Acinetobacter baumannii

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.

Escherichia coli, Acinetobacter baumannii, and Enterobacter cloacae strains with ATCC numbers were obtained from the American Type Culture Collection (ATCC). Acinetobacter baumannii and Klebsiella pneumoniae strains with KCTC numbers were obtained from the Korean Collection for Type Cultures (KCTC). Strains with names starting with F were isolated from clinical patients and were kind gifts from Professor Kwan Soo Ko (Sungkyunkwan University). Strains with names commencing with K were isolated from clinical patients and generously donated by Professor Min Sang Shin (Kyungbuk National University). For cloning purposes, Escherichia coli DH5a was used. For the overexpression of proteins, E. coli strains BL21(DE3) pLysS (Invitrogen, Seoul, Republic of Korea), or BL21(DE3) Star (Invitrogen, USA) were used. Bacteria were grown in either Luria Bertani (LB) medium (Duchefa Biochemie, Haarlem, The Netherlands) or CAA medium (5 g/L Casamino acids, 5.2 mM K₂HPO₄, and 1 mM MgSO₄).

The gram-negative bacterial strains Escherichia coli (KCTC 1682) and Acinetobacter baumannii (KCCM 40203) were purchased from the Korean Collection for Type Cultures (Korea) and the Korean Culture Center of Microorganisms (Korea), respectively. The MDR gram-negative strains Escherichia coli CCARM 1229, Escherichia coli CCARM 1238, Acinetobacter baumannii CCARM 12010, and Acinetobacter baumannii CCARM 12220 were obtained from the Culture Collection of Antibiotic-Resistant Microbes at Seoul Women’s University (South Korea).
The antibacterial properties of all peptides were determined by measuring the Minimum Inhibitory Concentration (MIC) as described previously [22 (link)]. In brief, the MIC was defined as the concentration of peptides that inhibited 99% of the bacterial growth. Two-fold serial dilutions of respective peptides were inoculated in 96-well plates in Mueller-Hinton medium, followed by the addition of bacterial suspensions (2 × 10⁵ CFU/ml) at the log growth phase. The plates were incubated at 37°C for 16 h, and absorbance was read at 600 nm using a SpectraMAX microplate reader (Molecular Devices, USA).