Decoding the Mystery of Antibiotic Persistence

This work combines microbiology, molecular biology, Next-Generation Sequencing and system biology approaches to explore the mechanism of antibiotic persistence: a multi-drug tolerant, non-dividing, and metabolically altered state present in a subpopulation of cells due to phenotypic diversity rather than genetic variation (i.e. mutations). Persister can survive lethal antibiotic state and resuscitate after the treatment period is over. They are considered as the major contributing factor behind recurring infections. They also have a high mutation rate, which increases the chances of bacteria gaining antibiotic resistance. The formation of this phenotypic variant (persister) threatens the therapeutic effectiveness of antibiotics and understanding how bacteria form these heterogenic populations is critical for the development of new therapies. The molecular mechanisms underlying persistence are varied and debated in the literature. To probe this phenomena, I have explored three fundamental questions in this study related to antibiotic persistence, 1) when do persister cells form, do they form in response to the stress or are some cells prepared for stress (Chapter 2); 2) how do they go into this metabolically altered state and maintain this state for long-term antibiotic treatment (Chapter 3); 3) Is persistence an essential mechanism for bacteria and if so, does a minimal cell form persisters (Chapter 4)? There is much controversy in the literature when do these phenotypic variants (persister) form. To understand this, I have taken a novel approach to explore bacterial heterogeneity using a powerful fluctuation test (FT) framework to infer transient cell states that arise via reversible and non-genetic mechanisms to find hidden features of bacterial persistence. The FT was first pioneered ~80 years ago when Luria & Delbrück demonstrated that genetic mutations arise randomly in the absence of selection but not in response to the selection. They were studying phage (bacterial virus) infection. At the time of their work, it was debated whether mutations leading to resistance were directly induced by the virus (Lamarckian theory), or if they arose randomly in the population before viral infection (Darwinian theory). Their result showed genetic mutations arise randomly in the absence of selection, validating the Darwinian theory of evolution and led to a Nobel Prize in 1969. Our bacterial persister work is quite similar. Instead of studying mutations, we are exploring the existence of primed cells (a heterogeneously distinct subpopulation) before treatment that allow for long-term survival after treatment. Using the FT, we tested the variation between clonal populations that originated from an identical clone and showed that a subset of the population has an epigenetic memory that primes cells for persistence. We support our results with experimental benchwork and mathematical models. I am the first to definitively demonstrate that some cells are prepared in the population through a stochastic mechanism that enters into persistence in response to stress. Our work highly suggests that phenotypic heterogeneity is not entirely a random event resulting from stochastic noise in gene expression. Instead, it can develop from a rare, transiently inherited gene expression event. Another most argued topic in persister research field is whether persister cells transcribe during this non-dividing state in response to stress. To understand this, we isolate RNA from persister subpopulation at different time-points and compare the persister transcriptome profile with before antibiotic treated culture. Our analysis of RNAsequencing data demonstrates that persisters actively transcribe and translate proteins and they use multiple regulatory pathways to enter and maintain this altered state during antibiotic treatment. From our transcriptome analysis, we have selected seven uncharacterized genes which upregulated in persister cell and observed their effect on persister survival by overexpressing or making knockouts of those genes. When we overexpressed the hypothetical genes, most of them cause the growth defect and some of them put the cells into sleepy state for several hours. Our knockout mutants decrease the persister level dramatically by ~4-6 fold and ~10-15 fold at 3 h and 6h of Amp treatment, respectively. However, at 24 h, it showed no significant difference in survival from wild type which again showed that the persister transcriptome adjusts its expression profile over time. This result demonstrated that persisters alter their transcriptome to maintain their survival over the long-term antibiotic treatment. This research is particularly important, because researchers in persister field still argued over this and often cited dormancy is the primary method of persister survival. Another exciting study I have done where we used a minimal cell Mycoplasma mycoides JCVI-syn3B, contains only 491 genes and most of them are essential genes. We demonstrated that this minimal cell persists against multiple antibiotics although it contains a few already identified persister-related genes, whereas lacking many systems previously linked to persistence (e.g. ribosome hibernation genes). We also found that Syn3B evolves antibiotic resistance to different types of antibiotics expeditiously. Overall, in this study I explored the regulatory mechanisms that allow persisters to survive long-term antibiotic treatments. This work will impact our fundamental knowledge about antibiotic survival and set the groundwork for developing more effective therapeutics for recurrent bacterial infections

Unified Policies, Healthier Journeys: Adressing the Used-Lead-Acid-Battery challenge in Bangladesh

E-rickshaws play significant roles in Bangladesh’s society and economy, but there are major concerns about the health and environmental impacts related to the system in which they operate, in particular how their batteries are sourced and disposed of. This report reviews the current state of e-rickshaws and the recycling of Used-Lead Acid Batteries (ULABs) in Bangladesh, focusing on the regulatory landscape and policies ensuring sustainable practices, particularly in the context of e-mobility. Existing policies aim to address challenges associated with informal Used Lead-Acid Batteries (ULABs) recycling, environmental pollution, and public health hazards. However, notable policy gaps and challenges persist, hindering the effective implementation of sustainable practices

Potentially mineralizable nitrogen in ten important sub - tropical soil series of Bangladesh

Knowledge on nitrogen (N) supplying capacity of inherent soil organic matter helps farmers and extension workers to determine the N fertilization rate. However, this information is very scanty particularly in developing countries like Bangladesh. Thus, this research work was conducted to estimate potentially mineralizable nitrogen (N) in ten important soil series of Bangladesh namely Jamalpur, Silmondi, Sonatala, Ghatail, Tejgaon, Chandra, Khilgao, Kalma, Brahmaputra and Sherpur. Net N mineralization in these soil series was measured during 100 days laboratory incubation at 200C at field capacity. Findings revealed that eight out of ten soil series had silt loam texture and the rest two were loams. All soils were slightly acidic in reaction. The organic carbon and total N contents of the soils varied from 5.24 to 17.4 and 0.53 to1.48 g kg soil-1, respectively with the C:N ratios of 9.89 to 11.76. The amount of net N mineralization and nitrification (NO3-1-N) increased linearly with time; however, ammonification (NH4+-N) did not follow any definite trend. Majority of mineralized N undergone nitrification falling within the ranged between 30 and 128 mg/kg soil/98 days. The net N mineralization (mg NH4+-N+NO3-1-N kg-1 soil) varied from 51 mg N kg-1 soil in Tejgaon soil series to 154 mg N kg-1 soil in Khilgaon soil series accounting for 4.3 to 10.8% of total N mineralized 120 days-1. The rate of N mineralization also varied widely from 1.19 to 0.28 (mg N kg-1 soil day-1) like the net N mineralization. The highest rate of N mineralization was observed in Khilgaon soil series followed by Chandra, Ghatail, Silmondi, Sherpur, Jamalpur, Sonatola, Kalma, Bramaputra silt and Tejgaon soil series. N mineralization was significant and positively correlated with organic carbon and total N and NH4+ -N contents of soils. Khilgaon and Chandra soil series have shown greater potentiality in supplying nitrogen under aerobic condition

Identification of pathogenic Escherichia coli strain from river and sewage water in Bangladesh

The study was conducted to isolate and identify the presence of Shiga toxin producing Escherichia coli (STEC) strains in water samples of the Old Brahmaputra River and Sewage water at Mymensingh Municipality. A total of 20 water samples were collected for this experiment. Samples were cultured on EMB agar, stained and PCR was done to detect the pathogenic E. coli. The Old Brahmaputra River is used as a sink of all types of municipal sewage, agricultural wastes, domestic wastes and religious ritual wastes through unplanned sewerage to the river water body. Low quality sanitation system and open defecation are also considerable problems to deteriorate river water quality. Due to accumulation of municipal untreated wastes to river body, it is possible to contain various pathogens. After investigation and identification, fourteen isolates of E. coli was found to contain stx-1 gene with none of stx-2 gene among twenty isolates which indicate pathogenic STEC. There may present major health risk to human and animal due to STEC. Several human diseases like mild diarrhea, bloody diarrhea or even severe hemolytic-uremic syndrome (HUS) may occur for drinking of untreated river water. Domestic ruminants can act as a reservoir for STEC and play a significant role in the epidemiology of human infections

Students' Perception of Educational Environment of Medical Colleges in Bangladesh

Background: Students' perceptions of their educational environment are a useful basis for modifying and improving the quality of educational environment. Educational environment is one of the most important factors determining the success of an effective curriculum. The quality of educational environment has been identified to be crucial for effective learning. Identifying the weakness of educational environment and understanding how students perceive the environment will help the institute to facilitate learning and to achieve better learning outcome. Objective: To explore students' perceptions of their educational environment and to find out gender differences in perception. Methods: It was a cross sectional descriptive study. Dundee Ready Education Environment Measure (DREEM) inventory was administered to 1903 medical students (studying in 3rd, 4th and 5th year MBBS course) in 15 medical colleges of Bangladesh adopting purposive sampling. Results: The total mean score for all students was found positive (110/200). Students' perceptions of learning was positive (28/48), perceptions of teachers was moving in right direction (24/44), students academic self perception was positive (19.5/32). Students' perceptions of atmosphere was expressed as many issues need to change (24/48) and social self perceptions was not a nice place (14/28). Female students’ perceptions were significantly higher than male students. Conclusion: Remedial measure should be needed in the subscales of students’ perceptions of atmosphere and social self perceptions for further improvement. Findings from this study may give guideline to curricular planner and faculties/administrators of medical college for further improvement of educational environment. Key words: perception; educational environment; medical college  DOI: 10.3329/bsmmuj.v3i2.7060BSMMU J 2010; 3(2): 97-10

Geographical Concentration of Rural Poverty in Bangladesh

This paper was presented at the dialogue on Mapping Poverty for Rural Bangladesh: Implications for Pro-poor Development. The dialogue was organised as part of CPD's ongoing agricultural policy research and advocacy activities with IRRI under the PETRRA project. The study reported geographical concentration of rural poverty in Bangladesh for 425 upazilas in 2000-01. The study measured and mapped incidence of poverty (using Headcount Index), intensity of poverty (using Poverty Gap Index) and severity of poverty (using Squared Poverty Gap Index). It has analyzed factors contributing to the spatial concentration of poverty. It is hoped that the findings of the study would be helpful in identifying target areas and priorities for agricultural R&D interventions and poverty reduction programmes.Poverty, Rural Poverty, Bangladesh

Formulation of value added beef meatball using tulsi (Ocimum sanctum) leaf extract as a source of natural antioxidant

The present study was undertaken to evaluate the effect of different levels of tulsi leaf extract on fresh and preserved beef meatballs. Four types of beef meatballs were formulated for this purpose. Meatballs were made with 0 (control), 0.1, 0.2 and 0.3% tulsi leaf extract, respectively and preserved at-20°C. Quality and safety evaluation of meatballs were determined by sensory, physicochemical, biochemical and microbiological tests. The analyses were conducted at 0, 15th, 30th and 60th days of interval. Considering CP, tenderness, juiciness, overall acceptability, cooking loss, Free Fatty Acid (FFA), Per oxide Value (POV) and Thiobarbituric Acid Reactive Substances (TBARS) value, it can be concluded that tulsi leaf extract @ 0.1, 0.2 and 0.3% can be used in the formulation of beef meatball. In case of sensory evaluation 0.2% tulsi leaf extract is appreciated but on the basis of nutrient quality, physicochemical properties, biochemical analysis and microbial analysis 0.3% tulsi leaf extract is more satisfactory as a source of natural antioxidant than that of other treatment groups. Therefore, it may be concluded that 0.3% tulsi leaf extract can be added as a functional ingredients in beef meatball

Amoxyclav Resistance Pattern and Aerobic Bacterial Profile in Diabetic Foot Infection Patients in Bangladesh

Introduction: The goal of this study was to determine the frequency of bacterial isolates cultured from diabetic foot infections and to assess their amoxyclav resistance and susceptibility.Methods: A total of 378 diabetic foot lesions were included in this prospective analysis. The antibiotic susceptibility pattern of bacteria isolated from foot lesions was assessed using the Kirby-Bauer disk diffusion method.Results: The most commonly isolated Gram-positive bacteria were Staphylococcus aureus, followed by Enterococcus spp. and CoNS. The most commonly isolated Gram-negative bacteria were Klebsiella spp., Pseudomonas spp., Proteus spp., Escherichia coli, Enterobacter spp., Citrobacter spp., Citrobacter freundii, and Proteus vulgaris. Amoxyclav was found to be 100.00% resistant against Pseudomonas aeruginosa and followed by Enterococcus spp. (89.50%), Proteus spp. (87.50%), Staphylococcus aureus (84.30%), Escherichia coli (81.50%), Klebsiella spp. (70.50%) and Enterobacter spp. (69.20%).Conclusion: The present study confirmed the prevalence of amoxyclav drug resistant pathogens (84.90%) in diabetic foot ulcers. The diverse bacteria infecting the wound must be evaluated, as well as the antibiotic susceptibility patterns of the isolates from the infected lesion. This information is critical for selecting the right medications, eliminating resistance trends, and lowering healthcare costs. Keywords: Diabetic Foot Infection, Polymicrobial Infections, Amoxyclav DOI: 10.7176/JHMN/94-03 Publication date:October 31st 202

Toward Genetic Engineering: Teaching Transformation Using the pGLO Plasmid in High School Classrooms

In this lesson plan, students will learn about genetic engineering and perform the same transformation protocol used by scientists on a daily basis around the world. Genetic engineering is a technique used for direct manipulation, alteration, or modification of genes or genomes of an organism to manipulate the phenotypes. The use of genetically modified organisms (GMOs) has emerged everywhere as a mainstay from agriculture to pharmaceuticals. Through this lesson, students will transform Escherichia coli with pGLO plasmid, which give a brilliant fluorescent green glow under UV light. Students will also learn the central dogma of molecular biology: DNA à RNA à Protein à Trait (Green Fluorescence), and the basics of gene regulation. This hands-on module is designed for 9th through 12th grade students. To adjust the difficulty level of the modules, some aspects can be modified or removed based on the grade. This module includes an instructional slide show, assessment options, and links to additional resources