PhD Thesis
Browse
15 results
Search Results
Item Bioprocess Development for Production of Proteases by Bacillus Licheniformis(University of Dhaka, 2019-06-17) Al Mamun, Md. ArafatBacterial proteases have enormous technical applications in food, pharmaceuticals and leather industries. Alkaline protease, however, has got important applications in eco-friendly leather processing. The bulk application of this hydrolyzing enzyme demands its cost effective production. Hence the present study was aimed at the development of a bioprocess for production of the alkaline protease by Bacillus licheniformis MZK05 strain which was previously isolated from tannery effluents and identified by 16S rRNA gene sequence analysis. Since the production of the enzyme by the wild BlMZK05 strain was not adequate in a liquid medium namely Alkaline Protease Producing Broth (APPB), strain improvement through random mutagenesis followed by optimization of the enzyme fermentation by statistical methods were investigated. The mutation protocol involved the treatment of the BlMZK05 cells of exponential phase with ultra violet (UV) irradiation and ethyl methanesulfonate (EMS) under varied combinations viz. combinatorial, single or sequential with different doses and exposure periods. The potential mutants were selected from colonies of distinct characteristics, their clear zone ratio of casein hydrolysis on Skim Milk Agar (SMA), production capacity of the enzyme in liquid APPB medium and test for prolonged reproducible enzyme production ability. The distinct colonies were selected after mutagenic treatments that resulted in varied level of death rates. The highest death rates were revealed with EMS treatments for overnight. Of 182 colonies with distinct phenotypic characteristics, 100 mutants which exhibited casein hydrolysis in varying ratio yielded the enzyme activity between 46 – 270 U/ml in liquid APPB medium in shake flasks at 37°C, pH 7.5 and 150 rpm. The mutants with rather larger, raised and irregular colony appearance with respect to the parental organism produced greater protease activity than the other mutants. Most of the mutants tested for the reproducibility of their enzyme production capability for a prolonged period of about twelve months, were found inconsistent as “rise and fall” except two mutants namely B. licheniformis MZK05M9 (BlM9) and B. licheniformis MZK05EO17 (BlEO17). These mutants exhibited persistent enzyme activities of 135±3 U/ml and 235± 4 U/ml, respectively. The enzymes of two mutants BlM9 and BlEO17 were thus evaluated for their possible applications in leather processing based on their hydrolyzing capability of nonstructural (albumin, globulin and elastin) and structural (collagen) leather proteins. Both the mutants were capable of hydrolyzing all but BlM9 did not act on collagen indicating the suitability of BlM9’s enzyme in leather manufacturing processes. In this regard, optimization of the fermentation of alkaline protease by BlM9 was performed using statistical methods which demonstrated effective medium components molasses as carbon source, soybean meal as nitrogen source and the salts NaCl, MgSO4.7H2O and K2HPO4 by Plackett-Burman design. The concentrations of these medium components were optimized by Response Surface Methodology (RSM) based on Central Composite Design (CCD) in shake culture at pH 7.5, agitation 150 rpm and temperature 37°C. The optimum values for the tested variables for the maximum alkaline protease production were found as molasses (0.92%), soybean Meal (0.79%), NaCl (0.125%), MgSO4.7H2O (0.125%) and K2HPO4 (0.59%) with the protease activity in the optimized medium 761 U/ml predicted by statistical software Minitab Version 17. The experimental value of the enzyme activity as obtained 765 U/ml was comparable and thus validated the predicted values of the software. The granular sizes of the soybean meal affected the enzyme production revealing 4.7 mm mesh size supported the enzyme production 5 % higher than that of the mixed sizes between 6 to 4 mm. Fermentation in 7.0 L bioreactor cultivation with optimized medium at pH 7.5 and 37°C under cascade control of dissolved oxygen concentration, a high level of enzyme activity 1020 ± 10 U/ml was obtained after 28 hrs at stationery phase. This enzyme activity was 1.7 fold higher than that found in Molasses Soybean meal medium optimized by “one variable-at-a time” method. The enzyme was stable up to 30 days at 4°C where as the enzyme lost its activity to 50% at 30°C after 40 days. However, it exhibited 100% stability up to 18 days at 30oC in the presence of 0.5% polyethylene glycol. The partially purified protease had a pH optimum of 8.5 and temperature optimum of 55°C and the molecular mass of the enzyme was 27.2 kDa as judged by SDS–PAGE. According to the inhibition profiles obtained with the various protease inhibitors, it was confirmed that the partially purified protease belongs to the serine protease type. The activity of partially purified enzyme was enhanced by calcium, magnesium, barium, potassium and manganese ions and strongly inhibited by mercury ion. In addition, the protease showed remarkable stability in the presence of 1% SDS; 1, 3 and 5% Triton X-100 and H2O2, which comprise the common bleach-based detergent formulation suggesting its possible usage in detergent as cleansing aids. The application of the enzyme in dehairing of animal skin in leather processing demonstrated that treatment with enzymes removed 85% of hair from goat skin after 24 hrs under mild shaking condition at room temperature where as the treatment with enzymes and 5% lime together resulted in 100% dehairing under similar conditions. Sodium sulfide along with lime also removed 100% hair faster (with 20 hrs) than other two treatments. The grain surface of the enzyme treated skin was smoother and silkier than that of the chemical treated skin as revealed by Scanning Electron Microscopy. The quality of the enzyme was found equally efficient to a commercial enzyme in bating of animal hide as proved by different physical tests such as tensile strength, percent of elongation, stitch tears strength, water vapor permeability, grain crack strength and tongue tear strength tests. In addition, the stability profile (pH, temperature and surfactants) also revealed its suitability for application in detergent industry. Thus these results will be a useful basis for commercial production of the alkaline protease by BlM9 in Bangladesh.Item Development of an edible vaccine against pneumococcal diseases using transgenic plant(University of Dhaka, 2019-03-24) Akter, ShahinaStreptococcus 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.Item Molecular Epidemiology of Drug Resistant Strains of Tuberculosis in Bangladesh(University of Dhaka, 2018-09-04) Hyder, Md Khurshid AlamTuberculosis (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.Item Epidemiology of Anthrax in Domestic Animals of Bangladesh(University of Dhaka, 2018-04-25) Rume, Farzana IslamItem Molecular and bioinformatic strategies in reducing the burden of influenza infections in slum areas of Dhaka(University of Dhaka, 2018-04-17) Islam, Mohammad ArifulInfluenza 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.Item Microbiological and physico-chemical assessment of surface water processed with low-cost and sustainable technology(University of Dhaka, 2018-02-01) Emon, Sharmin ZamanSurface 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.Item Molecular characterization of Burkholderia pseudomallei and its seroprevalence in Bangladesh(University of Dhaka, 2017-05-08) Jilani, Md. Shariful AlamThe 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.Item Characterization of indigenous bio-control agents against wilt complex pathogens of tomato(University of Dhaka, 2016-06-16) Sultana, NazneenThe 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.Item Characterization of cry genes and insecticidal proteins from indigenous bacillus thuringiensis to develop potential biopesticide(University of Dhaka, 2016-05-31) Shishir, Md. AsaduzzamanIndiscriminate 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.Item 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 AhmedPatients 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.