Cinnamic and gallic acids

First, cinnamic and gallic acids (LOBA Chemie, Boisar, India) stock solutions were prepared (Daneshfar et al., 2008 ; Bradley et al., 2015 (link)). In brief, 1.5 g of cinnamic acid was dissolved in 10 ml dimethyl sulfoxide (DMSO); then, distilled water was added to 100 ml, sonicated on water bath for 2 h at 80°C, and 0.1 N NaOH was added dropwise till complete solubilization, then distilled water was added to the final volume (200 ml). For gallic acid, 4 g was dissolved in 150 ml distilled water, sonicated on water bath for 30 min; then distilled water was added to the final volume (200 ml). Next, 100 μl of cation adjusted Mueller Hinton broth CAMHB (Hi-Media, Mumbai, India) was dispensed in each well of a 96-well microtiter plate. Then, twofold serial dilutions of cinnamic or gallic acid solutions were prepared in the culture media at a final volume of 100 μl. Afterward, the wells were inoculated with 100 μl of an overnight culture of the test isolate adjusted to 0.5 McFarland standard, and plates were incubated at 37°C for 18–24 h (Bardbari et al., 2018 (link)). The positive control wells were the bacteria in CAMHB and the used solvent without cinnamic nor gallic acids. While wells without bacterial inoculum served as negative controls. The MICs of cinnamic and gallic acids were recorded as the lowest concentrations that totally inhibit visible bacterial growth.

Amikacin was purchased from Eipico Co., Tenth of Ramadan City, Egypt; imipenem from Merck & Co., Kenilworth, NJ, USA; colistin, doxycyclin and levofloxacin from Sedico Co., Giza, Egypt. Cinnamic and gallic acids were obtained from Loba Chemie, Boisar, India, and dissolved in dimethyl sulfoxide DMSO (Fisher Scientific, Fair Lawn, NJ, USA) and distilled water, respectively. Cation-adjusted Mueller Hinton broth (CAMHB) and trypticase soy broth (TSB) were from Hi-Media, Mumbai, India.