Department of Microbiology

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Activation of DNA damage signaling by Shiga toxin(s) in mammalian cells
(© University of Dhaka, 2025-05-04) Begum, Kohinu
Adaptation and survival of Escherichia coli in tropical aquatic environments
(© University of Dhaka, 2025-05-04) Akter, Selina
Anti-malarial drug resistance and associated genetic polymorphism of Plasmodium falciparum in members of Armed Forces of Bangladesh working in endemic areas at home and abroad
(University of Dhaka, 2020-01-07) Amin, Md Nurul
Despite achieving a trend of reduction in global morbidity and mortality from malaria since beginning of 21st century the parasitic disease continues to have a devastating impact on people’s health and livelihoods in Asia and Africa. Plasmodium falciparum (pf), responsible for most malaria-related deaths among all 5 parasite species, has a long tradition of acquiring resistance against anti-malarial drugs. Development of pf resistance to artemisinin, the world’s most efficient antimalarial drug, is now threatening the achievements of global malaria control and elimination programmes. Armed forces’ personnel in South East Asia and Sub-Saharan Africa traditionally remain exposed to this volatile malarial situation as they usually need to operate in malaria endemic areas. Members of Bangladesh Armed Forces operate in endemic areas of both home and Africa because of their deployment in hilly areas at home and peace keeping missions in Africa. Not surprisingly, they need to travel between different endemic regions. Unfortunately, there is no data available on the genetic types of falciparum malaria they suffer from or probable resistant strains they might transmit between different endemic areas they travel. The present study is designed to investigate and compare molecular pattern and epidemiological aspects of anti-malarial drug resistance of P. falciparum in members of Armed Forces of Bangladesh working in endemic areas at home and Africa. A total of 252 ‘dried blood samples on filter paper’ were collected between November 2014 and February 2016, from P. falciparum positive Bangladeshi soldiers working in Chittagong Hill Tracts (CHT), Bangladesh and 5 Sub Saharan African Countries namely, Central African Republic (CAR), Democratic Republic of Congo (DRC), Liberia, Mali and Ivory Coast. These samples (94 from Bangladesh and 138 from African countries) were then transported to Microbiology Laboratories of University of Dhaka, where pf DNA extraction was done from all of them using QIAamp® DNA Mini Kit (Qiagen GmbH, Germany), following manufacturer’s protocol. Plasmodium species was confirmed by a nested PCR following standard protocol with minor modifications. Thereafter, a multiplexix nested PCR followed by restriction fragment length polymorphism (RFLP) method was employed to investigate the presence of chloroquine resistance marker ‘K76T mutation’ in P. falciparum chloroquine resistance transporters (pfcrt) gene and lumifantrine and mefloquine resistance marker ‘N86Y mutation’ in P. falciparum multidrug resistance1 (pfmdr1) gene. The propeller region of the kelch 13 (pfk13) gene in 29 Bangladeshi and 40 African samples was amplified by a nested PCR following a protocol developed by Pasteur Institute, Paris and Cambodia and then sequenced to see markers of artemisinin resistance. Some data on demography, clinical features, epidemiological aspects and Knowledge attitude and practice (KAP) were also collected from the respondent soldiers using a pretested structured questionnaire and a check list. All molecular and epidemiological data were entered and analyzed in statistical software IBM SPSS version 19. The P. falciparum DNA was confirmed in 35 (37.23%) Bangladeshi and 45 (28.48%) African samples. The ‘pfcrt (K76T) mutation’ that confers resistance to chloroquine, was detected in 93.10% Bangladeshi and 29.27% African samples. The ‘pfmdr1 (N86Y) mutation’ that confers resistance to lumifantrine and mefloquine, was detected in 20.69% Bangladeshi and only 2.44% African samples. None of the Bangladeshi samples had mutation in k13 propeller domain. On the other hand, 9 (22.50%) African samples exhibited pfk13 mutations including 5 non-synonymous and 3 synonymous mutations, reported for the first time. All of these new non-synonymous mutations namely A617P, Y616F, S491F, N458k and Y616F and synonymous mutations namely F614F, I616I and K503K were found in samples from D R Congo. Mutations F614F and A617P were detected in a single isolate and S491F was detected in two of the isolates. The most common African mutation A578S was detected in a sample from Ivory Coast. None of the pfk13 mutations, so far recognized to be associated/candidate or validated as artemisinin resistance marker by World Health Organization (WHO), was detected in this study. While analyzing clinical data it was found that, duration of fever, number of fever episodes, failure of drug, referral and length of hospital stay were associated with the 1st line antimalarial drug used (p ˂ 0.05). Epidemiological data in this study revealed a yearlyx incidence of 180 cases of malaria per thousand population under mefloquine prophylaxis in Liberia, although variables like duration of fever, drug failure and mutation in pfmdr1 were not associated with this prophylaxis in all the study areas. While analyzing data on knowledge attitude and practice (KAP) it is found that troops under mefloquine prophylaxis were more confident on the efficacy of this prophylactic drug (P=0.00). Majority (92%) of the troops could identify mosquito bite as the route of malaria transmission while 62.30% chose bed net as the most important preventive measure against malaria. The correctness of their understanding about malaria transmission, cause of resistance and preventive priorities, was associated with their source of information (p ˂ 0.05). More than 90% of the troops used bed net as a protective measure although only 19% of them used insecticide treated net. Regular use of protective clothing to prevent mosquito bite by the soldiers in different study areas varied between 70% and 95%. A routine practice of outdoor mosquito-cidal spray was reported by more than 80% troops working in different countries. Preventive behavior of the troops like use of protective clothing, insecticide treated bed net and mosquito repellent were found to be associated with their source of information (p ˂ 0.05). This study, first of its kind to be conducted in members of Bangladesh Armed Forces deployed in malaria endemic areas of both Bangladesh and Africa, attempted to investigate and compare molecular and epidemiological pattern of antimalarial drug resistance. It revealed 5 new non-synonymous and 3 synonymous mutations in pfk13 gene found in its African samples. Further study is required to see the relationship of these new mutations with delayed parasite clearance and eventually artemisinin resistance. Further study is also required to examine the potential transportability of drug resistant strains of pf malaria between endemic areas.
Antimicrobial activity and bio-potency of homoeopathy medicines against enteric pathogens
(University of Dhaka, 2019-12-30) Rahim, Md. Abdur
Homoeopathy medicines are used in many countries for the treatment of various human ailments. Homoeopathy medicines have no direct effect on some acute infectious diseases such as post operation infection, because it needs immediate recovery, but homoeopathy medicines may cure patients slowly forever. Homoeopathy medicines are very effective in case of chronic ailments. In the case where pathogens of infectious diseases become multidrug resistant, traditional antibiotics are failed to kill bacteria; in that case homoeopathy medicines may be an effective alternative, producing no toxic side effects & bringing about rapid recovery. Homoeopathy medicine actually gained its greatest popularity for its impressive successes in the treatment of infectious diseases in the19th century. It represents a different approach for understanding disease and health, for its potential of strengthening a person’s immune system that fights against infection. Homoeopathy medicines have been using worldwide, because of trust on homoeopathy medicines. Some works on antimicrobial activity of homoeopathy medicines had been done worldwide, but a few reports or no reports are available so fur in Bangladesh especially on enteric human pathogens. The study was carried out in vitro for antimicrobial activity of homoeopathy medicines against human enteric pathogens. Five homoeopathy medicines were used such as Mercurius Corrosivus-6 (Merc Cor-6), Mercurius Solubilis-6 (Merc Sol-6), Pyrogenium-6, Hepar Sulfuris calcareum-6 (Hepar Sulf-6), and Sulfur-6. These all medicines were applied against E. coli O157:H7, Salmonella typhi, Shigella dysentriae, Vibrio cholera. Staphylococcus aureus, Bacillus cereus and Micrococcus luteus had been treated with the five homoeopathy medicines for finding positive reference organism. Based on sensitivity to all five homoeopathy medicines Micrococcus luteus was selected as reference organism for further study. In vitro antibacterial activity of five homoeopathy medicines were done by Kirby Bauer method, of which, Merc-Sol-6 and Sulfur-6 medicines showed zone of inhibition against human pathogens studied. Merc-Sol-6 showed zones of inhibition against E. coli O157:H7 (13.1±0.20/50 µl/disc and 20.09±0.21/100 µl/disc), Shigella dysenterae (10.5±0.33/50 µl/disc and 15.45±0.15/100 µl/disc), Salmonella typhi (15.3±0.18/50 µl/disc and 19.13±0.30/100 µl/disc),Vibrio cholera (14.33±0.24/50 µl/disc and 19.46±0.20/100 µl/disc) and sulfur-6 showed zones of inhibition against E. coli O157:H7 (12.67±0.31/50 µl/disc and 20.13±0.30/100 µl/disc), Shigella dysenterae (10.33±0.20/50 µl/disc and 22.33±0.18/100 µl/disc), Salmonella typhi (14.33±0.28/50 µl/disc and 21.67±0.35/100 µl/disc), and Vibrio cholera (9.13±0.20/50 µl/disc and 18.05±0.08/100 µl/disc). MIC and MBC were done by broth dilution method where Merc-sol-6 displayed a strong antimicrobial activity with MIC and MBC values as low as 64 μl/ ml and 128 μl/ml, respectively against E. coli O157:H7 , Salmonella typhi where 226 μl/ ml and 512 μl/ml, respectively against Shigella dysenterae and Vibrio cholerae, on the other hand sulfur-6 displayed a strong antimicrobial activity with MIC and MBC values as low as 64 μl/ ml and 128 μl/ml, respectively against E. coli O157:H7 and Salmonella typhi and 226 μl/ ml and 512 μl/ml against Shigella dysenterae and 128 μl/ml and 256 μl/ml against Vibrio cholerae. Merc-Sol-6 and Sulfur-6 were Potentized/Dynamized to Merc-Sol-7 and Sulfur-7 and their antibacterial activity was determined. Merc-Sol-7 showed zones of inhibition against E. coli O157:H7 (9.40±0.10/50 µl/disc and 11.04±0.01/100 µl/disc), Shigella dysenterae (8.5±0.34/50 µl/disc and 10.25±0.06/100 µl/disc), Salmonella typhi (8.23±0.12/50 µl/disc and 10.13±0.31/100 µl/disc),Vibrio cholera (9.34±0.12/50 µl/disc and 10.21±0.20/100 µl/disc) and Sulfur-7 showed zones of inhibition against E. coli O157:H7 (9.37±0.01/50 µl/disc and 11.04±0.40/100 µl/disc), Shigella dysenterae (8.35±0.02/50 µl/disc and 10.34±0.08/100 µl/disc), Salmonella typhi (8.33±0.08/50 µl/disc and 9.38±0.06/100 µl/disc),Vibrio cholera (8.23±0.20/50 µl/disc and 11.05±0.08/100 µl/disc). These findings showed a little less activity than merc-sol-6 (E. coli O157:H7; 45.05%, Shigella dysenterae; 33.66%, Salmonella typhi; 47.05%, Vibrio cholera; 47.53%) and sulfur-6 (E. coli O157:H7; 45.16%, Shigella dysenterae; 53.74%, Salmonella typhi; 56.76%, Vibrio cholera; 38.78%), suggesting that activity of dynamized homoeopathy medicines sometime for some reasons would not be increased. Bio-potency of Merc-Sol-6 and Sulfur-6 were determined in comparison to Azithromycin. Bio-potency of Merc-Sol-6 showed 19.33%, 20.42%, 8.96%, 23.16%, and, 17.84% against M. luteus, E. coli O157:H7, Shigella dysenterae, Salmonella typhi, and Vibrio cholerae, respectively and Sulfur-6 showed 20.56%, 39.92%, 23.52%, 23.59% and 20.88%, against M. luteus, E. coli O157:H7, Shigella dysenterae, Salmonella typhi, and Vibrio cholerae, respectively. Comparative study of Merc-Sol-6 and Sulfur-6 to Azithromycin was carried out which showed 80.33%, 66.08%, 86.52%, and, 78.42 against E. coli O157:H7, Shigella dysenterae, Salmonella typhi, and Vibrio cholerae, respectively and Sulfur-6 showed 91.31%, 85.17%, 91.11%, and 81.67%, against E. coli O157:H7, Shigella dysenterae, Salmonella typhi, and Vibrio cholerae, respectively. From this investigation it is suggested that Merc-Sol-6 and Sulfur-6 can be used in controlling infectious diseases caused by E. coli O157:H7, Shigella dysentrae, Salmonella typhi, and Vibrio cholera. Extensive researches are to be necessary to find any other homoeopathy medicines against active against enteric and non-enteric human infectious diseases.
Assessment of microbiological quality of drinking water in an urban low-income settlement of Bangladesh: investigation for Escherichia coli and Vibrio cholerae
(University of Dhaka, 2020-01-01) Ferdous, Jannatul
Assessment of virulence properties and charcterization of environmental aeromonas spp by Biochemical, serological and molecular techniques
(© University of Dhaka, 2025-05-04) Rahman, Muhammed Zeaur
Biodiversity of intestinal microflora in severely malnourished children with cholera
(© University of Dhaka, 2025-05-04) Monira, Shirajum
Bioprocess Development for Production of Proteases by Bacillus Licheniformis
(University of Dhaka, 2019-06-17) Al Mamun, Md. Arafat
Bacterial 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.
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 fungi and its enzymes for allocation in retting of jute ribbons
(© University of Dhaka, 2025-05-04) Akhter, Firoza
Characterization of indigenous bio-control agents against wilt complex pathogens of tomato
(© University of Dhaka, 2025-05-04) Sultana, Nazneen
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.
Characterization of microbial agents in diabetic foot infection and its correlation with chronic subclinical inflammation
(© University of Dhaka, 2025-05-04) Karmaker, Mousumi
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.
Characterization of PE-PGRS Proteins of Mycobacterium tuberculosis and Molecular Studies of Strains from Bangladesh
(© University of Dhaka, 2025-05-04) Banu, Sayera
Characterization of the Immune Response to Vibrio cholerae 01 antigens and its impact on Vaccine development strategies
(© University of Dhaka, 2025-05-04) Ahmed, Firoz
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.
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.
Detection and molecular characterization of multi-drug resistant nonfermentative Gram-negative bacteria from clinical isolates
(University of Dhaka, 2021-08-24) Islam, Md. Nazrul
The increasing rate of carbapenem resistant nonfermentative Gram-negative bacteria is a serious global health threat. Almost all studied isolates of Pseudomonas aeruginosa and Acinetobacter baumannii showed resistance to carbapenem. Carbapenem is the last option for treating Multi-drug resistant (MDR) P. aeruginosa and A. Baumannii but resistance against this drug has been emerging. Two types of β-lactamases differentiated by their mode of target hydrolysis, serine β-lactamase and metallo-β-lactamase (MBL). These are major foci of the present study in nonfermentative Gram-negative isolates of P. aeruginosa and A. baumannii. When studying various virulence properties, a majority of both P. aeruginosa and A. baumannii isolates showed hemolytic activity. However, formation of capsule, invasiveness and cytotoxic activity were observed only in the case of A. baumannii. Among the 100 P. aeruginosa and A. baumannii isolates tested, 92% and 35% showed MBL positive, respectively. Verona Integron-encoded metallo-β-lactamase (VIM) is an important MBL which confers resistance to regular using antimicrobials and now a days carbapenems became resistant due to this enzyme. We obtained a total of 16 VIM encoded MBLs from 100 P. aeruginosa isolates, but none from a similar number of A. baumannii strains. The sequences of VIM gene from 16 strains together with 22 other similar sequences, retrieved from the NCBI GenBank database were used to construct the phylogenetic tree to understand the nearest neighbour of the study sequences. Genetic divergence and homogeneity of the sequences are apparent in the phylogenetic tree. For VIM type of MBL, P. aeruginosa phylogenetic analysis showed similarities with the strains of India, Thailand, Nepal, Egypt, Turkey, UK, USA and Tunisia (93-100%). These findings suggest that the blaVIM gene is similar to global circulating strains. From Bootstrap table it has been found that 90-100% nucleotide similarities exist among the 16 Bangladeshi strains. Comparable results of nucleotide and amino acid sequences of VIM showed differences with a reference strain retrieved from the NCBI for 4 Bangladeshi P. aeruginosa strains and among the changes, three were in important positions (72, 145 and 146) as these changes brought significant modification in amino acid sequences from polar/nonpolar to nonpolar/polar that might affect the enzymatic nature of VIM. We obtained 6 New Delhi Metallo-β-lactamase-1(NDM-1) positive isolates from 100 A. baumannii strains, but none from the same number of P. aeruginosa strains. Among these 6 blaNDM-1 genes, 5 sequences together with 17 other similar sequences retrieved from the NCBI GenBank database were used to construct the phylogenetic tree to understand the nearest neighbor of the study sequences. Genetic divergence and homogeneity of the sequences are apparent in the phylogenetic tree. Amongst the DNA sequences obtained in our study, the isolates MN226842, MN226843, MN226844 and MN226846 formed distinct lineages with the strains from India, while only isolate MN226845 shared similarity and was found to be closely related to the sequences with Iran, Egypt and Korea. From these findings, we can predict that the NDM-1 may have been transmitted in Bangladesh from India. Bootstrap table shows 100% nucleotide similarities among the 5 Bangladeshi strains. On the other hand, 10 strains from China showed 52% similarity. Comparable results of NDM-1 of A. baumannii showed no differences in nucleotide and amino acid sequences with a reference strain retrieved from the NCBI which suggest that these are clonal. The study reports on the presence of blaVIM and blaNDM-1 gene in nonfermentative Gram-negative bacteria in Bangladesh for the first time.