Escherichia coli in milk, is a serious public health concern as milk is considered a complete food and an important part of daily human diet worldwide, including in Bangladesh. However, there have been no reports on the molecular characterization and antibiotic resistance profile of extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk of healthy cows in Bangladesh. Therefore, this study aimed to detect and characterize ESBL-producing E. coli (ESBL-Ec) in milk samples from healthy cows in smallholder dairy farms in Mymensingh district, Bangladesh, and assess the potential risk of consuming this milk.The emergence of antimicrobial-resistant bacteria, such as Escherichia coli was isolated from the collected samples using standard methods. The detection of ESBL-Ec was performed phenotypically using cultural methods and genotypically by ESBL genetic determinants using multiplex polymerase chain reaction. Antimicrobial susceptibility testing of the ESBL-Ec isolates was performed using the disk diffusion method with 15 common antimicrobials.A total of 100 milk samples were collected from apparently healthy cows on smallholder dairy farms. E. coli. Among these, 41 (58.6%) strains were identified as ESBL-producing, both phenotypically and genotypically, with the presence of blaCTX-M, blaTEM, and blaSHV individually or combined (blaCTX-M plus blaTEM plus blaSHV). The antibiogram of these ESBL-positive isolates revealed high resistance against commonly used antibiotics, such as ampicillin, cefotaxime, and gentamicin (100%), azithromycin (88%), oxytetracycline (27%), nalidixic acid, cotrimoxazole/trimethoprim (24%), and streptomycin (22%). In addition, one isolate showed resistance to 4th generation of cephalosporin (cefepime). Most importantly, extensive multidrug resistance was found in many ESBL-Ec isolates. However, the isolates were highly sensitive to drugs such as ceftriaxone (100%) and imipenem (100%). This is the first study to detect ESBL-Ec in raw milk from healthy cows on smallholder dairy farms in Bangladesh.In this study, out of the 100 samples tested, 70 (70%) were found to be positive for E. coli isolated from raw milk of healthy cows tested positive for ESBL production and showed resistance to most commonly used antimicrobials which may be alarming for human health. A limitation of our study is that we had a small size of sample collected from one district in Bangladesh. Therefore, a larger sample size covering a wider geographic area, and using multi-locus sequence typing and whole genome sequencing could provide a more comprehensive understanding of the prevalence and characteristics of ESBL-Ec in Bangladesh.More than 58% of the  Escherichia coli harboring extended-spectrum beta-lactamase (ESBL) gene has been considered as a global threat to human health in the past decade [E. coli (ESBL-Ec) [E. coli is widely distributed in food and wild animals, hospital settings, humans, environment and food supply chain, etc. [blaCTX-M gene based on amino acid sequence homology  [blaCTX-M-15 member of blaCTX-M-1 group is the most dominant globally, including Bangladesh [blaCTX-M-1 and blaCTX-M-2 are prevalent [Enterobacteriaceae [Enterobacteriaceae bacteria, enterohemorrhagic E. coli strains can cause infections through milk, posing a significant health risk to humans [t decade , 2. The ESBL-Ec) . Extendein, etc. . There ain, etc. . GloballTX-M-25) . The blangladesh . In Bangrevalent , 8. Ceforevalent , 9. In Beriaceae . In addieriaceae . Among Eo humans . The preo humans , 13. Theo humans . Hence, The smallholder farming system contributes greatly to our national economy. However, farmers of smallholder dairy farms lack awareness about the rational use of antimicrobials, leading to frequent irrational use of antimicrobials as therapeutics or prophylaxis for dairy cows. Although there are a few reports on the prevalence of ESBL-Ec in drinking water , retail Therefore, this study was conducted to detect and characterize ESBL-Ec from healthy cow raw milk samples from smallholder dairy farms in Mymensingh district in Bangladesh.Ethical approval was not required for this study because animals were only subjected to milk collection. However, written informed consent was obtained from the owners for the participation of their animals in this study.The study was conducted from April 2021 to January 2022. A total of 100 raw milk samples were collected from healthy cows from smallholder dairy farms of 4 Upazilas  in Mymensingh district.Approximately, 15 mL of milk was collected in sterile plastic containers directly from the cow’s teat. The samples were transported to the laboratory of Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University while maintaining a cold chain within 5–6 h. All samples were tested on the same day they were received in the laboratory. The cows were healthy and did not show any signs of mastitis. Before collecting milk samples, the udder was thoroughly cleaned and wiped with a clean, dry towel, and each teat was disinfected using 70% alcohol.E. coli like colonies were collected from each sample.The milk samples were enriched using a modified protocol based on a previously described method . SpecifiE. coli [All the isolates were subjected to biochemical tests such as triple sugar iron, lysine indol motility, Simmon’s citrate test, and Voges–Proskauer test to identify E. coli .E. coli isolates was determined using the double-disk synergy test. The test involved using CTX and ceftazidime (CAZ) with or without clavulanic acid (CA), as recommended by the Clinical and Laboratory Standards Institute (CLSI) [The ESBL production of e (CLSI) . An isolet al. [blaTEM, blaSHV, blaCTX-M-1, and blaCTX-M-2) was performed by multiplex polymerase chain reaction (PCR) using primer set and PCR conditions [Bacterial DNA was extracted by boiling 1 mL of overnight culture, as described by Parvin et al. . Extendenditions . In brieE. coli colonies were collected and suspended in 5 mL of sterilized saline. The suspension was adjusted to achieve turbidity equivalent to 0.5 McFarland standards. An evenly distributed bacterial lawn was prepared on Mueller–Hinton agar plates using sterile cotton swabs. Antimicrobial disks were placed on each bacterial lawn. The inhibition zone of each antimicrobial agent was analyzed after 16–18 h of incubation at 37°C. Results were interpreted according to the CLSI guidelines [E. coli strain ATCC 25922 as a control strain. Multidrug resistance (MDR) was defined as resistance to at least one antimicrobial agent from three or more antimicrobial classes [The antimicrobial susceptibility of the ESBL-Ec isolates was tested by the disk diffusion method  using coidelines . The sus classes .Escherichia coli like colonies were isolated and identified from 70 out of 100 raw milk samples. The percentage of E. coli isolated from healthy cow raw milk in smallholder dairy farms from different upazilas was 67% in Mymensingh Sadar, 83% in Muktagacha, 75% in Phulpur, and 50% in Tarakanda , blaTEM, and blaSHV. The analysis of ESBL genotype exhibited that 54% (22/41) of the ESBL-Ec carried blaCTX-M, followed by blaTEM at 24% (10/41) and blaSHV at 5% (2/41), whereas the combination of blaCTX-M with blaTEM was observed in 5% (2/41) and blaTEM with blaSHV in 12% (5/41)  . The stuE. coli carrying one ESBL group or their combination, including blaCTX-M-1 (99.9%), blaCTX-M with blaTEM (100%), blaTEM (80%), blaTEM with blaSHV (80%), and blaSHV (100%) (E. coli harboring blaCTX-M-1 (27%), blaCTX-M with blaTEM (50%), blaTEM (30%), and blaTEM with blaSHV (20%) (All the isolates of ESBL-Ec obtained from raw milk showed resistance to AMP and CTX . The higV (100%) . Most imHV (20%) .We described the detection of ESBL-Ec in healthy cow raw milk for the first time in Bangladesh. The high prevalence of ESBL-Ec in raw milk and the multidrug-resistant nature of the isolates indicate a potential risk to human health. The consumption of raw milk should be avoided. The treatment of human illness caused by ESBL-Ec should be based on the antibiogram study findings to avoid further developing antimicrobial resistance.E. coli isolates, which is much higher than a previous report (38.0%) in Sudan [blaCTX-M (59%), blaTEM (41%), and blaSHV (17%). In contrast, in Bangladesh, 38.9% of E. coli isolated from milk of cows suffered with mastitis [blaTEM. This variation may be due to the difference in isolation protocol or sample sources. However, Batabyal et al. [blaCTX-M. The blaCTX-M gene in E. coli is thought to be circulating among dairy cattle in Bangladesh. Among blaCTX-M genes, the isolates carried blaCTX-M-1 gene-group (54%) and a combination of this gene with blaTEM (5%) but no isolates harbored blaCTX-M-2 gene-group. According to geographical distribution, the prevalence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 genes under blaCTX-M group have been reported in Asia [blaCTX-M-1 in E. coli from raw milk origin may be relevant. Moreover, many ESBL-Ec isolates harbored the combination of blaCTX-M plus blaTEM and blaTEM plus blaSHV genes. This indicates the increasing emergence of ESBL-Ec in Bangladesh, possibly due to travelers from abroad where ESBL-Ec is prevalent.Our study revealed that 70% of the raw milk carried in Sudan . The prein Sudan ‒24. Thesin Sudan . We founin Sudan , 26. Themastitis  and 100%l et al.  reported in Asia , 28. HenEnterobacteriaceae, causing gastrointestinal diseases in humans in many countries, including Bangladesh [E. coli isolated from raw milk. The high prevalence of MDR and XDR ESBL-Ec in this study highlights the potential for these bacteria to serve as a reservoir of resistance genes and pose a threat to public health [It is well known that the plasmids harboring ESBL genes can also carry resistant genes against many other antimicrobials such as aminoglycosides, sulfonamide chloramphenicol, and tetracycline . In thisngladesh , which mc health .The prevalence of ESBL-Ec in healthy cow raw milk is very high and consumption of raw milk should be avoided. Most of the ESBL-Ec we detected were MDR and XDR, which can easily be transmitted to humans as consumers and, thus, result in potential health hazards. Therefore, it is important to raise awareness among the public and stakeholders about the potential risks associated with the consumption of raw milk and need to adopt appropriate measures to prevent the spread of AMR pathogens. This is the first study to report the detection of ESBL-Ec in healthy cow raw milk in Bangladesh.AN, MMA, and AKMAR: Conceptualization. AN, AKMAI, MNI, MKK, and MSK: Methodology. AN, AKMAI, MNI, MKK, and MSK: Original draft preparation. MMA and AKMAR: Editing and supervision. All authors have read, reviewed, and approved the final manuscript.


Valeriana jatamansi,exhibited an association with beta-amyloid binding activity, a potential therapeutic approach for AD. From our study we could understand how these plants modulateour body to manage these diseases. However, further in vitro and in vivo validation is needed before commercial and public use of this data.Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and epilepsy, pose a growing global health challenge due to an agingpopulation. These conditions share common processes, including protein accumulation, oxidative stress, and neuro-inflammation, making their treatment complexand costly. Network pharmacology, an innovative approach integrating systems biology and computational biology, offers insights into multi-target formulationsand the repurposing of existing medications for neurodegenerative diseases. We shortlisted 730 bioactive compounds from 25 traditional Himalayan plants, assessedtheir drug-like properties using ADME criteria, and predicted their potential target proteins through reverse docking and pharmacophore mapping. Our studyidentified 287 compounds with high gastrointestinal absorption and good blood-brain barrier permeability. These compounds were subjected to target prediction,yielding a list of 171 potential target proteins. Functional annotation and pathway enrichment analysis highlighted their involvement in steroid hormone-relatedpathways, MAPK signaling, FOXO signaling, TNF signaling, VEGF signaling, and neurotrophin signaling. Importantly, one plant,  Neurodegenerative diseases are the progressive deteriorating conditions characterized by the death of nerve cells and supreme loss of neurons.Neurodegenerative diseases are age-related conditions that have an effect on the brain as well as the nerves found throughout the human body and the spinalcord. These age-dependent disorders have become progressively rife, partly as a result of the senior population has risen in recent years. Neurodegenerativediseases mainly include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), front temporaldementia and the spinocerebellar ataxias . NeurodeAmong all, presence of AD is considerably high and is increasing per annum. The global burden of AD is anticipated to accelerate from 26.6 million cases in2006 to 106.8 million by 2050 . AD is cUnfortunately, the complex etiology and pathogenesis of neurodegenerative diseases limit the efficacy of drugs for their treatment, and higCannabis sativa, Cyperus rotundus, Ravoulfia serpentina,and many others [Here, we studied twenty-five Himalayan plants mainly from Darjeeling and some Sikkim region which are traditionally used treating neural diseases likeepilepsy, headache, and other CNS related diseases. Traditionally many plants are used in different regions by local healers to treat many of these neurologicaldisorders. The sub-Himalayan region of Eastern-India is also rich in such traditional knowledge. Darjeeling, the northern most part of the state West Bengal,India, is situated in the foothills of the Himalayas. It is rich in culture and is inhabited by different tribes. These tribes have treated diseases like epilepsy,headache and other neurological disorders using plants such as y others ,9,10,11y others . TherefoAn extensive study was done to understand the traditionally used plants of Darjeeling Hills and Sikkim which are used for treating neural diseases likeepilepsy, headache and other CNS related diseases. Along with literature survey, we also talked with some of the locals of the region and validated thedocumented knowledge. A total of 25 plants from Himalayan region which are traditionally used in treating neural disease were selected using literature study.The bioactive compounds of the selected plants were obtained from Dr. Duke's Phytochemical and Ethnobotanical database .Dr. DukThe drug likeliness of the phytocompounds is determined on the basis of their ADME property. ADME  propertyevaluation helps to get an idea regarding how a chemical is processed by a living organism . Thus, wThe phytocompounds interact with numerous proteins of our body and modulate them. PharmMapper predicts potential target candidates for the given smallmolecules on the basis of reverse docking and pharmacophore mapping approach . The selTo understand the role of a set of genes in metabolic pathways, gene ontology analysis is important. The target-gene list of each plant was submitted inDAVID Bioinformatics Resources v6.8 (https://david.ncifcrf.gov/tools.jsp) for functional annotation and KEGG pathway enrichment analysis. It gives an insightin role of these proteins in different metabolic pathways. Gene ontology (GO) enrichment analysis comprised Biological Process (BP), Cell Component (CC), andMolecular Function (MF). The results with significant p-Value  were taken into consideration.A total of 25 plants from Himalayan region which are traditionally used in treating neural disease were selected using literature study. The plant list isprovided in the supplementary file.Total 1362 phytochemical compounds were obtained from the studied plants, out of which 730 compounds having canonical smiles and CID number. The ADMEproperties of the acquired compound were analyzed, which shows 287 compounds with High Gastrointestinal Absorption (GI) and good Blood Brain Barrier (BBB), 34compounds with low Gastrointestinal (GI) absorption but good Blood Brain Barrier (BBB) permeability, 139 Compounds with high Gastrointestinal absorption (GI) butno Blood Brain Barrier (BBB) permeability, and 273 compounds with low Gastrointestinal absorption (GI) and no Blood Brain Barrier permeability (BBB). Thecompounds having both high GI and good BBB permeability were considered and further analysis was carried out. A plant phytocompound network of the compoundswith best results is shown in Panax ginseng has the highest number of targets (51), followed by Curcuma longa and Cannabis sativa. Thecompound Yohimbine is the best compound as it has 15 targets. Functional annotation and pathway enrichment analysis of the targets show association of theplants with various pathways that are related to nervous system pathways in Homo sapiens were found to target several genes related to steroidhormone metabolic pathways. They show ability to modulate genes related to "steroid hormone mediated signalling pathway", "steroid hormone receptor activity","Steroid hormone biosynthesis" and "steroid binding" activity. The plants Centella asiatica and Ravoulfia serpentine mainlyact through these pathways only. Cannabis sativa and Panax ginseng too modulate a number of these pathways.The selected traditionally used plants for neural disorders , Parkinson's disease (PD), amyotrophic lateral sclerosis(ALS) and various types of cancers [Functional annotation and pathway enrichment analysis show association of the plants with MAPK signalling pathway, MAP kinase activity, activation ofMAPK activity, protein tyrosine kinase activity, etc. that play role in cell proliferation. The plants  cancers .Apart from MAPK signalling and kinase activity, the plants target various pathways like FoxO signaling pathway, VEGF signaling, Neurotrophin signallingpathway, etc. Foxo (Forkhead box) proteins help in regulation of growth factor and stress regulating factor. FOXO3 has been found to cause axonal degenerationupon withdrawal of neurotrophic factors and can influence both CNS and PNS . AbnormaThe plants also modulated TNF (Tumour necrosis factor) signalling pathway and VEGF signalling pathway. Both pathways are associated with neuroprotection inthe brain. Another modulated significant pathway is Neurotrophin signalling which pathway plays an important role for neural development and additionalhigher-order activities such as learning and memory . Thus, by regulating this pathway, development and enhancementof neural cells can be modulated, which may be helpful in preventing various neural diseases.V. jatamansitargets a different set of genes for treatment of neurological disorders, then the rest of the selected plants. Its phytocompounds interact with target proteinsthat have "beta-amyloid binding activity". It is known that beta-amyloid protein accumulation causes AD [Modulation of all these targets and pathways may be the mode of action for these plants that have been used to treat neurological diseases from timeimmemorial. These may also help in finding a new approach in treating such diseases. However, in our studies it was seen that the plant auses AD . Thus, tin vivo and in vitrostudies is essential to gain a deeper understanding of these results and their potential implications for the management of neurological disorders.In conclusion, while the human race has long been regarded as unique and superior among living organisms due to its intelligence and cognitive capabilities,the contemporary lifestyle and increasing levels of stress pose a significant threat to this exclusivity. Today's lifestyle significantly elevates the risk ofvarious cerebrovascular diseases, potentially contributing to degenerative forms of cognitive impairment, as indicated by researchers. Neurological disorders,as defined by the World Health Organization (WHO), encompass conditions affecting both the brain and the nervous system, which extends throughout the human body.Our study sheds light on the underlying mechanisms of traditionally employed plants in addressing these issues. Notably, steroid hormones play a direct role inthe aging process and the development of neural diseases, suggesting that the modulation of these pathways may be central to the action of these plants. Ourfindings suggest that the modulation of steroid hormone pathways and pathways related to cell viability may hold promise in managing cognitive diseasesassociated with neural cell damage. However, it is imperative to note that further validation through AS conceived the idea. TDL and SD collected data and executed the analysis. JL, MAA, SD & AS helped in data analysis using Bioinformatics tools. All theauthors have contributed in writing the manuscript and approved it.