Harnessing Molecular and Bioactivity Network Analysis to Prioritize Antibacterial Compound Isolation From Ant‐Associated Fungi

ABSTRACT Introduction Antimicrobial resistance is a global public health problem that requires the development of new bioactive compounds. In this context, metabolomic analyses can expedite the research of fungal metabolites as a valuable resource. Objectives To investigate the metabolic profiles and isolate antibacterial compounds from micromycetes associated with Mexican cloud forest ants by utilizing network analysis of their chemical and bioactivity data. Material and Methods 248 fungal strains isolated from six ant's species, soil of their anthills, and soil of the surroundings were evaluated for their in vitro inhibition growth of extensively drug‐resistant Acinetobacter baumannii and hypervirulent Klebsiella pneumoniae ; subsequently, their metabolites were dereplicated and analyzed by molecular networking and compound activity mapping from spectrometric data. Prioritization of some fungi for isolation of their major constituents was performed, and their structures were established by spectroscopic and spectrometric analysis and their bioactivity determined. Results From the fungal collection, 15 secondary metabolites ( 1 – 15 ) were dereplicated, and 10 compounds ( 16 – 25 ), including the new ( E )‐tridec‐7‐ene‐3,5,6,10‐tetraol ( 25 ), were isolated from Ascomycetes of Trichoderma , Cladosporium , and Clonostachys genera. Compounds 16 – 18 stood out for being bioactive. This study is the first report of antibacterial activity against A. baumannii for the tricyclic pyridin‐2‐ones deoxy‐PF1140 ( 16 ) and PF1140 ( 17 ), with minimum inhibitory concentration of 50 μg/mL. Conclusion Network analysis and dereplication proved effective in bioprospecting for antibacterial compounds, offering valuable insights into the chemical diversity of cloud forest soil fungi and their potential applications. Moreover, this study broadens the knowledge of fungal secondary metabolites linked to leafcutter, fire, and warrior ants.