Escherichia coli

We purchased Escherichia coli (KCTC1682), Salmonella typhimurium (KCTC 1926), Staphylococcus aureus (KCTC 1621), Bacillus subtilis (KCTC 1021), and Propionibacterium acnes (KCTC 3314, KCTC 3220, KCTC 5527, and KCTC 5933) from the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience & Biotechnology (Daejeon, Korea). S. aureus (CCARM 0027 and CCARM 3708) was obtained from the Culture Collection of Antimicrobial Resistant Microbes (Seoul, Korea). The MIC of phloretin against each of these bacteria was determined by broth microdilution assay [43 (link)]. We defined the MIC as the lowest concentration of phloretin that completely inhibited bacterial growth; this was calculated as the average of three independent measurements in the range of 0.5 to 512 µM. P. acnes was grown for 48 to 72 h at 37 °C on sheep blood agar under anaerobic conditions in a mixture of H₂ and CO₂ (95:5, v/v). Microbroth dilution using Muller–Hinton (MH) broth was used to determine the efficacy of phloretin against P. acnes. We diluted a bacterial suspension in MH broth to achieve turbidity equivalent to a McFarland standard of 0.5 (~1 × 10⁸ cells/mL), yielding 1 × 10⁴ cells/mL in each well after inoculation. Phloretin MICs were determined after incubation for 48 h at 37 °C.

Hen egg white lysozyme, lysostaphin, nisin, mutanolysin, and melittin were purchased from Sigma Aldrich. The following bacteria were used for antimicrobial activity assays and RNA isolation: Staphylococcus aureus ATCC 6538 (American Type Culture Collection, Manassas, VA, USA), Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC 29212, Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Streptococcus equi subsp. zooepidemicus ATCC 43079, Streptococcus iniae KCTC 3657 (Korean Collection for Type Cultures, Daejeon, Korea), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Salmonella typhimurium ATCC 14028. E. faecalis and streptococci were cultured in brain heart infusion broth (BD Bioscience) because of their slow growth in LB medium. All other bacteria were cultured in LB medium.

Four Gram-negative bacteria species (Escherichia coli KCTC 1682, Acinetobacter baumannii KCTC 2508, Pseudomonas aeruginosa KCTC 1637 and Salmonella typhimurium KCTC 1926) and three Gram-positive bacteria species (Staphylococcus aureus KCTC 1621, Bacillus subtilis KCTC 3028 and Staphylococcus epidermidis KCTC 1917) were procured from the Korean Collection for Type Cultures (KCTC). Multi-drug-resistant Gram-negative bacteria (Salmonella typhimurium CCARM 8003 and 8007, Escherichia coli CCARM 1229 and 1238, Pseudomonas aeruginosa CCARM 2002 and 2095 and Acinetobacter baumannii CCARM 12035 and 12036) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM). To determine MICs, bacteria were cultured in Luria-Bertani (LB) medium for overnight at 37 °C. An aliquot of the culture was incubated in 1% peptone media at 37 °C until mid-log phase. 100 μl of serial 2-fold diluted peptides were treated in 96 well plates and added to 100 μl of 2 × 10⁶ CFU/ml bacterial suspensions in 1% peptone media for 16 h at 37 °C. The lowest concentration of peptide that completely inhibited bacterial growth was determined to be the MIC.