Pramod Poudel

Multidrug‐resistant ( MDR ) Escherichia coli ( E. coli ) represents a significant public health concern, particularly when harboring virulence genes such as clbA, which encodes the genotoxin colibactin. This study assessed the distribution of the clbA gene among MDR E. coli isolates from normal individuals, cancer patients, and clinical patients, and examined its association with antimicrobial resistance patterns. A total of 115 MDR E. coli isolates were collected from January to December 2024 at two healthcare centers in Nepal. The clbA gene was detected in 13.0% (15/115) of isolates, with a significantly higher prevalence in clinical patients (25.0%) compared to cancer patients (8.6%) and normal individuals (5.0%) ( p = 0.0105). clbA‐positive isolates exhibited markedly increased resistance to critical antibiotics, including imipenem (100% vs. 15.0%, p = 0.003), meropenem (100% vs. 12.5%, p = 0.001), and amikacin (100% vs. 10.0%, p = 0.050), compared to clbA‐negative strains. These findings suggest that the presence of the colibactin‐encoding clbA gene in MDR E. coli is linked to heightened antimicrobial resistance, especially in clinical settings, underscoring the need for targeted molecular surveillance and infection control strategies.

In Nepal, improper storage of staple foods leads to contamination by aflatoxin-producing molds, posing serious health risks like liver cancer. Farmers, unaware of these dangers, often use unsafe chemicals for prevention, while biological control using Bacillus spp., a proven, safe, and multifunctional alternative, remains unexplored. This study aims to isolate native Bacillus species capable of inhibiting Aspergillus flavus growth and detoxifying aflatoxins, offering a sustainable solution. A total of 50 samples (40 soil samples, 5 compost and 5 food samples) were randomly collected from different places of Nepal. Bacillus. spp were selectively isolated by heating the samples at 80⁰C for 10 minutes. The isolates were identified by standard microbiological techniques. Growth inhibition of A. flavus by Bacillus. spp was done by dual-culture technique on potato dextrose agar. Isolates showing high inhibitory effect against A. flavus were further identified by 16S rRNA gene sequencing. Altogether 51 isolates were presumptively identified as Bacillus species. Five isolates, Bacillus subtilis, B. tequilensis, B. licheniformis, B. cereus, B. weidmannii effectively inhibited the growth of aflatoxigenic A. flavus. These potent Bacillus strains will have implication in the commercial production of bioactive anti-aflatoxigenic compound to minimize the postharvest contamination of the crops.

The rise of multidrug‐resistant (MDR) Escherichia coli ( E. coli ) poses a critical challenge due to limited treatment options. This study evaluates the anti‐MDR E. coli activity, phenolic and flavonoid content, antioxidant potential, and bioactive compounds in ethanolic leaf extracts of Azadirachta indica A. Juss. Among 115 MDR E. coli isolates, 61.7% showed susceptibility to the extract, with inhibition zones of 8–15 mm. The extract exhibited high phenolic (53.6 ± 0.444 mg GAE/g) and flavonoid (84.3 ± 0.889 mg QE/g) content, which may account for its strong antioxidant activity based on total antioxidant capacity (ascorbic acid IC ₅₀ : 9.0 ± 0.255  μ g/mL), although it showed only moderate free radical scavenging ability (DPPH IC ₅₀ : 406.69 ± 1.64  μ g/mL). High‐resolution liquid chromatography–mass spectrometry (HR‐LCMS) analysis identified 153 and 107 metabolites in negative and positive ESI modes, respectively, with several displaying antibacterial properties. The presence of alkaloids, phosphoramidates, and phenolic compounds supports its antimicrobial and antioxidant potential.

Introduction: The global issue of antimicrobial resistance to human health is becoming increasingly concerning. Escherichia coli (E. coli) is a widespread pathogen that causes a wide range of illnesses. This is because multidrug-resistant (MDR) E. coli bacteria have emerged, proliferated, and remained persistent. Antimicrobial resistance is more predominant among pathogenic organisms compared to the commensal ones. The correlation between resistance and virulence factors could be a result of the successive exposure of pathogenic organisms to antibiotics. Therefore, the goal of our study was to detect some virulence factors in multidrug-resistant E. coli that were isolated from fecal and other clinical samples. Methods: A laboratory-based cross-sectional study was conducted from October 2023 to March 2024, a period of six months. E. coli was isolated using standard microbiological methods from a range of clinical samples. Antibiotic susceptibility was done to identify MDR E. coli by Kirby-Bauer disk diffusion method. Different phenotypic assays were used to detect virulence factors. Results: Out of 318 isolated E. coli from different clinical samples, 160 (50.3%) were found to be multidrug-resistant. Higher distribution of MDR E. coli (51.9%) was found in extra-intestinal samples than fecal specimen. MDR E. coli strains were highly resistant to most antimicrobials. The most common virulence factor was cell surface hydrophobicity (100%) followed by motility (83.7%), biofilm production (36.2%), serum resistance (25%),hemolysin production (15%) and gelatinase production (2.5%). There were multiple virulence factors found in 156 (97.5%) MDR E. coli isolates. Conclusion: It was discovered that MDR E. coli strains obtained from patient intestinal and extra-intestinal samples were more virulent and more resistant to drugs.

Arsenic contamination in soil and water is one of the major environmental problems in multiple countries including Nepal imposing a serious threat to the ecosystem and public health. Many soil bacteria can detoxify arsenic, including genus Bacillus. With an objective to gauge the plant growth-promoting activities of arsenic-resistant Bacillus species, 36 samples (soil, rice, cauliflower, and beans) were collected from the Terai region of Nepal. For selective isolation of Bacillus species, each sample was heated at 80°C for 15min before the inoculation into nutrient agar (NA). Following the standard protocol, arsenic-resistant Bacillus species were screened using NA supplemented with 100ppm sodium arsenate and sodium arsenite. Among 158 randomly selected isolates, only five isolates were able to tolerate sodium arsenite concentration up to 600ppm. Notably, all five isolates were able to produce indole acetic acid (IAA), a plant hormone, and solubilize phosphate. Based on biochemical analysis and 16S rRNA gene sequencing, isolates N4-1, RW, KR7-12, Bhw1-4, and BW2-2 were identified as B. subtilis subsp. stercosis, B. flexus, B. licheniformis, B. cereus, and B. flexus, respectively. To the best of our knowledge, this is the first study showing the presence of arsenic-resistant B. flexus in Nepalese soil with plant growth-promoting traits. Possible utilization of these Bacillus strains could facilitate the novel bioremediation pathway to reduce the toxic effect of arsenic from the soil and water in the Terai region of Nepal.

Medicinal plants have been used from the beginning of human civilization against various health complications. Dengue virus (DENV) has emerged as one of the most widespread viruses in tropical and subtropical countries. Yet no clinically approved antiviral drug is available to combat DENV infection. Consequently, the search for novel antidengue agents from medicinal plants has assumed more insistence than in previous days. This study has focused on 31 potential antidengue molecules from secondary metabolites to examine their inhibitory activity against DENV nonstructural proteins through molecular docking and pharmacokinetics studies. In this research, the wet lab experiments were tested on a computational platform. Agathisflavone and pectolinarin are the top-scored inhibitors of DENV NS2B/NS3 protease and NS5 polymerase, respectively. Epigallocatechin gallate, Pinostrobin, Panduratin A, and Pectolinarin could be potential lead compounds against NS2B/NS3 protease, while acacetin-7-O-rutinoside against NS5 polymerase. Moreover, agathisflavone (LD₅₀=1430mg/kg) and pectolinarin (LD₅₀=5000mg/kg) exhibited less toxicity than nelfinavir (LD₅₀=600mg/kg) and balapiravir (LD₅₀=824mg/kg), and the reference drugs. Further research on clinical trials is required to analyze the therapeutic efficacy of these metabolites to develop new potential drug candidates against different serotypes of DENV.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has been a global concern. While there have been some vaccines and drugs, the rapid emergence of variants due to mutations has threatened public health. As the de novo drug development process is expensive and time-consuming, repurposing existing antiviral drugs against SARS-CoV-2 is an alternative and promising approach to mitigate the current situation. Several studies have indicated that some natural products exhibit inhibitory activities against SARS-CoV-2. This study is aimed at analyzing the potential of natural alkaloids, using various computational tools, as drug candidates against SARS-CoV-2. The molecular docking analysis predicted that naturally occurring alkaloids can bind with RNA-dependent RNA-polymerase (RdRP). The QSAR analysis was conducted by using the way2drug/PASS online web resource, and the pharmacokinetics and toxicity properties of these alkaloids were predicted using pkCSM, SwissADME, and ProTox-II webserver. Among the different alkaloids studied, neferine and berbamine were repurposed as potential drug candidates based on their binding affinity and interactions with RdRP. Further, molecular dynamics simulation of 90 ns revealed the conformational stability of the neferine-RdRP complex.

The multidrug- or extensively drug-resistant (MDR/XDR) Pseudomonas aeruginosa carrying some virulence genes has become a global public health threat. However, in Nepal, there is no existing report showing the prevalence of oprL and toxA virulence genes among the clinical isolates of P. aeruginosa. Therefore, this study was conducted for the first time in the country to detect the virulence genes (oprL and toxA) and antibiotic susceptibility pattern of P. aeruginosa. A total of 7,898 clinical specimens were investigated following the standard microbiological procedures. The antibiotic susceptibility testing was examined by the modified disc diffusion method, and virulence genes oprL and toxA of P. aeruginosa were assessed using multiplex PCR. Among the analyzed specimens, 87 isolates were identified to be P. aeruginosa of which 38 (43.68) isolates were reported as MDR. A higher ratio of P. aeruginosa was detected from urine samples 40 (45.98), outpatients specimens 63 (72.4), and in patients of the age group of 6079 years 36 (41.37). P. aeruginosa was more prevalent in males 56 (64.36) than in female patients 31 (35.63). Polymyxin (83.90) was the most effective antibiotic. P. aeruginosa (100) isolates harboured the oprL gene, while 95.4 of isolates were positive for the toxA gene. Identification of virulence genes such as oprL and toxA carrying isolates along with the multidrug resistance warrants the need for strategic interventions to prevent the emergence and spread of antimicrobial resistance (AMR). The findings could assist in increasing awareness about antibiotic resistance and suggest the judicious prescription of antibiotics to treat the patients in clinical settings of Nepal.