Isolation and Characterization of Galloylglucoses Effective against Multidrug-Resistant Strains of Escherichia coli and Klebsiella pneumoniae

The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid-liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-beta-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-beta-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-beta-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-beta-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2-256 mu g/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.