Preparation of cLFchimera and antibiotics concentrations
The cLFchimera recombinant peptide was prepared from our previously study. A concentration of 1000 mg/ml of cLFchimera peptide was prepared in a sterile culture medium, filtered with 0.22 µm and used as a stock to prepare other dilutions . The antibiotics including, gentamicin, ceftazidime and cefazolin, which were prepared from Jaber Ebne Hayyan Pharmaceutical Company,Tehran, Iran used according to ClSI .
Preparation of inoculum
The bacterial strains were procured from microbial collection, Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad. Strains included were Pseudomonas aeruginosa PTCC 1707, Escherichia coli ATCC 25922, and Salmonella typhi PTCC 1609. Before the antimicrobial tests, The microbial strains were cultured 24 hours To standardize the microbial strains from the 0.5 McFarland standard, it was used according to its administration instructions, which was equivalent to 1.5 × 108 CFU/mL of microorganism .
Determination of minimum inhibitory concentration
MIC was performed using the micro broth dilution method as suggested by the Clinical and Laboratory Standards Institution . Dilutions of cLFchimera (1, 2, 4, 8, 16, 32, 64, 128, 256, 512 µg/ml L) were prepared in sterile Muller Hinton Broth (MHB) (Sigma-Aldrich). 10 µL of microbial suspensions with an optical density of 630 nm (OD630) equal to 0.08–0.13, was added to 190 µL each dilution in the 96 microwell plates. The microwell plates were incubated at 37 ˚C for 24 h. To determine the MICs, the absorbance was considered at 630 nm by ELISA reader model BioTek ELx808. The above protocol was repeated for each microorganism at same concentrations using gentamicin, ceftazidime and cefazolin, separately. Growth medium without inoculum was used for negative controls . In order to confirm the results, the experiments were repeated in three times.
Determination of minimum bactericidal concentration
10 µL of the culture from each well including cLFchimera and Antibiotics in which the microbial growth was not observed, was cultured on Mueller Hinton Agar (Sigma-Aldrich). Then, the plates were incubated at 37 °C for 24 h and MBC was defined as the lowest concentration at which no growth of microorganism was observed . For confirm the results, the experiments were repeated in three times.
Synergistic Interaction between cLFchimera and Antibiotic by Checkerboard Assay
The synergistic interaction between cLFchimera peptide and any of the antibiotics, were measured using checkerboard method [11, 12]. Then, seven number of two-fold serial dilutions (from 2MIC to MIC/32) of the cLFchimera and the antibiotics were prepared; according to obtained MIC in the previous section for each microorganism. To obtain a fixed amount of both antimicrobial peptides, equal amount (25 µl) of each dilution were poured into 96-well microplates. Therefore, each row (and column) contained a fixed amount of the first agent and increasing amounts of the second one. A total of 50 µl of fresh bacteria suspension (108 CFU/ml) were added to each wells and cultured at 37˚C. The Fraction Inhibitory Concentration index (FICI) was calculated using the following formula:
The above formula, MICA and MICB belong to compound A and B respectively. MICA/B belongs to the MIC of compound A in combination with B. Total synergism (FICI ≤ 0.5), partial synergism (0.5 < FICI ≤0.75), Indifference (0.75 < FICI ≤2) or antagonism (FICI >2) between the two compounds were obtained using FICI .
Antimicrobial Dynamics of the Antimicrobial Agents
The antibacterial dynamics were determined using a liquid culture inhibition assay according Liu at al. with minor modifications . A total of 50 µl of fresh bacteria (106-108 CFU/ml) were added to the wells of 96-well microplates, also 50 µl of the antimicrobial agent (at MICs or FICs concentrations), was added to each well. The bacterial strains were cultured at 37˚C for 24 h and the absorbance was measured at 630 nm for 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 hours after incubation. 50 µl of the microbial suspensions without antimicrobial agents was used as a control group. The antibacterial activity was calculated using the following formula:
The above formula A0 is the absorbance of the control group and A is the absorbance of the tested group.
The effect of cLFchimera and any of the antibiotics were evaluated separately and in combination on the growth of microbial strains using draw a survival curve . The final concentration of suspension of the strains (adjusted to 106-108 CFU/ml) were added to the wells of 96-well microplates, and 50 µl of the antimicrobial agent (at MICs or FICs concentrations), was added to each well. The bacterial strains were cultured at 37˚C for 36 h. After incubating, a 50 µl liquid from each dilution were spread on the surface of the agar plates and were incubated at 37 °C for 24 h, eventually, the number of CFU/ml was counted. It is worth noting 50 µl of the microbial suspensions without antimicrobial agents was used as a control group. Finally, survival curves were constructed by plotting the log number of CFU/ml against time (h).
Release of cytoplasmic material absorbing at 260 nm
The release of cytoplasmic material absorbing at 260 nm was measured according to the method described by Fadli et al. (2012). Viable cells in their exponential phase were collected using centrifugation (4000 rpm for 15 min.), washed three times, and resuspended in a saline buffer solution. Three milliliters of cell suspension of approximately 108 UFC/ml were incubated, under agitation,for 1 h at 37 ◦C in the presence of the antimicrobial agent (at MICs or FICs concentrations). After incubation, cells were centrifuged at 4000 rpm for 20 min, and the absorbance (260 nm) of the supernatant was determined using a WPA Lightwave S2000 UV/Vis Spectrophotometer (Richmond Scientific Ltd, England). The untreated cells (control) were corrected with buffer saline.
Scanning Electron Microscopy
The bacterial strains cultured to the logarithmic phase in 100 ml of MHB at 37 C. The suspension was divided into four portions. Antimicrobials were added to three of the portions at at MICs or FICs concentrations. The remaining portion was left untreated as a control. The resuspension was incubated at 37 C for 3 h, and subsequently the cells from all four tubes were harvested through centrifugation and fixed with 2.5% glutaraldehyde overnight at 4 C. Subsequently, the cells were dehydrated using sequential ethanol concentrations ranging from 30 to 100%. The samples were gold covered through cathodic spraying. The morphology of the bacterial cells was observed through scanning electron microscopy (SEM, LAO-1450VP, Germany)  .