Hydrolysable tannins from Pelargonium graveolens overcome bacterial virulence and resistance through membrane and motility disruption.

Pelargonium graveolens L'Hér. (Geraniaceae) is traditionally used for the treatment of microbial infections. Its flowers exhibit antibacterial activity, but the bioactive constituents and their modes of action against multidrug-resistant (MDR) pathogens remain insufficiently characterised. This study aimed to identify the antibacterial compounds from P. graveolens flowers and to elucidate their in vitro effects on virulence-related bacterial phenotypes against clinically relevant MDR bacteria. A methanolic flower extract was subjected to HPLC-based activity profiling, followed by preparative chromatographic purification. The gallotannins obtained from the active fractions were then evaluated against Gram-positive and Gram-negative MDR clinical isolates harbouring mecA/C, erm, blaOXA-48-like, blaCTX-M resistance genes and Panton-Valentine leukocidin (PVL). Antibacterial activity (MIC, IC50), biofilm formation, motility (swimming, swarming), efflux pump activity, membrane permeability and depolarization were assessed using biochemical, fluorescence-based and microscopic assays including cryogenic transmission electron microscopy (cryo-TEM). Penta-, hexa-, hepta-, octa- and nonagalloylglucose were identified as the main antibacterial constituents with broad-spectrum activity observed for pentagalloylglucose and higher-order gallotannins. Mechanistic investigations showed that at a concentration of 250 μg/mL, gallotannins significantly impaired bacterial motility, reduced biofilm formation under the tested experimental conditions in a strain-dependent manner, enhanced membrane permeability and depolarization, and strongly inhibited efflux pump activity, exceeding the response induced by the reference proton motive force disruptor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in some strains. Gallotannins from P. graveolens exhibited multiple in vitro antibacterial and anti-virulence-related effects, including disruption of membrane-associated functions, motility and efflux-mediated resistance, highlighting their potential as complementary antimicrobial candidates against toxin-producing MDR pathogens and setting the stage for further studies on cytotoxicity towards mammalian cells and selectivity to confirm their therapeutic relevance.