Staphylococcus aureus antibiofilm agents from Combretum species (Combretaceae) by metabolomics profiling

About 80 % of all chronic bacterial infections are related to biofilms. Among several bacteria capable of developing biofilms, Staphylococcus aureus and Pseudomonas aeruginosa are recognized as two major opportunistic pathogens associated with biofilm infections, especially those related to medical devices. In light of the exciting progress of metabolomics within the field of natural products, and given the pressing need for new alternatives to control bacterial pathogens, this study applied metabolomic profiling and statistical analyses to identify S. aureus or P. aeruginosa biofilm inhibitors from three Brazilian Combretum species. The extracts were obtained from leaves and bark of C. leprosum, C. laxum, and C. lanceolatum, by solvent extraction with hexane (Hx), dichloromethane (Dcm), and hydromethanolic (Me70). Me70 extracts from leaves of all tested species were active against S. aureus biofilm formation (inhibition rate of 92 % for C. lanceolatum, 80 % for C. leprosum, and 65 % for C. laxum), but they did not inhibit bacterial growth. Other extracts from C. leprosum also presented similar results, decreasing S. aureus biofilm formation without affecting cell growth (inhibition of 90 % for bark Me70 extract, 72 % for bark Dcm extract, and 69 % for leaf Hx extract). Dcm extract from C. leprosum leaves decreased biofilm formation in 69 % while having antibacterial activity (85 % inhibition of bacterial growth). None of the extracts were active against P. aeruginosa. Metabolomic analysis by LC-MS followed by statistical evaluation clearly evidenced chemical differences associated with the activity level of the extracts. Univariate analysis by Pearson's r correlation suggested thirty-five metabolites with positive correlation with S. aureus antibiofilm activity, including alkaloids, tannins, phenolic acids, sugars, and glycosylated and non-glycosylated flavonoids. We obtained in vitro confirmation that ellagic acid is a key component for antibiofilm activity by Combretum extracts against S. aureus, showing circa 70% inhibition. It is likely that ellagic acid prevents bacterial-material surfaces interaction, since it led to increased hydrophobicity in S. aureus surface at the same concentrations that affected biofilm formation (up to 0.1563 µM). Importantly, ellagic acid, and the 4 most active Combretum crude extracts, are not toxic as determined using Galleria mellonella larvae model. The lower selective pressure toward bacterial resistance is an attracti