Evaluation of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of polyphenols against gram-positive and gram-negative bacteria

The molecules listed in Tables 1 and ​and22 were assayed for their antibacterial activity against both gram-positive and gram-negative foodborne pathogens. Among gram-positive bacteria, we selected Listeria monocytogenes Scott A, Staphylococcus aureus ATCC 25923, Enterococcus faecium DSM 20477, Enterococcus faecalis DSM 20478, and Bacillus cereus DSM 9378. Among the gram-negative bacteria, we considered Pseudomonas aeruginosa ATCC 27853, Escherichia coli DSM 682, E. coli DSM 8579, Salmonella enterica DSM 9386, and Proteus hauseri DSM 30118. The antibacterial activity was determined using the standard microdilution method for drug susceptibility testing⁴⁵. The strains were cultivated aerobically in tryptic soy broth (TSB, Sigma Aldrich, Italy) for 24 h at 30–37 °C, except E. faecium DSM 20477 and E. faecalis DSM 20478, which were cultivated in microaerophilic conditions for 24 h at 30 °C. Briefly, the test was carried out in 96-well plates in a final volume of 200 µl in the presence of 10 increasing concentrations of each antimicrobial, ranging from 1 up to 512 µg/ml. The molecules were resuspended in dimethyl sulfoxide (DMSO, Sigma Aldrich, Italy) at a concentration of 4.096 mg/mL and then diluted 1:2 in sterile broth. The inoculum was prepared by diluting the cell suspension from an overnight culture in sterile TSB to obtain a turbidity equivalent to the McFarland 0.5 standard. Then, plates were incubated for 24 h at 30–37 °C. All plates included at least one well as a positive growth control (TSB with and without DMSO 12.5% v/v and the inoculum) and a negative growth control (TSB without inoculum), to exclude any microbial contamination⁴⁵. As reference biocide, we selected chlorhexidine for its broad spectrum of activity against Gram positive and Gram negative bacteria and its known ability to disrupt the bacterial cell membrane. The MIC is the lowest concentration of the antimicrobial that completely inhibits growth. In addition, we determined the MBC for each molecule tested. After shaking the 96-well plate to resuspend the cell pellet, MBC determination was performed by subculturing 10 μL from each well where no visible microbial growth occurred. After 48 h of incubation, the antimicrobial dilutions yielding three colonies or less were scored as the MBC for the starting inocula of 10⁵ CFU/mL. The experiments were performed in triplicate. The MIC and MBC experiments were performed according to the CLSI (Clinical and Laboratory Standards Institute) methods for dilution antimicrobial susceptibility tests for aerobic bacteria (approved standard, Wayne, PA, USA: CLSI; 2009).