Antibacterial Activity of Extract, Fractions, and Compounds from <em>Termitomyces clypeatus</em> R. Heim (Lyophyllaceae) Against Multidrug-Resistant Bacteria Overexpressing Efflux Pumps

Background/Objectives: Microbial resistance to antibiotics has become a major global public health problem, threatening the effectiveness of current therapeutic strategies. The present study seeks to investigate natural compounds originating from fungal sources for their ability to interfere with efflux pump–mediated resistance in multidrug-resistant (MDR) bacteria, with the overarching goal of uncovering new candidates for antimicrobial therapeutic development. A chemical investigation of the ethanol extract of Termitomyces clypeatus was carried out to isolate and identify its constituents. Methods: Structural elucidation of the isolated metabolites was achieved through 1D and 2D NMR spectroscopy supported by mass spectrometric data. The crude extract and the purified compounds were then evaluated for their antibacterial activities individually, in the presence of an efflux pump inhibitor, and in combination with three antibiotics, using standardized microdilution assays. Results: Chromatographic separation of the extract yielded eleven known compounds including three sphingolipids: (9Z,12Z)-N-(1,3,4-trihydroxyoctadecan-2-yl)octadeca-9,12-dienamide (1), 2-hydroxy-N-(1,3,4-trihydroxyoctadecan-2-yl)hexadecanamide (2), and cerebroside B (3); four steroids: ergosterol (4), cerevisterol (5), ergosterol peroxide (6), and 5α,6α-epoxy-(22E,24R)-ergosta-8(14),22-diene-3β,7α-diol (7); one alkaloid: piperine (8); one carbohydrate: D-mannitol (9); and two phthalates: dimethyl phthalate (10) and bis(2-ethylhexyl) terephthalate (11). GC–MS analysis led to the identification of eight fatty acid derivatives (12–19). Sub-fraction A, along with compounds 3, 4, and 8 exhibited notable antibacterial activity against some tested strains with MIC values of 64 μg/mL. These compounds were identified as substrates of bacterial efflux pumps, and their presence enhanced the antibacterial effects of ciprofloxacin, doxycycline, and amikacin. Conclusion: The findings of the present work indicate that Termitomyces clypeatus contains antibacterial compounds with potential therapeutic value, both as standalone agents and as adjuvants that enhance the activity of conventional antibiotics.