeshi patients. Strains having the above STs were isolated from patients with abscess in different organs (liver, soft tissue, lungs). It is to be noted that in the present study, ST 56 (5 cases), ST 1007 (4 cases) and ST 1005 were the most frequently isolated types. ST 56 which was present in 5 clinical isolates out of 22, was the most common variant present in Bangladesh, followed by ST 1007 revealed from 4 cases and ST 1005, found in 2 clinical and 2 environmental isolates. Presence of ST 1005 in soil of Gazipur district as well as its presence in melioidosis patients from the same district indicated soil as the source and reservoir. All the strains containing ST 56 was isolated from patients with septicemia. ST 56 was also detected earlier in 1999 from a Bangladeshi patient living abroad. Apart from the four novel STs described above, all other STs that have been detected in the present study are also present in Thailand, Cambodia, China and Vietnam and other neighboring countries. The present study has demonstrated that a large proportion of people residing in the rural area of four districts are exposed to the organism as determined by serology and have a potential for developing overt diseases during their lifetime. The study has also identified for the first time the presence of B. pseudomallei in the soil samples of Bangladesh and determined soil as the source of B. pseudomallei infection in this region. All B. pseudomallei isolated in Bangladesh posses YLF gene, which confirms their Asian origin. As YLF strains are more virulent than BTFC strains, so people in this region are at higher risk of severe form of infection. MLST study has revealed that a number of novel STs of B. pseudomallei exist in Bangladesh. Presence of unique STs of B. pseudomallei in our environment demands further study to understand their importance to the biology of these organisms. Presence of same ST from the soil and clinical isolates indicates soil as the source and reservoir of this organism. It is likely that human and/or animal movements between these areas played pivotal role in the dissemination of B. pseudomallei, however analysis of broader range of isolates from these region are required for confirmation. Further large scale study is necessary to find out the magnitude of the infection in different areas of Bangladesh and its different reservoirs in the environment along with phylogenetic distribution.
Prevalence of Mycobacterium tuberculosis in prostatic tissues of patients suspected with different prostatic lesion specially cancer
(University of Dhaka, 2017-11-06) Mumu, Mahbuba Ashrafi
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis is a global health problem. According to the World Health Organization (WHO) 9 million people developed TB and 1.5 million died from the disease in 2013. Bangladesh stands 6th among 22 high TB burden countries. Pulmonary tuberculosis is the most common form of the disease; however, 20-25% of cases are extra-pulmonary in nature. Genitourinary tuberculosis accounts for 5-10% of extra-pulmonary cases in developed countries and 15-20% of cases in developing countries. Tuberculosis of the prostate gland is seen in 2.6% of genitourinary system. Studies have shown that approximately 20% of all human cancers in adults result from chronic infection and inflammatory states. Chronic prostate inflammation accelerates initiation of prostate cancer originating from basal cells and accelerates prostate cancer progression. There are reports describing tuberculosis of testis and prostate mimicking testicular cancer and prostatitis caused by M. tuberculosis infection serving as a predisposing factor for prostate cancer. This study aims to investigate whether there is any association between tuberculosis of prostate and development of prostatic lesions especially cancer in a cross section of Bangladeshi population. The study was carried out with 85 prostatic biopsy samples collected by trans-urethral resection of prostate (TURP) from patients and also two known TB positive lymph nodes. In addition to prostatic biopsy, venous blood samples were collected from respective individuals for estimation of prostate specific antigen (PSA) which is an aid for diagnosis of prostatic carcinoma. Histopathological diagnosis of 85 patients revealed nodular hyperplasia with chronic prostatitis (NHCP) in 56%, prostatic intraepithelial neoplasia (PIN) in 30.6%, granulomatous prostatitis (GnP) in 3.5% and cancer in 9.4% patients. The mean age of the cancer patients is 70 years. Most of the cancer patients belong to Dhaka division and smoking habit. None of the 85 biopsy sample revealed the presence of M. tuberculosis when analyzed by Ziehl-Neelsen (Z-N) stain and polymerase chain reaction (PCR). The failure of detection of M. tuberculosis from formalin fixed paraffin embedded tissue by conventional PCR prompted us to use Gene Xpert MTB/RIF which is a real-time hemi nested PCR test that simultaneously identifies M. tuberculosis and detects rifampicin resistance directly from clinical specimens. Two samples from each nodular hyperplasia with chronic prostatitis (NHCP), granulomatous prostatitis (GnP) and prostatic intraepithelial neoplasia (PIN) states, all 8 prostatic adenocarcinoma tissue samples were tested for presence of mycobacterial genomic DNA by the Gene Xpert MTB/RIF real time PCR. All these samples came out negative for mycobacterial DNA. Very importantly, the two positive control samples (lymph node tissues from confirmed TB cases) were positive in the Gene Xpert MTB/RIF assay. The finding of the positive results coming out from paraffin-embedded formalin-fixed lymph node tissue samples having confirmed M. tuberculosis infections validates that the absence of response for M. tuberculosis infection in Gene Xpert analysis from paraffin-embedded formalin-fixed prostatic tissues indicates that the prostate tissue samples used in this study did not harbour M. tuberculosis indicating that infection of prostate glands by M. tuberculosis. These findings indicate that tuberculous prostatitis is rare in a cross section of Bangladeshi population investigated in this study. This finding highlights the need of performing sensitive molecular test such as Gene Xpert in formalin fixed paraffin embedded tissue in ruling out whether a suspected patient is infected with M. tuberculosis or not.
Microbiological and physico-chemical assessment of surface water processed with low-cost and sustainable technology
(University of Dhaka, 2018-02-01) Emon, Sharmin Zaman
Surface water has been used for drinking purpose from ancient times. However, with increasing population and human activity this water became contaminated with pathogens, disease vectors or unacceptable levels of toxins or suspended solids. In Bangladesh, tube wells came as a solution, but soon it turned out to be a menace as it was contaminated with arsenic in many areas. Cities and towns are supplied with expensive large scale water treatment plants but this is not viable for the majority of the population in Bangladesh who live in isolated villages. A similar situation exists in most of the low resource countries of the world. Therefore, small domestic scale solutions for drinking water are needed for villages which are low-cost, simple technology and easy to maintain. Surface water is free of arsenic since it comes mostly from rain and melted snow. As this water seeps through different layers of the ground it picks up different minerals to become ground water aquifers; poisonous arsenic will also be present in the intermediate rocks. Since arsenic is difficult to remove in a domestic scale arrangement while destroying enteropathogenic microorganisms is simpler. So, use of arsenic free surface water treated using different simple techniques could be more desirable for the rural areas of the low resource countries like Bangladesh. From the perspective of human consumption, surface water is contaminated with enteropathogenic microorganisms, including those of diarrhea, cholera, typhoid, paratyphoid, jaundice etc., which need to be removed to make the water safe for drinking. So, the present study was basically taken up with the aim to study and evaluate the success of three new technologies that can disinfect surface water from diarrhoeal disease causing microorganisms. First one is low-cost solar pasteurization device developed by a group of scientists at the Department of Biomedical Physics & Technology (BMPT), University of Dhaka, that can be heated up water to much more than 60 0 C needed to destroy enteropathogenic microorganisms. This solar pasteurizer designed at BMPT automatically allows UV to act on the treated water. So, UV of the sun contributes in destroying diarrhoeagenic microorganisms. Water samples from seven different ponds, lakes and rivers of Bangladesh were subjected to treatment using this device and the highest temperature achieved was 84°C after 4 hours of exposure to sunshine. Regardless of sampling sites, highest reduction of aerobic bacterial population was 4.1±0.7 log CFU/ml recorded in non-selective medium. In another study, a simple and inexpensive water purification method was sought using natural coagulant (moringa seed powder) and antibacterial agents (scallop powder) followed by natural bio-sand filtration. Surface water collected from different sources (e.g. pond, lake and river) were treated with combined moringa seed powder (0.04%) and scallop powder (0.01%) (MOSP) for 30 minutes showed a clear water layer at the top and a sediment layer at the bottom. The clear water was then passed through natural bio-sand filter and the resulting water was found drinkable. Regardless of sampling sites, highest bacterial population reduction of 5.8±0.9 log CFU/ml was recorded in non-selective medium. The microbiological and physico-chemical parameters of the water treated with above two technologies showed non-significant differences compared to the United States Environmental Protection Agency (USEPA) drinking water quality parameters. Spiked study and molecular techniques using Polymerase Chain Reaction (PCR) of uidA gene of E. coli also confirmed the effectiveness of these developed technologies. The shelf-life study of the treated water demonstrated that it can be stored at room temperature up to 6 months without compromising the quality, indicating the usefulness of these two technologies in drinking water scarcity areas of the world, because the ingredients used are readily available, inexpensive, user friendly and natural. On the other hand, the sediment or sludge produced in the second technique could be used in agriculture field after proper application of compost. In this study, third technique was using of metals like brass and its constituents, copper and zinc. The results were obtained depending on the mode of the experiment. With brass filings (granules) in a small amount of water such that the water remains entirely within the brass filings, the bacterial count was found undetectable after about 30 minutes of treatment. On having three plates of these three metals at the bottom of three individual plastic containers containing the water under test and shaken continuously for 30 minutes each, if the water volume was small, having a few mm depth above the plates, brass could reduce the bacterial count to non-detectable values. The counts reduced to some extent by copper followed by zinc but were still detectable. When water volume was large with about 50 mm depth above the metal plates, the count reduced for copper to a large extent, but not much for zinc and brass. However, when the treated samples were stored at room temperature, all resident bacteria along with coliform including E. coli increased to almost original values after 4 hours. This result indicates that the resident microorganisms were possibly injured due to the effect of copper which resuscitated on storage. Thus, the use of copper, brass or zinc plates were not able to destroy bacterial population with practical volumes of water and therefore, this study was not extended further. The present work has shown that the solar pasteurization and the technique of using moringa and scallop powder followed by bio-sand filtration are indeed successful in providing safe drinking water in rural areas of the world and despite many similar water purification systems available commercially, these two new techniques would be the simplest, inexpensive and environmental friendly. These simple and easy treatment and filtration methods are particularly helpful for flood prone areas of Bangladesh, where there is a scarcity of drinking water during flood. The people can easily get safe drinking water if training on these techniques are provided to the village people so that they themselves can make the devices when needed, thus empowering themselves.
Molecular and bioinformatic strategies in reducing the burden of influenza infections in slum areas of Dhaka
(University of Dhaka, 2018-04-17) Islam, Mohammad Ariful
Influenza epidemics are caused by rapid evolution of the viral genotypes and play a significant role in the annual mortality and morbidity due to respiratory tract infections in tropical countries like Bangladesh. In low-income countries, influenza associated hospitalization is more prevalent in impoverished population such as slums. In Bangladesh, data on influenza infections in slums of densely populated Dhaka city is limited. Aims of this study were to detect circulatory influenza strains in slums, genetic characterization by molecular and serological methods, assessment of antiviral drug susceptibility of the virus and prediction of putative candidate epitope by in silico approach for alternate vaccine design. In this study course, 993 nasal and throat swabs were collected from patients presented influenza-like illness (ILI) from Rayerbazar, Hazaraibagh, Mohammadpur slums in Southeast Dhaka, Bangladesh between June 2012 and August 2013. Influenza viruses were detected using real time RT-PCR. One hundred one (10%) samples were identified as influenza positive, 47 influenza A (19 A/H1N1pdm09 and 26 A/H3N2) and 54 influenza B viruses including both B/Yamagata and B/Victoria lineages. Influenza positive samples were antigenically characterized by Hemagglutination Inhibition Assay (HAI) which revealed that Bangladeshi strains were antigenically similar to the WHO recommended vaccine strain for the Northern Hemisphere. Complete genome sequencing of two representative influenza A strains were conducted by sanger method and analyzed by BLAST, BioEdit, and Mega tools. Based on complete genome sequence, A/H1N1pdm and A/H3N2 strains were almost identical to other contemporary Bangladeshi strains as well as the globally circulating strains. In the phylogenetic analysis, HA gene of A/H1N1pdm and A/H3N2 were clustered in clade 6B and 3C.3, respectively. Compared to the vaccine strain, A/H1N1pdm showed at least three mutations, K163Q, S185T, and S203T, respectively in the HA antigenic sites (Sa, Sb, and Ca) while receptor binding sites remained conserved. Mutations were also observed at residue E374K of HA which is essential for membrane fusion. N-linked glycosylation sites were conserved in HA gene but one alteration at position 42 in NA gene was identified. The A/H3N2 showed two mutations at the antigenic site A with substitutions T144A and B, R158G in HA while receptor binding sites remained conserved. No strains had mutations that have been reported as responsible for enhanced virulence. Antiviral assay was performed in confluent monolayer of MDCK cells in 96-well plate in duplicate by MTT assay. Both strains were susceptible to the antiviral drugs routinely Abstract xvi | P a g e prescribed. Candidate epitope prediction was performed through in silico approach and putative peptide vaccine was determined. This study demonstrates several intriguing findings towards the understanding of genetic diversity of influenza viruses in Bangladesh and prediction of alternate vaccine approaches to reduce the burden of the disease. First, at least four types of influenza viruses were circulating during the study period which justifies that a quadrivalent influenza vaccine formulation that includes influenza A, influenza B, and both lineages of influenza B viruses could be more effective to reduce influenza disease burden in the country. Second, although Bangladeshi strains and WHO recommended vaccine strains are antigenically similar, several mutations were identified. It is interesting to see whether these mutations have any impact on vaccine efficacy. Third, all slum strains were found to be sensitive to the drugs routinely used for influenza treatment. The strains were analyzed based on known molecular markers for the drug resistance which could be useful for the clinical management of the patients particularly during pandemic situation. Fourth, prediction of HA and NA based candidate epitopes on the study strains could facilitate alternate vaccine approach. Notably, in Bangladesh the influenza vaccination has not been implemented in the national vaccination schedule and the vaccine effectiveness among Bangladeshi population is remained unknown. Therefore, assessment of current vaccine effectiveness as well as efforts to assess alternate vaccine approaches are required. In summary, the findings of this study contribute to understanding the characterization of slum influenza viruses that will be useful for routine surveillance, potential drug recommendation. Moreover, candidate epitope prediction will guide for alternate and improved vaccine for the control of influenza in future.
Epidemiology of Anthrax in Domestic Animals of Bangladesh
(University of Dhaka, 2018-04-25) Rume, Farzana Islam
Molecular Epidemiology of Drug Resistant Strains of Tuberculosis in Bangladesh
(University of Dhaka, 2018-09-04) Hyder, Md Khurshid Alam
Tuberculosis (TB) is one of the top 10 causes of death worldwide. According to the Global TB Report globally in 2015, there were an estimated 10.4 million incident cases of Tuberculosis (TB) of which 5.9 million (56%) were among men, 3.5 million (34%) among women and 1.0 million (10%) among children. In 2015, the gap between notifications of new cases and the estimated number of incident was 4.3 million (missing cases) reflecting a mixture of undetected cases and under-reporting of detected TB cases. Bangladesh is one of the world’s most densely populated countries, with 161 million people. In recent years, Bangladesh has made significant progress in child and maternal mortality reduction, life expectancy and poverty reduction. However, Bangladesh still faces serious health challenges and Tuberculosis is one of them. According to WHO Global TB report 2016, with annual occurrence of 362,000 new Tuberculosis cases Bangladesh is one of the world’s 30 high TB burden countries. About 73,000 people die annually due to Tuberculosis. The National TB Programme (NTP) of Bangladesh and its partners have maintained good “basic TB control services”, with reasonable case detection and excellent treatment outcomes. The Government of Bangladesh, the Global Fund, USAID and other partners have kept their financial commitment to TB control during recent years, enabling NTP to consolidate its activities and address challenges of TB/HIV, multidrug resistant (MDR) TB, intensified case-finding in high-risk groups and vulnerable populations, and use of new technologies. There has been consistent increase in case notification especially among new and relapse cases since 2013. Bangladesh is the first country in the Region to introduce a shorter treatment regimen for MDR-TB and is achieving high cure rate for MDR-TB patients (75%). This study was aimed to early diagnosis of susceptible TB and prevents drug-resistant TB and improves the overall understanding of genotpypic diversity of MTBC strains circulating in Bangladesh. The research was conducted in NTRL, NIDH, Mohakhali and Department of Microbiology, University of Dhaka, Dhaka, Bangladesh and total 371 sputum samples from tuberculosis reference laboratory were collected. We have conducted two state-of-the-art molecular techniques: i) Spacer oligotyping/spoligotyping and ii) MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats). In this study we found that most prevalent tuberculosis clade in Bangladesh is the Beijing clade and constitutes about 33.15% which is identified through Spoligotyping. Two different spoligotypes of Beijing clade including SIT 941 (9.19%) and SIT 1 (23.78%) was found. Evidence of presence of SIT 941 indicates its recent rapid spread in Bangladesh and it might be due to the extensive coverage of BCG vaccines. Moreover, two different types of MIRU-VNTR patterns were observed for the same spoligotype. This is possible as discriminatory power of MIRU-VNTR technique is more than spoligotyping technique. Although, MIRU-VNTR results could not be imparted with spoligotyping data, the spoligotyping technique alone was sufficient to identify predominant clades like Beijing, T1 and EAI in Bangladeshi population.
Development of an edible vaccine against pneumococcal diseases using transgenic plant
(University of Dhaka, 2019-03-24) Akter, Shahina
Streptococcus pneumoniae, also known as pneumococcus, is one of the leading causes of respiratory tract infections, meningitis and septicemia in Bangladesh. It is a major cause of morbidity and mortality, especially in children and the elderly. The emergence and progressive increase in incidence of antibiotic resistance coupled with the less-thanadequate polysaccharide vaccine warrants further investigations of protective pneumococcal protein antigens. In recent years, the pneumococcal surface protein A (PspA) has been suggested to be a promising candidate, alone or with other immunogens, to be included in a future vaccine. On the other hand, plant-based edible vaccines are recombinant protein vaccines. Ideally, the choice of plant species used to produce the selected antigen should allow for oral delivery in the form of an edible vaccine. Plant-based edible vaccines have been introduced as a revolutionary cost-effective vaccination modality. It offers a number of advantages over traditional vaccines: they eliminate highcost purification processes, exhibit long-term stability at room temperature, reduce the risk of disease caused by killed/attenuated organisms and are resistant to gastrointestinal enzymatic degradation. In this study, immune-informatics tools have been used to identify in silico four predicted epitope peptides within PspA. This computational approach was adopted to identify a multi epitope vaccine candidate against PspA that could be suitable to trigger a significant immune response. Sequences of the spike proteins were collected from a protein database and analyzed with an in silico tool, to identify the most immunogenic protein. B cell immunity were checked for the peptides to ensure that they had the capacity to induce both humoral and cell-mediated immunity. Four epitope peptide sequences of 33, 10, 14 and 7 amino acids were found as the most potential B cell epitopes. Furthermore, conservancy analysis was also done using in silico tools and showed a high conservancy for all epitopes. The sequences of S. pneumoniae epitopes were analyzed and placed in fusion with alpha zein or the N-terminal 90 amino acids of the 27KD gamma zein, respectively. Three constructs were produced using three different inserts namely alpha, gamma and Ds-red construct. Agrobacterium strain LBA4404 containing plasmid harboring nptII (neomycin phosphotransferase) was used for selection in all cases for the integration of gene of interest. Antigens of S. pneumoniae fused to either α-zein, γ-zein or Ds-red zein in Nicotiana tabacum and Lycopersicon esculentum were generated using Agrobacteriummediated genetic transformation protocols, and several positive transgenic plants were identified for each of the three constructs. The preparation of the constructs were successfully done. As a prerequisite of the transformation protocol, an efficient in vitro regeneration system was established for both Nicotiana tabacum and Lycopersicon esculentum and the transformation through A. tumefaciens were confirmed by kanamycin selection and PCR analysis with the specific primers. Transgenic plant material containing epitope sequences of PspA were the superior means of inducing a primary immune response. Mice fed transgenic leaves produced PspAspecific antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long lasting secondary antibody response. It have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. Immunized (fed transgenic leaves) and control mice were challenged with Streptococcus pneumoniae serotype 7F, and on average 88% immunized mice survived while 50% survived in case of control. The demonstrated success of oral immunization for pneumococcal with an ‘‘edible vaccine’’ provides a strategy for contributing a means to achieve global immunization for pneumococcal prevention and eradication. These immunogenic hot spot within PspA has the potential to serve as an attractive candidate for the development of a novel pneumococcal vaccine.