Polyphenols or phenolic compounds are groups of secondary metabolites widely distributed in plants and found in olive mill wastewater (OMW). Phenolic compounds as well as OMW extracts were evaluated in vitro for their antimicrobial activity against Gram-positive (Streptococcus pyogenes and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae). Most of the tested phenols were not effective against the four bacterial strains when tested as single compounds at concentrations of up to 1000 μg mL−1. Hydroxytyrosol at 400 μg mL−1 caused complete growth inhibition of the four strains. Gallic acid was effective at 200, and 400 μg mL−1 against S. aureus, and S. pyogenes, respectively, but not against the gram negative bacteria. An OMW fraction called AntiSolvent was obtained after the addition of ethanol to the crude OMW. HPLC analysis of AntiSolvent fraction revealed that this fraction contains mainly hydroxytyrosol (10.3%), verbascoside (7.4%), and tyrosol (2.6%). The combinations of AntiSolvent/gallic acid were tested using the low minimal inhibitory concentrations which revealed that 50/100–100/100 μg mL−1 caused complete growth inhibition of the four strains. These results suggest that OMW specific fractions augmented with natural phenolic ingredients may be utilized as a source of bioactive compounds to control pathogenic bacteria.

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