AvH-GroupLinkagePrg

Isolation and characterization of natural antibiotics from the high-altitude Actinomycetes against selected multidrug-resistant WHO prioritized human pathogenic bacteria by using HRMS based de-replication and genome mining

01/07/2026 to 30/06/2029

This research project is a continuation of presently running research project funded by the University Grants Commission, Government of Nepal towards the antibiotic drugs discovery using high altitude soil Actinomycetes. Human pathogenic bacteria that are classified by the WHO as PRIORITY 1 pathogens comprise carbapenem-resistant bacteria of the genera Acinetobacter, Pseudomonas and Enterobacteriaceae. Since they are a serious threat to the human health care systems worldwide, the WHO officially has announced the strong need for the research and development of new antibiotics. Actinomycetes from the extreme environments are considered promising sources of potent antibiotics. As a part of establishment of cryo-preservation based Actinomycetes culture collection at the Tribhuvan University, a total of 200 soil samples were collected from the Annapurna region and 405 soil samples were collected from the Everest region totaling 605 samples. So far, more than 1500 Actinomycetes colonies have been isolated and preserved as glycerol stock. These 1500 isolates were isolated from 250 soil samples collected from the Mount Annapurna (2800 to 4200 m asl) and Mount Everest (3800-5600 m asl) regions. Out of so far isolated 1500 isolated colonies, bacterial extracts of 850 colonies were prepared by using solid phase extraction of bacterial culture broth and screened for their antibiotic potential against human pathogenic two gram positive strains, Staphylococcus aureus ATCC 25923, Streptococcus pneumonia ACTC 49619 , and three gram negative strains, Escherechia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603. Out of 850 colonies tested, 161 strains have shown antibacterial activity against at least one targeted pathogen. The extract of 96 colonies were active against S. aureus, 26 colonies were active against E. coli, 39 colonies were active against P. aeruginosa, 61 colonies were active against S. pneumonia and 52 were active against K. pneumoniae. During this research project period, the isolated selected most active 100 strains will be characterized by using gene sequencing of the 16S rRNA gene. The active extract will be subjected to de-replication analysis using HPLC-HRMS experiments and GNPS platform. Thus obtained, potential selected isolates will be subjected to mass culture and antibiotic molecules will be isolated by using bioassay-guided fractionation and various chromatographic techniques including size exclusion chromatography, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Finally, the obtained active pure fractions will be characterized by using various spectroscopy experiments such as UV, IR, NMR, and X-Ray crystallography. Whole genome sequencing of two selected Actinomycetes strains among others will be done and genome mining analysis will be carried out to analyze the responsible gene clusters and biosynthetic pathways of identified antibiotic molecules. With the resources and capacity developed with this research project, we will continue antibiotics drugs discovery research from the active Actinomycetes collected in the Actinomycetes culture collection of the Central Department of Botany, Tribhuvan University, Kathmandu, Nepal in the future.