Materials and bacterial strains
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), chloramphenicol (CHL), ciprofloxacin (CIP), oxacillin (OXA), pepsin (EC 3.4.23.1, Sigma), trypsin (EC 3.4.21.4, Sigma), α-chymotrypsin (EC 3.4.21.1, Sigma), and proteinase K (EC 3.4.21.64, Sigma) were supplied from Sigma-Aldrich (St. Louis, MO, USA). HyClone Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from SeouLin Bioscience (Seoul, Korea). Multidrug-resistant Pseudomonas aeruginosa (MDRPA) (CCARM 2109) and multidrug-resistant Escherichia coli (MDREC) (CCARM 1229) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) of Seoul Women’s University in Korea. Pseudomonas aeruginosa (KCTC 1637) were procured from the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB).
Antimicrobial activity assay (MIC determination)
The minimal inhibitory concentrations (MICs) of SAMP-5 and melittin against MDREC and MDRPA were determined via the microbroth dilution method according to Clinical and Laboratory Standards Institute guidelines (Chou et al. 2016). In brief, mid-logarithmic phase of bacteria were diluted with Mueller-Hinton broth (MHB) (Difco, USA) and added to a microtiter plate (2 × 106 CFU/well). A two-fold serial dilution of samples was subsequently added, and the plate was incubated for 24 h at 37 °C. The experiment was performed in triplicate using three replicates for each sample and each bacterium. The lowest peptide concentration which gave no visible growth is determined as the MIC value.
Protease resistance assay
MDRPA (CCARM 2109) was grown overnight to stationary phase at 37 °C in 10 mL of Luria-Bertani (LB) medium (Difco, USA). The overnight cultures were 10-fold diluted in fresh LB broth and incubated for additional 3 h at 37 °C to obtain mid-log phase organisms. A bacteria suspension (2 × 106 CFU/mL in LB) was mixed with 0.7% agarose, poured into a 10-cm Petri dish, and dispersed rapidly. Five microliters of an aqueous SAMP-5 and melittin stock solution (10 mg/mL) were added to 25 μL of trypsin (pH 7.4), α-chymotrypsin (pH 7.4), pepsin (pH 2.0), and proteinase K (pH 7.4) stock solution (0.2 mg/mL) in 50 mM Tris-HCl buffer, and incubated at 37 °C for 4 h. The reaction was stopped by freezing with liquid nitrogen, after which 30 μL of aliquots were added to each circle paper (6 mm in diameter) placed on the agarose plates, and then incubated at 37 °C overnight. The diameters of the bacterial clearance zones surrounding the circle paper were measured for the quantitation of inhibitory activities.
Cytotoxicity assay
To determine the cytotoxicity of SAMP-5, we used the MTT dye reduction assay against RAW264.7 (mouse macrophage), NIH-3T3 (mouse embryonic fibroblast), and SH-SY5Y (human bone marrow) cells as previously described (Rajasekaran et al. 2019). Briefly, the cells (2 × 104 cells/well in DMEM supplemented with 10% FBS) were placed into 96-well plates and incubated for 18–24 h in the presence of 5% CO2 at 37 °C. The cells were treated with different concentrations (1 μg/ml to 128 μg/ml) of the peptides for 24 h. Then, 20 μl MTT (5 mg/ml) reagent in DMEM was incubated for 3 h and formed formazan crystals were dissolved in 200 μl DMSO. Cell viability was calculated by measuring absorbance at 570 nm by a microplate ELISA reader.
Resistance assay
To assess the drug resistance inducing ability of SAMP-5, P. aeruginosa (KCTC 1637) was chosen as the model bacteria for further research by sequential passaging method (Kim et al. 2020). Briefly, the MIC were determined according the antimicrobial activity assay, as described above. Then, the bacteria from the sub-MIC (0.5 × MIC) well were cultured overnight in fresh MHB medium and re-measured MICs. The next sub-MIC (0.5 × MIC) inoculum continued to re-measure MICs and was repeated for 7 days. Ciprofloxacin served as control in parallel cultures.
Checkerboard assay
The checkerboard titration method is performed to evaluate the combinatorial effects of SAMP-5 and conventional antibiotics (chloramphenicol, ciprofloxacin, and oxacillin) as described elsewhere (Wu et al. 2017; Qu et al. 2020). First, 2-fold serial dilutions of SAMP-5 and each antibiotic were prepared. Subsequently, 50 μl of each of different concentrations of SAMP-5 and each antibiotic was mixed and added into 100 μl of bacterial solution (containing approximately 0.5–1 × 106 CFU/mL) in each well of 96-well plate. The plates were then incubated in a shaking incubator at 37 °C for 24 h. Bacterial growth was assessed spectrophotometrically at A600nm using microplate ELISA reader (EL800, Bio-Tek instrument). The fractional inhibitory concentration (FIC) index (FICI) was calculated as follows: FICI = [(MIC of SAMP-5 in combination)/(MIC of SAMP-5 alone)] + [(MIC of antibiotic in combination)/(MIC of antibiotic alone)]. Where FICI ≤ 0.5 is considered to indicate synergy; 0.5 < FICI ≤ 1.0 is considered additive; 1.0 < FICI ≤ 4.0 is considered indifferent; and FICI > 4.0 is considered antagonism.
